research.vu.nl dissertation.pdf · VRIJE UNIVERSITEIT Osteoinductive bone substitutes ACADEMISCH...

Osteoinductive Bone Substitutes

Tie Liu

The following institutions generously funded printing of this thesis:

Academic Centre for Dentistry Amsterdam

VU University Amsterdam

Tie Liu

Osteoinductive bone substitutes

Thesis Amsterdam – With ref. – With Summary in Dutch

ISBN: 978-90-5383014-7

Copyright © 2013 by Tie Liu. All Rights Reserved.

No part of this book may be reproduced, stored in a retrievable system, or transmitted in

any form or by any means, mechanical, photo-copying, recording or otherwise, without

the prior written permission of the holder of copyright.

VRIJE UNIVERSITEIT

Osteoinductive bone substitutes

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aan

de Vrije Universiteit Amsterdam,

op gezag van de rector magnificus

prof.dr. F.A. van der Duyn Schouten,

in het openbaar te verdedigen

ten overstaan van de promotiecommissie

van de Faculteit der Tandheelkunde

op woensdag 18 september 2013 om 11.45 uur

in de aula van de universiteit,

De Boelelaan 1105

door

Tie Liu

geboren te Zhejiang, China

promotoren: prof.dr. D. Wismeijer

prof.dr. Z. Gu

copromotor: dr. Y. Liu

Dedicated to my wife Qian Lu

to my parents

Publications

1. Tie Liu, Bing Xia, Zhiyuan Gu. Inferior alveolar canal course: a radiographic study.

Clinical Oral Implant Research, 2009; 20: 1212–1218.

2. Tie Liu, Gang Wu, Daniel Wismeijer, Zhiyuan Gu and Yuelian Liu. Deproteinized

bovine bone functionalized with the slow delivery of BMP-2 for the repair of

critical-sized bone defects in sheep. Bone. 2013, 56: 110–118.

3. Xin Zhang&, Tie Liu

&, Yuanliang Huang, Daniel Wismeijer, and Yuelian Liu.

Icariin: Does It Have An Osteoinductive Potential for Bone Tissue Engineering?

Phytotherapy Research. 2013 Jul 4. doi: 10.1002/ptr.5027. [Published online]

(& contributed equally)

4. Tie Liu, Gang Wu, Yuanna Zheng, Daniel Wismeijer, Vincent Everts, and Yuelian

Liu. Cell-mediated BMP-2 release from a novel dual drug delivery system promotes

bone formation. Clinical Oral Implant Research 2013. [under revision]

5. Yuanna Zheng, Gang Wu, Tie Liu, Yi Liu, Daniel Wismeijer, and Yuelian Liu. A

novel BMP2-coprecipitated, layer-by-layer assembled biomimetic calcium

phosphate particle: a biodegradable and highly-efficient osteoinducer. Clinical

Implant Dentistry and Related Research, 2013 Mar 4. doi: 10.1111/cid.12050.

[Published online]

6. Jingxiao Wang, Yuanna Zheng, Juan Zhao, Tie Liu, Lixia Gao, Zhiyuan Gu and

Gang Wu. Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone

defects: a micro-CT evaluation. Journal of Clinical Periodontology 2012

Jan;39(1):98-105.

7. Tie Liu, Gang Wu, Yuanna Zheng, Daniel Wismeijer, and Yuelian Liu. A

biomimetic osteoinducer enhances the therapeutic effects of deproteinized bovine

bone in a sheep critical-sized bone defect (Ø8×13mm) model. 2013 [submitted].

8. Tie Liu, Gang Wu, Daniel Wismeijer, and Yuelian Liu. Osteoinductive biomimetic

bone substitute for the repair of critical-sized bone defects in sheep. 2013

[submitted].

9. Tie Liu, Gang Wu, Yuanna Zheng, Afsheen Tabassum, Daniel Wismeijer, Vincent

Everts, and Yuelian Liu. A single biomimetic calcium phosphate granule as a model

to deliver proteins. 2013 [submitted].

10. Tie Liu, Sven Bakx, Gang Wu, Leo van Ruijven, Daniel Wismeijer, and Yuelian

Liu. Cone-beam CT and micro-CT analysis of deproteinized bovine bone for the

repair of critical-sized bone defects in sheep. 2013 [in preparation].

11. Yuanan Zheng, Tie Liu, Zhiyuan Gu. Investigation of changes of articles on China

national academic stomatological conferences in last two decades. Stomatology

2007 (12): 643-645. (Chinese)

12. Qian Lu, Tie Liu. Progressive Studies on Effects of Traditional Chinese Medicines

on Differentiation, Proliferation and Bone Formation Gene Expression of

Osteoblasts. Journal of Zhejiang Chinese Medical University 2012(5): 609-612.

(Chinese)

CONTENT

Chapter 1 General introduction ……………………………………………….…1

Chapter 2 Cell-mediated BMP-2 release from a novel dual drug delivery system

promotes bone formation ...…………………………….….………….9

Chapter 3 Preparation and characteristics of osteoinductive biomimetic calcium

phosphate material: in vitro and in vivo study ……………………...29

Chapter 4 Osteoinductive biomimetic bone substitute for the repair of

critical-sized bone defects in sheep ...……………………………….47

Chapter 5 A novel BMP2-coprecipitated, layer-by-layer assembled biomimetic

calcium phosphate particle: a biodegradable and highly-efficient

osteoinducer ..…………………………………...…………………...65

Chapter 6 A biomimetic osteoinducer enhances the therapeutic effects of

deproteinized bovine bone in a sheep critical-sized bone defect

(Ø8×13mm) model ……………………………………………….…83

Chapter 7 Deproteinized bovine bone functionalized with the slow delivery of

BMP-2 for the repair of critical-sized bone defects in

sheep ……………………………………………………………….101

Chapter 8 Low-dose rhBMP2/7 heterodimer to reconstruct peri-implant bone

defects: a micro-CT evaluation ……………………….…………....123

Chapter 9 Icariin: does it have an osteoinductive potential for bone tissue

engineering? …………………………………………………….….137

Chapter 10 General discussion ...……………………………………………….161

Chapter 11 General summary ….……………………………………………….167

Dutch summary….………………………………………...…….….172

Acknowledgements ………..………………………………………177

Curriculum vitae ………………………...…………………………179

Abbreviations

ACP amorphous calcium phosphate

BCP biphasic calcium phosphate

BioCaP biomimetic calcium phosphate

BMP bone morphogenetic protein

BMP2-cop.BioCaP BMP-2-coprecipitated biomimetic calcium phosphate

BMSCs bone marrow stem cells

BSA bovine serum albumin

CaP calcium phosphate

CDHA calcium deficient hydroxyapatite

CPS calcium phosphate supersaturated solution

CSBD critical-sized bone defect

DBB deproteinized bovine bone

EDX energy-dispersive x-ray spectroscopy

FBGC foreign body giant cell

HA hydroxyapatite

ICA icariin

MNC multinucleated giant cell

OCP octacalcium phosphate

OPG panoramic radiograph

PBMCs peripheral blood mononuclear cell

PMMA poly methylene methacrylate

RANKL receptor activator for nuclear factor-κB ligand

SBF supersaturated body fluids

SEM scanning electron microscopy

TB trabecular bone

TCM traditional Chinese medicine

TCP tricalcium phosphate

TRACP tartrate-resistant acid phosphatase

VEGF vascular endothelial growth factor

XRD X-ray diffraction

1

Chapter 1

General Introduction

Chapter 1

2

GENERAL INTRODUCTION

The treatment of bone fractures and defects requires adequate volume of bone tissue

which is of paramount importance to achieve an excellent restoration. When the bone

defects are too large to be self-healed, bone grafting is required in order to fill the defect

[1, 2]. Bone grafts fill voids and serve as scaffolds to provide support, and therefore may

enhance the biological repair of the defect. Critical-sized bone defect (CSBD) is defined

as the intraosseous wound with the smallest size, which cannot spontaneously heal

completely without intervention [3]. Bone healing heals through the generation of new

bone rather than by forming fibrotic tissue. Usually, the fibrous connective tissue

regenerates faster than bone tissue and becomes dominant within the CSBD because of

the faster migration mechanism of fibroblasts compared to osteoblasts.

Bone grafting, as a common surgical procedure, is carried out in approximately 10%

of all skeletal reconstructive surgery cases [4]. Worldwide, more than 2.2 million grafting

procedures are performed annually [1, 5]. In most patients, the intervention therapies can

be unproblematically executed and the outcome is generally excellent [6, 7]. However,

there are still a significant number of eligible individuals with the existence of

well-recognized risk factors such as diabetes, local osteoporosis and metabolic bone

disorder. These risk factors are associated with poor activity of bone formation [2, 8].

Nevertheless, the expectations of patients and surgeons alike are continually rising, both

aspiring to a curtailment of the recovery phase and the postoperative period of functional

incapacity [9]. Consequently, these clinical, social and economic pressures make it

absolutely necessary to develop a simple, efficacious and cost-effective bone substitute to

expedite and augment bone formation.

Bone regeneration

Bone regeneration in large bone defects requires four critical elements: (i) osteogenic

cells (e.g. progenitor cells or osteoblasts); (ii) osteoinductive signals (growth factors); (iii)

an biocompatible, biodegradable and osteoconductive matrix (scaffold); and (iv)

adequate blood and nutrient supply [10]. Therefore, bone grafts are often associated with

the terms biocompatibility, biodegradability, osteoconductivity and osteoinductivity.

Good biocompatibility refers to the ability of a biomaterial to perform its desired function

with respect to a medical therapy, without eliciting any undesirable local or systemic

effects in the recipient or beneficiary of that therapy, but generating the most appropriate

beneficial cellular or tissue response in that specific situation, and optimizing the

clinically relevant performance of that therapy [11]. Ideal biodegradability refers to that

bone substitute can be degraded in short time, enabling bone remodeling, and

concomitantly replaced by bone tissue [12]. Osteoconductivity is the ability of the graft

to function as a scaffold to permit bone growth on its surface or for ingrowth of new bone

[13]. Osteoinductivity is the ability of a graft to stimulate primitive, undifferentiated and

pluripotent cells to develop into the bone-forming cell lineage, and consequently to

promote bone formation [13, 14].

Therefore, the perfect bone substitute should be osteoconductive, osteoinductive,

biocompatible and biodegradable. It should induce minimal or no fibrotic reaction,

General introduction

3

undergo remodeling and support new bone formation. From a mechanical point of view

bone substitutes should have similar strengths to that of the bone being replaced. Finally,

it should be cost-effective and ought to be available in the amount required.

Bone substitutes

Autografts

Autologous bone, mostly harvested from the iliac crest, is regarded as the gold standard

since it provides an osteoconductive 3-demensional scaffold for bone ingrowth,

osteogenic cells and osteoinductive growth factors [15]. However, the harvesting of

autologous bone prolongs the surgery and the graft amount may be insufficient.

Autograft harvesting is also associated with an 8-39% risk of complications, e.g.

infection, hematoma, nerve injury, cosmetic disadvantages, pain and morbidity of the

donor site [16]. Moreover, the irregular rate of resorption of the autologous bone may

require secondary corrective surgery or compromise the restoration rate [17]. It has been

reported that resorption rates for endochondral bone is up to 75% and rates of 20%-30%

for membranous bone autografts [18, 19].

Allografts

The allogeneic bone graft is obtained either from cadavers or living individuals from the

same species [20, 21]. It provides a good, natural, and bony scaffold. However,

allogeneic bone is still associated with risks such as disease transmission [22], variable

host immune response [23], toxicity associated with sterilization [24], and limited

supplies [25]. In some countries, the allografts are culturally unacceptable.

Xenografts

Xenografts are composed of tissue taken from another species (i.e. from an animal source,

usually bovine). The use of xenografts has the potential to reduce morbidity as harvest of

autogenous bone is unnecessary. The antigenic potential of xenografts can be diminished

or eliminated by chemical treatment. One of the most widely used xenograft in clinical

dentistry is deproteinized bovine bone (DBB, Bio-Oss®, Geistlich, Switzerland). It is

derived from a bovine source and is treated by a chemical extraction process to remove

all the organic components and pathogens [17]. In terms of its inorganic composition and

its isomeric crystalline dimensions, DBB has a physical and chemical structure similar to

that of natural bone [26]. It shows osteoconductive properties when it is in close contact

with the newly formed bone [27]. However, it was reported that DBB delays the early

bone formation [28] and lacks sufficient intrinsic osteoinductivity [29].

Synthetic calcium phosphate bone substitute

Technological evolution and better understanding of bone-healing mechanism resulted in

the development of numerous alternative bone substitutes. Calcium phosphate (CaP)

grafts have been widely used for bone regeneration in most trauma and orthopedic

surgery procedures when grafting is necessary to restore bone defects. The calcium

phosphate materials are available with different application forms, e.g. pastes, granules,

blocks, composites. Based upon their chemical composition, calcium phosphates can be

Chapter 1

4

classified as either hydroxyapatite (HA), beta-tricalcium phosphate (β-TCP), biphasic

calcium phosphate (BCP), amorphous calcium phosphate (ACP), carbonated apatite (CA)

or calcium deficient HA (CDHA) [4]. A further subdivision can be made between

ceramics and cements. A ceramic is defined as an inorganic, non-metallic solid prepared

by sintering [30]. The sintering process removes volatile chemicals and increases crystal

size, resulting in a porous and solid material. Cements consist of a mixture of calcium

phosphates which can be applied as a paste and harden in situ due to precipitation

reactions. Calcium phosphate cements/ceramics can be applied as carriers for drugs. In

general, ceramics show a higher initial release than cements that have a more sustained

release pattern.

It has been aware that the low degradability of sintered CaP material, and in particular

HA or CA forms a problem. It has been well known that the high porosity of implants has

benefits for bone formation inside the implant and increases degradation [12]. Porosity in

the CaP material can be introduced by leaching and sintering out of salt crystals or

polymeric microparticles/mold after which the CaP material remains. Complete

resorption in most cases is very difficult due to the crystalline architecture. The

combination of CaP and polymer can form a suitable scaffold for cells and serve as a

delivery vehicle of osteoinductive drugs. The addition of biodegradable polymers can

improve the degradability of the CaP materials and alter their mechanical/physical

properties.

The ideal osteoinductive bone substitute

Although most bone substitutes are osteoconductive to bone-forming osteoblasts, only a

limited number of osteoinductive materials are currently available on the market with

FDA approval [31]. It was reported that tricalcium phosphate (TCP) is osteoinductive as

a synthetic alternative to autologous bone grafting [32]. However, a fundamental

understanding of the term, osteoinductivity, is of critical importance. Osteoinductivity is

the ability of the material to induce de novo bone formation. The osteoinduction

phenomenon could be divided into 3 principles [31]: (1) mesenchymal cell recruitment;

(2) mesenchymal differentiation to bone-forming osteoblasts; and (3) ectopic bone

formation in vivo. The ability for a bone graft to induce new bone formation in an

intraosseous defect does not fully reflect its true osteoinductive property. The

osteoinductive property of a material is usually demonstrated by bone formation after

implantation in ectopic/nonosseous sites. There are two kinds of nonosseous sites that

can be used to test osteoinduction in vivo. One is to implant subcutaneously, and the

other is to implant into intramuscular site [33]. However, the subcutaneous and

intramuscular sites are different. The difference in osteoinduction could be related to the

partial pressure of oxygen or the blood supply in the intramuscular and subcutaneous

sites, and that immature mesenchymal cells in the muscle could more easily differentiate

into osteoblasts, leading to osteoinduction [34]. It was also demonstrated that even a

small amount (5μg) of recombinant human bone morphogenetic protein-2 (rhBMP-2)

induces new bone in the subcutaneous tissue, which has a lesser blood flow than the

muscle [34].


5

Biomimetic calcium phosphate for bone regeneration

Recently, the biomimetic calcium phosphate coating has been developed very well for the

slow delivery of growth factors. This coating usually included two layers: an amorphous

layer and a crystalline layer, both of which are calcium phosphate. The amorphous layer,

serving as a seeding layer, can strongly deposit on the underlying materials. Thereafter,

the crystalline layer, octacalcium phosphate serving as a three-dimensional reservoir for

carrying protein/drugs, can grow on the seeding layer. Therefore, this biomimetic coating

can be applied on a variety of materials such as titanium implant [35], polymer [36],

ceramic [37], zirconia [38], and deproteinized bovine bone [39]. At the same time,

growth factor or drugs have been incorporated into this coating, such as antibiotics, bone

morphogenetic protein-2 (BMP-2), and vascular endothelial growth factor (VEGF).

These bioactive agents incorporated in the latticework of crystalline calcium phosphate

of the coating presented a slow and sustained release manner [39, 40]. Such a slow

release has been shown to be beneficial for the effect of different growth factors such as

BMP-2 and VEGF. The slow delivery of BMP-2 from the coating enhances

osteoinduction [41], and the slow delivery of VEGF promotes vascularisation [40].

Objectives of the thesis

The general aim of this thesis includes 5 aspects:

1. To develop a biomimetic calcium phosphate (BioCaP) bone substitute as a dual

delivery model with two protein-delivery modes: one mode by which protein was

incorporated in the interior of BioCaP; and one by which protein was coated on the

outside of BioCaP. We hypothesize that using this model the release of the protein can be

sequential and slow, and that the two delivery modes of BMP-2 could efficiently

accelerate bone formation.

2. To develop particles of biomimetic BMP-2-coprecipitated calcium phosphate

(BMP2-cop.BioCaP). We hypothesize that these particles could serve as an independent

and biodegradable osteoinducer.

3. To evaluate the therapeutic effect of the deproteinized bovine bone functionalized

with coating-incorporated BMP-2 in the repair of critical-sized bone defect in sheep.

4. To delineate the dynamic micro-architectures of bone induced by low-dose bone

morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects compared to

BMP2 and BMP7 homodimer.

5. To determine the present evidence of the osteoinductive potential of a Chinese

traditional medicine, icariin.

Outline of this thesis

As an alternative of autograft, a biomimetic calcium phosphate (BioCaP) bone substitute

was developed with two protein-delivery modes: 1) internally-incorporated mode: protein

was incorporated in the interior of BioCaP; and 2) coating-incorporated mode: protein

was coated on the outside of BioCaP. Slow release of bioactive agents from bone

substitutes plays an important role in the treatment of bone defects. Resorbing cells such

as osteoclasts may accelerate the degradation of bone substitutes so as to elevate the

protein release. The cell-mediated protein release of the two modes of BioCaP was

Chapter 1

6

investigated. The in vivo bone formation and cell response were evaluated by histological

and histomorphometric analysis in an ectopic rat model (Chapter 2 and 3).

In addition, we evaluated the physical and chemical properties of BioCaP and the

ability for protein loading and release in a long period. A Micro-CT method for the

evaluation of graft material and bone has been applied by using a unique “onion-peeling”

algorithm and specific threshold settings (Chapter 3).

Furthermore, we hypothesized that BioCaP can be a synthetic alternative to

autologous bone grafting. The aim of Chapter 4 is to investigate the therapeutic

effectiveness of BioCaP with or without the two modes of BMP-2 in repairing a large

cylindrical bone defect in sheep.

To repair large-size bone defects, most bone-defect-filling materials in clinic need to

obtain osteoinductivity either by mixing them with particulate autologous bone or

adsorbing BMP-2. However, both approaches encounter various limitations. In Chapter 5,

we hypothesized that our novel particles of biomimetic BMP-2-coprecipitated calcium

phosphate (BMP2-cop.BioCaP) could serve as an independent and biodegradable

osteoinducer to induce bone formation efficiently for the bone-defect-filling materials,

e.g. deproteinized bovine bone (DBB). To enhance the therapeutic effect of DBB for

bone defect repair, BMP2-cop.BioCaP particles was mixed with DBB. In Chapter 6, we

investigated the therapeutic effect of BMP2-cop.BioCaP mixed with DBB in the

treatment of critical-sized bone defects in sheep.

As an alternative to an autologous bone graft, deproteinized bovine bone (DBB) is

widely used in clinical dentistry. Although DBB provides an osteoconductive scaffold, it

is not capable of enhancing bone regeneration because it is not sufficiently osteoinductive.

In order to render DBB osteoinductive, BMP-2 has previously been incorporated into a

three dimensional reservoir (a biomimetic calcium phosphate coating) on DBB, because

it can effectively promote the osteogenic response by the slow delivery of BMP-2. We

investigated the therapeutic effectiveness of such BMP-2/coating functionalized DBB

granules in repairing large cylindrical (critical-sized) bone defects in sheep (Chapter 7).

Heterodimeric BMPs exhibited several- or dozens-fold more effect than the respective

homodimers in inducing in vitro osteoblastogenesis. In Chapter 8, we hypothesized that

BMP2/7 heterodimer could facilitate more rapid bone regeneration in better quality than

BMP2 and BMP7 homodimers in a peri-implant bone defect model in minipigs.

Traditional Chinese Medicines (TCMs) have been recommended for bone

regeneration and repair for thousands of years. Icariin, a typical flavonol glycoside, has

been extracted from the Herba Epimedii (a native herb). Icariin can be locally delivered

by biomaterials and has an osteoinductive potential for bone tissue engineering. The

review in Chapter 9 focuses on the performance of icariin in bone tissue engineering and

blended the information from icariin with the current knowledge relevant to molecular

mechanisms and signal pathways. The osteoinductive potential and low price of icariin

make it a very attractive candidate as a substitute of expensive osteoinductive protein −

BMPs, or as a promoter to enhance the therapeutic effects of BMPs.


7

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9

Chapter 2

Cell-mediated BMP-2 release from a novel dual

drug delivery system promotes bone formation

Tie Liu, Gang Wu, Yuanna Zheng, Daniel Wismeijer, Vincent Everts, and Yuelian Liu.

Clinical Oral Implant Research, under revision, 2013.

Chapter 2

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ABSTRACT

Objectives: In this study, a novel biomimetic calcium phosphate bone substitute (BioCaP)

is introduced as a dual drug release system with two drug/protein delivery modes: protein

is incorporated into (i) the interior of BioCaP (an internal depot); and (ii) a superficial

calcium phosphate coating on BioCaP (a surface coated depot). Our aim is to investigate

each of the two delivery modes of BioCaP. Our hypotheses are that (i) both of the drug

delivery modes, in in vitro as well as in vivo environment, can achieve a sustained

cell-mediated protein release; and (ii) BioCaP with these two delivery modes with

incorporated bone morphogenetic protein-2 (BMP-2) promotes bone formation.

Materials and Methods: Tablets of BioCaP were prepared with different carrying modes

using bovine serum albumin (BSA) as model protein. The release of this protein was

analyzed. Next, granules of BioCaP with different carrying modes of BMP-2 were

implanted subcutaneously in rats. Samples were collected after five weeks for

histomorphometric analysis.

Results: In vitro data showed that the internal and surface coated depots of BSA resulted

in a sustained osteoclast-mediated release, while the adsorbed BSA was rapidly released

and this release was not affected by osteoclasts. In vivo data showed that the volume

densities of bone, bone marrow, and blood vessels were significantly higher in samples

where BMP-2 was incorporated internally or in the coating compared with granules with

adsorbed growth factor. Osteoclast-like cells were associated with the granules and

resorption lacunae were frequently observed.

Conclusion: It is shown that different modes of incorporation of BMP-2 on and in

BioCaP granules have a beneficial effect on the formation of ectopic bone. This dual

drug release system makes BioCaP granule a promising tool for delivering multiple

therapeutic agents for different clinical applications.

Keywords: Biomimetic; Calcium phosphate; Protein release; Osteoclast; Bone

regeneration; BMP-2.

INTRODUCTION

Calcium phosphate (CaP)-based biomaterials are widely used for the regeneration of

bone defects because of their similarity to bone, good biocompatibility,

osteoconductivity and unlimited availability [1-5]. Currently, the major focus is to

utilize CaP biomaterials as a drug delivery system by integration of different bioactive

agents [6, 7]. By integrating bioactive agents, CaP biomaterials can acquire additional

properties such as anti-infection [8], osteoinduction [9, 10], and anti-cancer properties

[11, 12]. The therapeutic effect of these bioactive agents is highly dependent on their

release kinetics [12, 13]. Usually, superficial adsorption results in a rapid and passive

release which limits the effectiveness of many bioactive agents [7, 14-16], while a

controlled release can optimize the therapeutic effects [11, 17, 18].

A controlled release of protein from CaP biomaterials can be achieved in various

ways. Biomimetic coating is one of the attractive approaches for achieving a controlled

release of protein/drug [17] and this coating seems to provide better results than

materials, e.g. (bio) polymers that are physically or chemically mixed with a

Cell-mediated BMP-2 release from BioCaP

11

ceramic/cement compound [5, 19, 20]. This biomimetic approach is to precipitate

protein and CaP together in simulated body fluids under physiological conditions (37°C)

[21, 22]. Consequently, a thin layer of biomimetic CaP coating with incorporated protein

is formed on biomaterials.

The protein release kinetics from carriers has usually been investigated by

incubating them in physiological solutions such as cell culture media, phosphate

buffered saline or simulated body fluid [5, 15, 23]. However, once a biomaterial is

introduced into the body multiple factors might affect the protein release, such as

cellular invasion and interstitial body fluid flow [24]. Therefore, in vivo, apart from the

solubility of the biomaterials (physicochemical dissolution), cell-mediated resorption

also plays a critical role in the degradation of the biomaterials [25-27]. This influences

the protein release [28]. The cells involved in the degradation are mainly osteoclasts,

foreign body giant cells, macrophages, and monocytes [24, 29]. It is important to

understand how and to what extent these cells might influence the protein release from a

drug-delivery system [28, 30]. Such a study may provide a guideline to predict the in

vivo protein release kinetics.

Recently, we have made a breakthrough in modifying the biomimetic coating

approach. We have for the first time developed a novel biomimetic CaP bone substitute

(BioCaP) as a dual release system. In this system protein and calcium phosphate were

precipitated together to form BioCaP granules in which a depot of protein was

incorporated in the center of the granules as an internal depot. Next, protein and calcium

phosphate were co-precipitated onto the surface of these granules, thus creating a

surface coated depot. This dual system provides an ideal model for delivery of different

protein/drugs in two phases, an initial slow delivery phase (surface coated depot) and a

delayed phase (internal depot). By adopting this system, a single drug can be

administered in a more consistent manner due to this dual phase release or two different

drugs can be administered simultaneously. Therefore, BioCaP granule might be

considered as a promising tool for the orderly delivery of multiple therapeutic agents,

such as antibiotics, osteogenic agents, and anti-cancer drugs for different clinical

applications.

There is, however, a need to study the delivery modes of this biomaterial to evaluate

the amount and extent of cell mediated drug release during each phase. Our aim in the

present study is to investigate each of the two delivery modes of BioCaP. Our

hypotheses are that (i) both of the drug delivery modes, in in vitro as well as in vivo

environment, can achieve a sustained cell-mediated protein release; and (ii) BioCaP

with these two delivery modes with incorporated bone morphogenetic protein-2 (BMP-2)

promotes bone formation. For this purpose, BioCaP tablets will be coated with labelled

bovine serum albumin (BSA) to analyze protein release in vitro and BioCaP granules

will be coated (incorporated) with BMP-2, and implanted subcutaneously in rats to

analyse their capacity to induce bone formation in vivo.

MATERIALS AND METHODS

In vitro investigation

Fabrication of BioCaP

According to the biomimetic coating principle [21, 22], a supersaturated CaP

Chapter 2

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solution [200 mM HCl, 20 mM CaCl2·2H2O, 680 mM NaCl, 10 mM Na2HPO4, and 250

mM Tris (pH 7.4)] was incubated in a shaking water bath (50 agitations/min) at 37°C.

Protein was added to this CaP solution and co-precipitated (incorporated) into the

interior of BioCaP (viz., internal depot of protein). After 24 hours of incubation, the

precipitation was retrieved, gently washed by Milli-Q water, filtered and compressed to

form a tablet (diameter: 5mm; thickness: 0.4 mm) using a vacuum exhaust filtering

method with a vacuum filter (0.22-μm pore, Corning, NY, USA) and an air pump. After

drying at room temperature, BioCaP tablets were used as such or ground into granules

of different sizes. For sterilization, all the solutions were filtered with the vacuum filter

(0.22-μm pore) before co-precipitation. All the procedures were performed under aseptic

conditions.

In this study, BioCaP tablets were used to investigate the in vitro cell-mediated

release kinetics because cell seeding was easy on these tablets with their flat surface,

and BioCaP granules were used for the in vivo investigation. The tablets and granules

have the same physicochemical properties. It has been proven that the tablets and

granules have the same surface structure by scanning electron microscopy, and the

protein loading did not affect the surface structure of BioCaP.

Biomimetic coating procedure

To introduce the surface coating of protein, the BioCaP was immersed in the CaP

coating solution [40 mM HCl, 4 mM CaCl2·2H2O, 136 mM NaCl, 2 mM Na2HPO4, and

50 mM Tris (pH 7.4); total volume of 20 ml] for 24 hours at 37°C according to a

biomimetic coating protocol [21, 31]. Protein was added to this CaP solution and

thereafter co-precipitated in the CaP coating on the surface of BioCaP (viz., surface

coated depot of protein).

Distribution of protein

To confirm that the protein was incorporated into BioCaP, four BioCaP tablets were

prepared with an internal or surface coated depot of bovine serum albumin which was

labelled with fluorescein-isothiocyanate (FITC-BSA, 5.0μg/ml, Sigma, St. Louis, MO,

USA). The distribution of FITC-BSA in BioCaP tablets was studied by analysing cross

sections of the tablets. The samples were embedded in methylmethacrylate, sectioned,

and ground [23]. 80-µm-thick sections were prepared and examined with a fluorescence

microscopy (Leica, Wetzlar, Germany). The surface and cross-section morphology of

BioCaP tablets was also investigated by scanning electron microscopy (SEM, XL20,

FEI Company, The Netherlands) at an accelerating voltage of 10 kV.

Cell-mediated protein release kinetics

Two different concentrations of BSA were incorporated into BioCaP tablets to study

the protein release. Apart from FITC-BSA with a concentration of 5.0μg/ml, BSA

labelled with Alexa Fluor® 555 (Alexa-BSA, invitrogen, Carlsbad, CA, USA) was used

at a concentration of 0.5μg/ml, since FITC-BSA release from the samples with 0.5μg/ml

is out of the detection range. According to the manufacture’s protocol Alexa-BSA is

more easily detected at lower concentrations than FITC-BSA.

Six groups were established for in vitro cell-mediated release: (1) BioCaP tablets

with an internal depot of FITC-BSA; (2) BioCaP tablets with an internal depot of


13

Alexa-BSA; (3) BioCaP tablets with a surface coated depot of FITC-BSA; (4) BioCaP

tablets with a surface coated depot of Alexa-BSA; (5) BioCaP tablets with adsorbed

FITC-BSA; and (6) BioCaP tablets with adsorbed Alexa-BSA. To adsorb BSA, BioCaP

tablets were immersed in an aqueous protein solution (total volume of 20 ml per tablet)

for 24 h at 37°C in plastic tubes.

Passive and cell-mediated release of the variously labelled BSAs from the BioCaP

tablets was monitored over a period of 16 days. For a cell-mediated release, bone

marrow cells (BMC) were harvested from femurs and tibias of 6-week-old male mice

and 1×106 BMCs were seeded on the tablets. The tablets were cultured in duplicate in

α-MEM (Gibco BRL) containing 10% fetal calf serum (FCS) and 1%

penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively) with M-CSF (25 ng/ml,

R&D Systems, Minneapolis, MN, USA) and RANKL (40 ng/ml, PreProtech, Rocky

Hill, NJ, USA) [32]. BioCaP tablets without BMCs were incubated in α-MEM culture

media to investigate the passive (spontaneous) release of BSAs. The culture medium

was refreshed at 3-day intervals and used for spectrophotometric analysis (n=6 per time

point) in a Fluorimeter (Spectramax M2, Molecular Devices, CA, USA), using 490 nm

excitation and 504 nm emission wavelengths for FITC-BSA and using 540 nm

excitation and 570 nm emission wavelengths for Alexa-BSA. Fluorescence readings

were converted into the amount of protein by using a standard curve that was generated

from a dilution series of labelled BSA prepared in 2 mL PBS. In this study, we chose

osteoclasts for the analysis, since it has been demonstrated that the

monocytes/macrophages has no significant effects on the release of

coating-incorporated protein (Wernike et al. 2010a).

At the end of the release experiments, the residual BSA in the BioCaP tablets was

determined by dissolving the materials in 0.5 M ethylenediamine tetraacetic acid (EDTA,

pH 8.0). The percentage of BSA released from the BioCaP tablets was calculated using

the formula: [amount of the released fraction of BSA / total amount of BSA (amount of

the released fraction + amount of the residual BSA of BioCaP tablets) ×100]. All cell

culture experiments were performed at least three times.

Tartrate resistant acid phosphatase (TRACP), which is a marker enzyme for

osteoclasts, was used to identify the presence and number of these cells in the cultures.

The tablets with cultured cells were washed with phosphate buffered saline (PBS) and

fixed in 4% PBS buffered formaldehyde for 5 min and then stained for TRACP activity

using the leucocyte acid phosphatase kit (Sigma). The nuclei were stained by incubating

the cell cultures with diamidino-2-phenylindole-dihydrochloride (DAPI) in PBS. The

number of TRACP+ cells with three or more DAPI

+ nuclei was counted.

Tablets with cells were also fixed, dried, and sputter-coated for SEM investigation

[28]. To monitor the dissolution of BioCaP, the BioCaP tablets with or without cultured

cells were gently washed with water, dried and weighted at each time point.

In vivo investigation Experimental animal model

Adult male wistar rats (200–220g) were used as an animal model for ectopic bone

formation.[33] A total of 30 rats were used, which was approved by Ethical Committee of

School of Stomatology, Zhejiang Chinese Medical University. All the animal experiments

were carried out according to the ethics laws and regulations of China. Throughout the

Chapter 2

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study, the rats were treated following the guidelines of animal care established by

Zhejiang Chinese Medical University

Grouping

Two experimental and three control groups were analyzed (n=6 animals per group).

The groups were: (1) BioCaP granules with an internal depot of BMP-2 (BioCaP int.

BMP-2, experimental); (2) BioCaP granules with a surface coated depot of BMP-2

(BioCaP surf. BMP-2, experimental); (3) BioCaP granules with adsorbed BMP-2

(BioCaP ads. BMP-2, control); (4) BioCaP granules without a CaP coating or BMP-2

(BioCaP, control); and (5) BioCaP granules with a CaP coating but no BMP-2 (BioCaP

CaP, control).

Human recombinant BMP-2 (INFUSE® Bone Graft, Medtronic, USA) was

introduced into the CaP solution or the coating solution at a concentration of 1 μg/ml.

The amount of incorporated BMP-2 was determined using the ELISA technique [23].

About 35-μg of BMP-2 was finally incorporated into each sample of group (1) and (2).

Hence, for group (3) as a control, 35-μg of BMP-2 was likewise loaded (0.22g of BioCaP

granules) by adsorption [23].

Surgery and histology

The surgery was performed under conditions of general anaesthesia using Sumianxin

II (purchased from the Military Veterinary Institute, Quartermaster University of PLA,

Chang Chun, China). Two samples of 0.22g of BioCaP granules were implanted in dorsal

subcutaneous pockets in each rat, one on the left side and one on the right according to a

random protocol as used in the previous studies [23, 34]. The samples were trapped by

suturing the incision. .

Five weeks after implantation, the samples were collected, fixed and embedded as

previously reported [13, 35]. Applying a systematic random sampling [36], the samples

were sawn vertical to the short axis, into 10–12 slices of 600 μm-thickness, 1 mm apart.

All the slices of each sample were separately mounted on Plexiglas holders and polished.

The thickness of the final histologic sections after polishing is about 500μm. Then they

were surface-stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue [23]

for the histological and histomorphometric analysis.

Histomorphometric analysis

In this study, the space under the fibrous capsule that embraced the whole block of

implants (subcapsular space) was taken as the reference space, as described in a previous

study [23]. The reference space was estimated using Cavalieri’s methodology [37]. This

involves measuring the cross sectional area of a defined number of tissue sections

separated at a fixed distance through the reference volume. The cross sectional area of

each section was estimated using a point-counting technique [38].

The volume densities of both the unmineralized and mineralized newly formed bone,

bone marrow, blood vessels, fibrous capsular tissue, and multinucleated giant cells (on

the surface of BioCaP) within the reference space were assessed using the point-counting

technique [38]. The unmineralized bone is defined as the new bone with less density than

the mineralized bone [13]. The volume density of a component (Va) is defined as its

volume (Vb) per unit volume of reference space (Vc): Va=Vb/Vc.


15

To evaluate the degradation of BioCaP, the volume of BioCaP before implantation

(Da) and after 5 weeks of implantation (Db) was measured by the same histological

method. Six samples were used for the measurement of the BioCaP volume before

implantation. The percentage of non-degraded BioCaP (Dc) was defined as:

Dc=(Da/Db)×100%.

Statistical analysis

All data were presented as mean values and standard deviation (SD). The data were

evaluated statistically using by a one way analysis of variance (ANOVA) using SPSS

statistical software (version 16.0 for Windows). Post-hoc comparisons were made using

Bonferroni's corrections with the level of significance set at p< 0.05.

RESULTS

In vitro results

Surface topography and protein distribution

There was no significant difference of the surface topography between BioCaP

granule and table (Fig. 1). The cross section of BioCaP tablets showed that the

FITC-BSA incorporated internally was distributed throughout the whole volume of the

tablet in a net like configuration (Fig. 2A), while the surface coated FITC-BSA was

found only in the crystalline surface coating of the tablet (Fig. 2B). The representative

SEM micrographs depicted the cross section of BioCaP tablet with the internally

incorporated FITC-BSA (Fig. 2C) and the crystalline coating with incorporated

FITC-BSA (Fig. 2D).

Figure 1. SEM micrographs of the surface of BioCaP granule (A) and tablet (B).

Chapter 2

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Figure 2. Fluorescence micrographs of BioCaP tablets with two different protein-carrying modes:

A: FITC-BSA (green) distributed throughout the tablets (an internal depot); B: FITC-BSA

distributed in the crystalline coating layer (a surface-coated depot); C: Scanning electron

micrographs of sectioned BioCaP tablet (the center part); D: Scanning electron micrographs of a

section of the crystalline coating layer on the surface of BioCaP tablet. Bar= 100μm in (A). Bar=

20μm in (B) and (D). Bar= 10μm in (C).

Passive release of BSAs from BioCaP tablets

The passive release, that is without cells, of BSAs [FITC-BSA (5.0μg/ml) and

Alexa-BSA (0.5μg/ml)] from BioCaP tablets was monitored over a period of 16 days

(Fig. 3). A burst release occurred in all groups within the first 4 days of incubation. The

internal or the surface coated depot had a significantly lower burst release than the

adsorbed fraction in the 4 day period. No significant difference was found between the

internal and the surface coated depots of protein. The adsorbed depot of BSAs was

released rapidly within 16 days, whereas the release of the internal or surface coated

depots occurred at a steady rate after the 4th day up to the 16th day. Alexa-BSA had a

significantly lower burst release than FITC-BSA within 4 days in all groups.


17

Figure 3. Graphs show the release percentage profiles of FITC-BSA (5 μg/ml) and Alexa-BSA

(0.5 μg/ml) with/without cells from an internal (A and D), a surface-coated (B and E), and an

adsorbed depot (C and F) respectively. Mean values are represented ± SD (n = 6 for each group).

#p< 0.001.

Cell-mediated release of BSAs from BioCaP tablets

The cell-mediated release of BSAs from BioCaP tablets was monitored over a period

of 16 days. The samples with the internal or the surface coated depot showed a sustained

cell-mediated of BSAs release (Fig. 3A, B, D, E), while the samples with the adsorbed

depot showed a rapid release (Fig. 3C, F).

The cell-mediated release of BSAs from an internal depot was significantly higher

than the passive (without cells) release from day 7 until day 16 (Fig. 3A, D). At the 16

day time point, the initial amount of FITC-BSA and Alexa-BSA had been decreased by

55% and 25%, respectively. The cell-mediated release of BSAs from a surface coated

tablet was also significantly higher than the passive release from day 4 until day 16 (Fig.

3B, E). After 16 days the initial amount of FITC-BSA and Alexa-BSA was decreased by

45% and 40%, respectively. Cells appeared to have no influence on the protein release

from the adsorbed depot (Fig. 3C, F). At day 16, the initial adsorbed amount of

FITC-BSA and Alexa-BSA was decreased by 90% and 80%, respectively.

Osteoclast formation and the degradation of BioCaP tablet

Representative cultures of cells on BioCaP without and with coating are shown in

Fig. 4A and B after staining for TRACP and DAPI respectively. No significant

differences were found between the two differently labelled BSAs as regards osteoclast

formation or the degradation of BioCaP tablets. The number of TRACP positive

multinucleated osteoclasts on the BioCaP tablet with FITC-BSA is shown in Fig. 4C.

The number of osteoclasts on the uncoated BioCaP tablet, with an internal depot of BSA,

was significantly lower than on the BSA coated BioCaP tablet on day 4 and 7 (Fig. 4C).

Chapter 2

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From day 7, osteoclast numbers gradually decreased on uncoated or coated BioCaP

tablet.

The weight of coated and uncoated BioCaP tablets was monitored over a period of

16 days (Fig. 4D). During the 16 day incubation period, the weight of uncoated BioCaP

tablets in the absence of cells significantly decreased from 35.23 ± 0.33mg to 28.86 ±

0.68mg, while the weight of the coated BioCaP tablet showed no significant changes.

When cells were seeded on the uncoated BioCaP tablets, there was a significant decline

of the weight of BioCaP from day 7 compared with the group without cells. The cells

did not influence the weight of the coated BioCaP.

Scanning electron microscopy (SEM) showed the surface morphology of BioCaP

tablet with internal or surface coated FITC-BSA in Fig. 5A and 5C respectively. The

incorporation of protein did not significantly change the surface morphology of BioCaP.

Actively resorbing osteoclasts associated with typical resorption lacunae were observed

on BioCaP with internal or surface coated FITC-BSA in Fig. 5B and 5D respectively.

Figure 4. Tartrate-resistant acid phosphatase (TRACP) -positive and multinucleated osteoclasts

(arrows) generated from murine bone marrow cells (BMCs) are visible on A: BioCaP tablet and B:

the CaP coating of BioCaP tablet. C: The graph depicts that the number of TRACP+

multinucleated cells were significantly higher for BioCaP with coating at day 4 and day 7 as

compared to non-coated BioCaP tablets. D: The graph shows the weight of BioCaP tablets during

the cell culture. The presence of cells resulted in a significant decline of the weight of BioCaP


19

without coating from day 7 compared to the no cell group. The weight of BioCaP with coating

showed no significant changes. Mean values are represented ± SD (n = 6 for each group). *p<

0.05; #p< 0.001. Bar=50μm in (A) and (B).

Figure 5. Scanning electron microscopy was performed to analyze the surface morphology of

BioCaP tablets and resorbing osteoclasts. A: BioCaP tablet bearing internal depot of FITC-BSA;

B: Osteoclasts seemed to stick in their resorption lacunae on BioCaP (arrow head); C: BioCaP

tablet bearing a surface-coated depot of FITC-BSA; D: Osteoclasts seemed to stick in their

resorption lacunae on the coating of BioCaP (arrow head).

In vivo results

Formation of bone, bone marrow, blood vessels, and fibrous tissue

Five weeks after implantation in rats, bone and bone marrow were associated only

with BMP-2-functionalized BioCaP granules. This was found for all types of granules

that contained BMP-2 (see Figs 6A-F). In the two groups without BMP-2 neither bone

nor bone marrow was found (Fig. 6G, H).

The volume density of unmineralized and mineralized bone (Fig. 7A), bone marrow

(Fig. 7B) and blood vessels (Fig. 7C) was significantly higher in association with

BioCaP granules with internal or surface coated BMP-2 as compared to granules with

adsorbed BMP-2. The volume density of fibrous capsular tissue, however, was

significantly higher in the adsorption group (Fig. 7D).

Chapter 2

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Figure 6. Light micrographs of cross-sections through BioCaP granules, 5 weeks after

subcutaneous implantation in rats. BioCaP granules with an internal depot of BMP-2 (A), a

surface-coated depot of BMP-2 (B), or an adsorbed depot of BMP-2 (C). Newly formed

mineralized bone (reddish, white asterisk) and unmineralized bone (purple, black asterisk) has

been formed between the BMP-2-functionalized BioCaP granules or deposited on these BioCaP

granules. The bone quantity in (C) was smaller than in (A) and (B). Higher-magnification of

BioCaP granules with an internal depot of BMP-2 (D), a surface-coated depot of BMP-2 (E), or

an adsorbed depot of BMP-2 (F). BioCaP was observed in close contact with bone (white asterisk)

and bone marrow (M) in (D) and (E). Osteoblasts (arrow), fibrous capsular tissue (FT) and blood

vessels (arrow head) were also observed. The bone in (F) displayed unmineralized appearance

(black asterisk). Neither bone nor bone marrow was formed on or around the non-functionalized

BioCaP granules that had a CaP coating but no BMP-2 (G) or those without coating or BMP-2 (H).

The sections were stained with McNeal’s Tetrachrome, basic Fuchsine, and Toluidine Blue. Bar=

500μm in (A), (B), (C), (G), and (H). Bar= 100μm in (D), (E), and (F).

BioCaP degradation

The histomorphometric evaluation of the degradation of BioCaP revealed that the

degradation was higher in the groups with adsorbed or in those without BMP-2 (Fig.

7E). About 60% of BioCaP granules were degraded in these two groups compared with

about 20% for BioCaP in the other three groups.


21

Figure 7. Graphs depicting the volume density of unmineralized and mineralized bone (A), the

bone marrow (B), blood vessels (C), and fibrous capsular tissue (D), and the percentage of

non-degraded BioCaP granules (E). Mean values are represented ± SD (n = 6 for each group). *p<

0.05; +p< 0.01; #p< 0.001.

The response of the cells

Multinucleated osteoclast-like cells were observed not only on the surface of BioCaP

granules (Fig. 8A ,B), but also on the newly-formed bone (Fig. 8A, C). The cells were

associated with resorption pits on the surface of BioCaP (Fig. 8B). The volume density

of multinucleated osteoclast-like cells on the surface of BioCaP granules was

significantly higher in association with BioCaP with adsorbed BMP-2 compared with

Chapter 2

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BioCaP with internal or surface coated BMP-2 (Fig. 8D).

Figure 8. Light micrographs of a cross-section through BioCaP granules with internal depot of

BMP-2 after 5 weeks of subcutaneous implantation in rats (A). Multinucleated giant cells could

be observed on the BioCaP surface (arrow, B) or on the bone surface (arrow, C) at higher

magnification. Newly formed bone (asterisk); bone marrow (M); blood vessels (V), and fibrous

capsulate tissues (FT). The sections were stained with McNeal’s Tetrachrome, basic Fuchsine, and

Toluidine Blue. Bar= 100μm in (A). Bar= 50μm in (B) and (C). Graphs depicting the volume

density of multinucleated giant cells on the BioCaP surface (D). Mean values are represented ±

SD (n = 6 for each group). +p< 0.01; #p< 0.001.

DISCUSSION

The main objective of the present study is to investigate BioCaP as a potential dual

drug/protein delivery system for the slow and sustained delivery of different bioactive

agents. The data from the present study confirmed that a gradual, sustained and

cell-mediated release of bioactive agents can be achieved in vitro as well as in vivo

using BioCaP. Moreover, we showed that the BioCaP granules containing BMP-2 are

osteoinductive. In addition, the histological analysis confirmed the excellent

biocompatibility of BioCaP.

In order to analyse in vitro cell-mediated protein release we used labelled BSA

instead of the very costly BMP-2 [28, 39]. BSA has been often utilized as a substitute

for BMP-2 [22, 23] because the release kinetics of BSA and BMP-2 are similar [40-42].

In the present study, BSA presented a sustained cell-mediated release from the two

delivery modes of BioCaP, thus suggesting that the BMP-2 release from BioCaP might

be sustained as well.

It is known that the adsorption of BMP-2 on materials is always associated with a

high-dose burst release. This results in a poor osteoinduction [23, 43]. In the current

study, the histomorphometric analysis demonstrated that the two modes in which

BMP-2 was incorporated into the BioCaP granules resulted in a better osteoinduction

than BMP-2 adsorbed onto the granules. The two groups not only induced more bone,

bone marrow and blood vessels, but also less fibrous capsular tissue was found. Since

bone marrow and blood vessels are important sources of oxygen, nutrients, signalling


23

molecules, and pluripotent progenitor cells for osseous tissue [44], the presence of these

structures and cells in the experimental groups helps to enhance bone formation.

Previous studies have demonstrated that the slow release of BMP-2 enhances

osteoinductivity [13], and leads to a higher osteoinductive efficiency than the adsorption

[23]. Therefore, given the in vivo results, we speculate that the two modes resulted in a

sustained release of BMP-2, since they achieved a better osteoinduction than the

adsorbed mode. However, when BioCaP was implanted in this ectopic rat model,

endogenous protein including BMPs can be adhered on the graft surface and thus induce

different cell-mediated resorption processes. The BMP-2 release may result in a

completely different protein release pattern. The detection of the BMP-2 release in vivo

was studied apart from this study (unpublished data).

The degradability of a CaP-based material is very important for the in vivo longevity

and efficacy of its biological effects [45]. In the present study, the findings indicate that

BioCaP is biodegradable. The material degradation is associated with its dissolubility

and the cell-mediated resorption [30]. The degradation rate of BioCaP granules was

significantly lower for those in which BMP-2 was incorporated internally compared

with those with adsorbed BMP-2 or with those with no BMP-2. A reason for this could

be that newly formed bone tissues covered the surface of the granules, thereby

preventing their degradation. In addition, both in vitro and in vivo findings indicate that

the coating can prevent and delay the degradation of underlying BioCaP, even though

the coating is biodegradable [13, 34].

The coating and BioCaP were all biomimetically formed by precipitation of calcium

phosphate. However, their surface structures were totally different. The coating had a

crystalline surface, while the surface of BioCaP seemed to be amorphous. A higher

number of osteoclasts were found on the coated BioCaP tablets compared with the

BioCaP without coating. This indicates that the different physicochemical properties of

BioCaP and the coating may affect the formation of osteoclasts.

Moreover, in vivo the two different modes of BMP-2 delivery resulted in a

significantly lower number of multinucleated cells compared with the adsorption mode.

It has been shown that BMP-2 may exert a dual concentration dependent effect [13, 46].

At low doses, it stimulates the recruitment, proliferation and differentiation of

osteoprogenitor cells, whereas at high doses, it induces the recruitment, formation and

activation of osteoclasts. In a previous study we have demonstrated that a slow and

steady release of BMP-2 from the coating suppresses the formation of multinucleated

cells [23]. We assume that not only the physicochemical property of materials affects

the formation of multinucleated cells but also the way BMP-2 is released and its local

dose influences this process.

Our previous study has demonstrated that BMP-2 incorporated into biomimetic

coatings can retain its biological activity [31]. However, the BMP-2 activity might be

influenced by the digestion of multinucleated osteoclast-like cells. The previous study

showed that the growth factors released through osteoclast-mediated manner could

significantly promoted osteoblastogenesis in vitro [47]. Therefore, we may assume the

proteinaceous BMP-2 may maintain its activity in this study. However, further

investigations are needed to prove the percentages of BMP-2 activity.

In this study, the rodent ectopic model may be sensitive to BMP-2. Therefore, larger

animals may need more BMP-2 to respond appropriately. In our on-going study, we

Chapter 2

24

implanted BioCaP with the two delivery modes of BMP-2 into the bone defects in sheep

compared with the therapeutic effect of autologous bone.

One concern associated with the use of BioCaP is its biocompatibility. Histological

analysis revealed that the newly formed bone was deposited directly on the BioCaP

surface and bone marrow was in close contact with BioCaP. This suggests that the used

BioCaP granules are highly biocompatible. Our previous study has demonstrated that

the coating was highly biocompatible and osteoconductive [34].

Our findings strongly suggest that BioCaP granules can be used as an attractive

protein delivery vehicle. This seems particularly true for the granules in which the

growth factor was incorporated internally. In addition, the use of the coating can offer an

alternative for slow release. By combining the two protein carrying modes, BioCaP can

be a dual release system for a sequential delivery of different proteins/drugs. This

combination could be applicable for a variety of clinical applications. For example,

osteogenic agents can be incorporated into the interior of BioCaP, and at the same time

antibiotics can be incorporated into the surface coating. This could be considered as a

new strategy for the treatment of bone defects caused by peri-implantitis.

In conclusion, it was shown that BioCaP with an internal or surface coated depot of

protein has the capacity to maintain a slow and sustained protein release in the presence

of osteoclasts in vitro. Both modes of delivering of BMP-2 with the use of BioCaP make

these granules efficient osteoinductive compounds and suppress the formation of

multinucleated giant cells in vivo. The in vivo detection of the BMP-2 release by using a

radioactive labelling method was studied apart from this study (unpublished data). The

dual drug release system renders BioCaP granules promising tools for an orderly

delivery of multiple therapeutic agents, such as antibiotics, osteogenic agents, and

anti-cancer drugs for different clinical applications.

ACKNOWLEDGMENTS

We would like to thank Dr. Afsheen Tabassum for the assistance and thank Prof. Dr.

Tony Hearn as a native speaker for editing the grammar.


25

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Chapter 2

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29

Chapter 3

Preparation and characteristics of osteoinductive

biomimetic calcium phosphate material:

in vitro and in vivo study

Tie Liu, Gang Wu, Yuanna Zheng, Afsheen Tabassum, Daniel Wismeijer,

Vincent Everts, and Yuelian Liu.

Submitted, 2013.

Chapter 3

30

ABSTRACT

Objectives: A biomimetic calcium phosphate (BioCaP) bone substitute was developed

with two protein-delivery modes: one mode by which protein was incorporated in the

interior of BioCaP (internally-incorporated mode); and one by which protein was coated

on the outside of BioCaP (coating-incorporated mode). The aim of this study was to

prepare and evaluate the physical and chemical properties of BioCaP, and the ability for

protein loading and release in a long period, and using micro-CT analysis to evaluate the

degradation of BioCaP and bone formation.

Material and methods: BioCaP was prepared by refining a well-established biomimetic

protocol. The compressive strength of BioCaP was assessed by a compressive strength

machine. The structure and morphology of BioCaP was analyzed by X-ray diffraction

(XRD) analysis and scanning electron microscope (SEM). The two phases of precipitated

BioCaP were visualized using SEM. An energy dispersive X-ray (EDX) source was used

for the chemical composition analysis. The protein release was analyzed in vitro (35

days). Human osteoclasts were used for testing the cell-based degradation of BioCaP. A

micro-CT method for the evaluation of graft material and bone was applied by using an

“onion-peeling” algorithm and specific threshold settings in an ectopic rat model.

Results: BioCaP exhibited bone-like mechanical strength and the characteristics of

calcium-deficient apatite. The Ca/P molar ratio of BioCaP was about 1.48. The granules

with internally- or coating-incorporated protein exhibited a slow release in vitro.

Osteoclasts seeded on the granules were shown to resorb the BioCaP. Micro-CT analysis

showed three-dimensional reconstructions of BioCaP and new bone formation in vivo.

Conclusion: BioCaP granules were developed as an osteoinductive bone substitute and a

vehicle for protein/drug slow release. The micro-CT method provides an alternative for

the evaluation of bone substitute and bone formation.

Keywords: Biomimetic calcium phosphate material; Protein slow release; Human

osteoclasts; Micro-CT; BMP-2

INTRODUCTION

Large-size bone defects, which exceed the self-healing capacity of bone tissue, can be

treated using autografts, allografts, xenografts, and synthetic materials [1, 2]. Autografts

(gold standard) can be seen as an osteoconductive scaffold, containing osteoinductive

cytokines and osteogenic cells. However, it is always associated with limited availability

as well as with donor-site pain and morbidity [3]. At present, synthetic calcium phosphate

(CaP)-based bone substitutes have become widely used in the clinic [4, 5]. However,

most of these clinically used CaP bone substitutes lack intrinsic osteoinductivity, which is

an essential property to realize osseous restoration of large-size bone defects [2]. The

osteoinductivity of such CaP material can be conferred by using osteogenic growth

factors such as bone morphogenetic protein-2 (BMP-2). However, the rather harsh and

non-physiological conditions during the production process preclude the incorporation of

biological active proteinaceous molecules into the interior of CaP material. The usual

way to circumvent this is to adsorb osteogenic agents onto the CaP surface [6, 7].

However, such adsorbed molecules are frequently associated with a rapid and burst

Preparation and characteristics of BioCaP

31

release, which results in a poor osteogenic potential [8-10].

Using a biomimetic mineralization approach, growth factors were co-precipitated into

the latticework of crystalline calcium phosphate on titanium implants or other

biomaterials and were shown to be released locally in a slow manner [2, 11]. Such a slow

release has been shown to be beneficial for the effect of different growth factors such as

BMP-2 and vascular endothelial growth factor (VEGF). The slow delivery of BMP-2

enhanced osteoinduction [12], and the slow delivery of VEGF promoted vascularization

[13]. Furthermore, since bone regeneration is a coordinated cascade of events regulated

by several growth factors, the local sequential delivery of VEGF and BMP-2 could

enhance bone formation compared with BMP-2 alone [14]. Therefore, there is a need to

develop a carrier material that has the capacity to sequentially and slowly deliver growth

factors.

Previous study has introduced the calcium-phosphate (BioCaP) granular bone

substitute that can act as a dual delivery model [15]. We incorporated protein in two ways:

in the interior of the granules and in their surface coating. These two modes of

incorporation of BMP-2 rendered BioCaP osteoinductive efficiently. However, the

physical and chemical properties of BioCaP is unclear. The purpose of the present study

was i) to evaluate the BioCaP physicochemical properties such as mechanical strength,

dissolution and degradability; ii) to investigate the in vitro release kinetics of the two

protein-delivery modes of BioCaP during a period of 35 days; iii) using human

osteoclasts to evaluate the cell-based resorption of BioCaP; and iv) using micro-CT to

distinguish BioCaP and new bone and determine whether BioCaP with these two delivery

modes of BMP-2 can efficiently induce ectopic bone formation in rats.


Fabrication of biomimetic calcium phosphate (BioCaP) bone substitute

BioCaP was fabricated by refining a well-established biomimetic mineralization

technique, which has been described in a previous study [15]. Briefly, a CaP solution

(200 mM HCl, 20 mM CaCl2·2H2O, 680 mM NaCl, and 10 mM Na2HPO4) buffered by

TRIS (250 mM) to a pH of 7.4. Rapid precipitation appeared at pH of 6.25. In order to

sterilize the CaP solution it was filtered with a vacuum filter (0.22-μm pore) before

buffering. All the following procedures were performed under aseptic conditions. After

buffering, the solution was incubated in a shaking water bath (50 agitations/min) at 37°C

for 24 hours. Thereafter the solution was removed. The precipitated material was gently

washed by Milli-Q water, filtered and compressed to a block using a vacuum exhaust

filtering method with a filter (0.22-μm pore, Corning, NY, USA) and a vacuum pump.

Before drying, the block can be shaped differently such as in a tablet or cylinder shape.

After drying at room temperature for 2 hours, the hardened block can be ground and

filtered to obtain different sizes of granules using metallic mesh filters. The protein

introduced into the CaP solution can be co-precipitated into the interior of BioCaP, viz.,

the internally-incorporated depot of protein (Fig. 1).

Biomimetic coating procedure and protein incorporation

The superficial coating was deposited on BioCaP according to the procedure described

before [16]. Briefly, BioCaP was incubated in the coating solution [40 mM HCl, 4 mM

Chapter 3

32

CaCl2·2H2O, 136 mM NaCl, 2 mM Na2HPO4, and 50 mM TRIS (pH 7.4); total volume

of 20 ml] in a shaking water bath (50 agitations/min) at 37°C for 24 hours. The protein

present in this coating solution can be co-precipitated into the coating on the BioCaP

surface, viz., the coating-incorporated depot of protein (Fig. 1).

Figure 1. Schematic illustration of BioCaP granules with two cytokine-carrying (delivery) modes:

the internally- or the coating- incorporated modes.

Physicochemical properties of BioCaP bone substitute

The compressive strength of BioCaP was assessed. BioCaP was made into cylinders

(diameter, 5mm; height, 8 mm; n=6). Each cylinder was crushed at a crosshead speed of

1 mm/min, using a compressive strength machine (Instron 6022, High Wycombe, Bucks,

U.K).

BioCaP granules with no protein or those with internally-incorporated bovine serum

albumin (BSA, 1µg/ml or 20µg/ml, Sigma, St. Louis, MO, USA) or BMP-2 (1µg/ml,

INFUSE® Bone Graft, Medtronic, USA) were ground into fine powder for X-ray

diffraction (XRD) analysis. XRD patterns of the samples were recorded with a vertically

mounted diffractometer system (Bruker D8 Advance, Bruker AXS, Germany), using

Ni-filtered Cu Kβ radiation generated at 40 kV and 40 mA. Specimens were scanned

from 5° to 60° 2θ (where θ is the Bragg angle) in continuous mode.


33

The morphology of precipitated BioCaP (BioCaP dried on a glass plate at room

temperature) and granules with or without protein was visualised using a scanning

electron microscope (SEM, XL20, FEI Company, the Netherlands), under an accelerating

voltage of 10 kV after being sputter-coated with gold. An energy dispersive X-ray

(Voyager, Eindhoven, The Netherlands) source was attached to the apparatus for the

chemical composition analysis.

To confirm whether protein was incorporated into the interior of the BioCaP granules

or in its coating, the presence and distribution of BSA labelled with

fluorescein-isothiocyanate (FITC-BSA, Sigma, St. Louis, MO, USA) in BioCaP granules

was analysed by using cross sections of the granules. The samples were embedded in

methylmethacrylate, sectioned, and ground [11]. A series of 80-µm-thick sections were

prepared for analysis by fluorescence microscopy. Micrographs were taken with a digital

camera (Leica, Wetzlar, Germany) mounted on an inverted light microscope (Leica)

equipped with a fluorescence lamp.

Protein and Ca2+

release in vitro

To study the protein release from BioCaP granules, bovine serum albumin (BSA) labelled

with fluorescein-isothiocyanate (FITC-BSA, Sigma, St. Louis, MO, USA) or with Alexa

Fluor® 555 (Alexa-BSA, invitrogen, Carlsbad, CA, USA) was used as cost effective

alternative of BMP-2. Previous studies have indicated the similarity of the release

kinetics of BSA and BMP-2 [17-19]. The BSA and Ca2+

release kinetics from BioCaP

granules with the internally-incorporated, coating-incorporated, or adsorbed delivery

modes (n=6 per group) were investigated by soaking them in phosphate-buffered saline

(PBS) at pH of 7.4. BioCaP granules with adsorbed BSA (as control) were prepared by

immersing them in an aqueous protein solution (total volume of 20 ml) for 24 h at 37°C

in plastic tubes. According to the manufacturer’s protocol, Alexa-BSA is more easily

detected at lower concentration than FITC-BSA. Therefore, 20μg/ml of FITC-BSA and

1μg/ml of Alexa-BSA were used for loading. Each sample (0.05 g BioCaP granules per

sample) was placed in a 2-ml sealed Eppendorf tube containing 2 ml PBS. The tubes

were incubated for up to 35 days in a shaking water bath (50 agitations/min) at 37°C. The

PBS was refreshed at each time point (hour 3, 6, day 1, 2, 3, 5, 7, 10, 13, 17, 22, 28, and

35) and triplicate 200-µl aliquots of the PBS were withdrawn for spectrophotometric

analysis in a Fluorimeter (Spectramax M2, Molecular Devices, CA, USA). Fluorescence

readings were converted into amounts of protein using a standard curve that was

generated from a dilution series of BSA prepared in 2 ml PBS. Meanwhile, Ca2+

release

was monitored by measuring Ca2+

concentration in the 200-µl aliquots of the PBS using

atomic adsorption spectrometry (Analyst 100, PerkinElmer, USA). At the end of the

release experiments, the residual BSA in BioCaP was determined by dissolving the

materials in 20 ml of 0.5 M ethylene diaminetetraacetic acid (EDTA, pH 8.0) for

spectrophotometric analysis. The percentage of BSA released from the BioCaP was

calculated according to the formula:

% BSA= (amount of the released BSA fraction / total loaded BSA)×100 %

Osteoclast-based resorption assay

The resorbability of BioCaP was tested using a cell-based resorption assay [20]. BioCaP

tablets were used to investigate the resorption. Briefly, human peripheral blood

Chapter 3

34

mononuclear cells (PBMCs), isolated from whole blood with Ficoll–Paque density

gradient, were seeded on 800-μm-thick BioCaP tablets with or without coating (without

protein) in 96-well plates at a density of 106 cells per well. The PBMCs were cultured

with RANKL (40 ng/ml, PreProtech, Rocky Hill, NJ, USA) and M-CSF (25 ng/ml, R&D

Systems, Minneapolis, MN, USA) as described preciously [21]. Cells were cultured in

duplicate, and the culture media were refreshed twice a week.

The formation of osteoclasts was assessed by analyzing tartrate-resistant acid

phosphatase (TRACP) positive multinucleated cells and by SEM after 21 days of

culturing on BioCaP tablets [21, 22]. The cells were washed with PBS and fixed in 4%

PBS-buffered formaldehyde for 5 min and stained for TRACP activity using the

leucocyte acid phosphatase kit (Sigma). The cells were fixed, dried, and sputter-coated

for SEM investigation. After 24 days of culturing, cells were removed by using

demineralised water and 10% NH3OH and the formation of lacunae was detected by

SEM. All in vitro cell culture experiments were performed at least three times.

In-vivo investigation

As an experimental animal model, we used adult male wistar rats (200–220g). The

animal experiments were approved by the Ethics Committee of Zhejiang Chinese

Medical University, China.

Four groups were established (n=6 animals per group):

(1) BioCaP with internally-incorporated BMP-2;

(2) BioCaP with coating-incorporated BMP-2;

(3) BioCaP with directly-adsorbed BMP-2 (as control); and

(4) BioCaP without BMP-2 (BioCaP material only; as control).

Each sample consisted of 0.22g BioCaP granules (0.25-1mm). The samples were

prepared as described in a previous study [15]. Two samples per rat were randomly

implanted in dorsal subcutaneous pockets (one on the left side and one on the right) [15].

Five weeks after implantation, the samples were retrieved, chemically fixed and

embedded as previously reported [23].

Three-dimensional (3D) reconstructions of the samples were obtained using a

high-resolution micro-CT system (μCT 40, Scanco Medical AG, Bassersdorf,

Switzerland). To this end, samples were fixed in synthetic foam and placed vertically in a

polyetherimide holder and scanned at a 18 μm isotropic voxel size, 70 kV source voltage,

and 113 μA current. Grey values, depending on radiopacity of the scanned material, were

converted into corresponding values of degree of mineralization by the analysis software

(Scanco Medical AG). A distinction could be made between the newly formed bone and

BioCaP, since the mineralization degree of BioCaP, was significantly higher than the

mineralization degree of bone. A method for the separation of graft material and newly

formed bone was applied according to the previous study by using an “onion-peeling”

algorithm (Scanco Medical AG) and specific threshold settings [24]. By using this

method, the micro-CT results were comparable with histomorphometrical results [24].

Briefly, a low threshold of 467.1 mg hydroxyapatite (HA)/cm3 to distinguish bone tissue

from connective tissue and bone marrow, and the grey values were scaled from 1 to 1000

and the threshold was set at 158 to distinguish BioCaP from bone tissue. These two

thresholds were calculated by averaging the thresholds determined in 3 slices of three

samples by two independent observers. The samples were analysed for bone volume


35

(BV), bone density (BV/TV), material volume (MV), and bone mineral density (BMD).

For the measuring of the volume of BioCaP before implantation (time 0), six

chemically fixed and plastic-embedded samples (0.22g of BioCaP granules per sample)

were specifically reserved and evaluated by micro-CT.


All data are presented as a mean ± SD. The data were statistically evaluated by a one-way

analysis of the variance (ANOVA) using SPSS statistical software (version 16.0 for

Windows). Post-hoc comparisons were made using Bonferroni's corrections. The

significance level was set at p < 0.05.

RESULTS

In vitro characterization of BioCaP granules

The biomimetic precipitation of BioCaP consisted of two different phases: the initial

quick precipitation forming super-fine crystalline structures (Fig. 2A), and the subsequent

slow precipitation forming micro-crystalline spheres with 2-10 µm in diameter (Fig. 2B).

The BioCaP granules were shown in Fig. 2C. The compressive strength value of BioCaP

was 4.58±0.31 MPa. The energy dispersive X-ray analysis revealed that the Ca/P molar

ratio of BioCaP was about 1.48. The contents of BioCaP include Ca (16.7%), P (24.8%),

O (52.8%), Na (1.9%), and Cl (3.8%) (Fig. 3). BioCaP powder exhibited a unique

diffraction peak at 2θ = 26°, 32°, and 46° in the XRD spectra (Fig. 4). The incorporation

of BSA or BMP-2 elicited no profound change in the major diffraction spectrum. The

surface morphology of the BioCaP and the coating was shown in Fig. 5A and Fig.5B,

respectively. The loading of 1μg/ml BMP-2, 1μg/ml Alexa-BSA, or 20μg/ml FITC-BSA

had no effect on the surface structure of BioCaP or the crystalline coating. ELISA results

showed that BMP-2 with the internally-incorporated mode had a significantly higher

loading efficiency (40.3 ± 0.9%) than with the coating-incorporated mode (35.1 ± 1.3%).

By the internally-incorporated mode, protein (green signal) was distributed

homogeneously throughout the BioCaP granules (Fig. 6Aa and Ab). By the

coating-incorporated mode, protein (green signal)was distributed uniformly throughout

the coating layer (Fig. 6Ac). The coating showed at higher magnification a crystalline

structure (Fig. 6Ad). The mean thickness of the coating was 21.02±7.53 μm.

Figure 2. SEM micrographs of two phases of the precipitation of BioCaP (A and B) and

BioCaP granules (C). The first phase was the initial precipitation (A). The second phase

of precipitation (B; white pane) came from the incubation period containing small

particles with tiny crystals (particle size of 2-10 µm).

Chapter 3

36

Figure 3. Energy Dispersive X-Ray Spectroscopy analysis of the chemical content of BioCaP.

Figure 4. XRD patterns showed BioCaP bearing no protein (a), 1µg/ml of BSA (b), 20µg/ml of

BSA (c), and 1µg/ml of BMP-2 (d). The protein-carrying mode was the internally-incorporated

mode.


37

Figure 5. SEM micrographs of the surface of BioCaP granules bearing an internally-incorporated

(A) or a coating-incorporated (B) depot of FITC-BSA. After 35 days immersion in PBS (pH 7.4,

37°C), SEM micrographs showed the surface of BioCaP granules bearing an

internally-incorporated (C) or a coating-incorporated (D) depot of FITC-BSA

The adsorbed FITC- and Alexa-BSA was released rapidly, being completely

exhausted after 10 or 13 days (Fig. 6B). However, both FITC- and Alexa-BSA revealed a

low burst release and subsequently a sustained release from the internally- or

coating-incorporated depot in BioCaP granules. In the internally-incorporated depot,

about 10-14% of BSA released from BioCaP granules in an initial burst release stage

(within the first 24 hours). Subsequently BSA was gradually released at a steady rate

until the 35th day (Fig. 6B). The initial burst release of coating-incorporated depot of

BSA was about 18-20% and was also followed by a sustained release (Fig. 6B).

Meanwhile, Ca2+

also revealed a low burst release and subsequently a sustained release

from BioCaP with or without protein (Fig. 6C). The presence of BSA did not affect the

Ca2+

release (Fig. 6C in which only Ca2+

release from BioCaP with FITC-BSA is shown).

After 35 days, the total amount of released Ca2+

from BioCaP with coating was

significantly lower than from granules without coating (p<0.05). In the end, the surface

morphology of BioCaP without coating had not changed (Fig. 5C), while BioCaP with

coating showed that most crystals of the coating had been dissolved (Fig. 5D).

Chapter 3

38

Figure 6. Fluorescence micrographs of cross-sections of BioCaP granules at low and high

magnifications: the internally-incorporated depot of FITC-BSA (Aa and Ab, green signal) or the

coating-incorporated depot of FITC-BSA (Ac and Ad, green signal). FITC-BSA has been

incorporated into the interior of BioCaP granules (black arrow) or the coating (white arrow).

Graphs depicting the cumulative protein release kinetics from BioCaP granules (FITC-BSA:

20μg/ml; Alexa-BSA: 1μg/ml) soaked in PBS (pH 7.4, 37°C) for 35 days (B), and the cumulative

Ca2+ release kinetics of BioCaP granules with or without protein (C). Mean values (n=6 per group)

are represented together with the standard deviation. Time points: hour 3, 6, day 1, 2, 3, 5, 7, 10, 13,

17, 22, 28 and 35.

Figure 7. TRACP staining of osteoclasts (white arrows) on BioCaP (A) or its coating (D); SEM


39

micrographs of osteoclasts on BioCaP (B) or its coating (E), and the resorption by osteoclasts on

BioCaP (C) or its coating (F).

Tartrate-resistant acid phosphatase-positive osteoclasts generated from human

peripheral blood mononuclear cells were grown on BioCaP tablets (Fig. 7A and D).

Representative SEM micrographs of osteoclasts showed their attachment to the material

(Fig. 7B and E). Resorption pits were clearly discernible after 24 days of culture (Fig. 7C

and F).


At the time of their sacrifice (5-week juncture), all animals were in good health, and no

complications had become manifest during the postoperative period. The histological

analysis showed no inflammatory activity in all the samples.

Three-dimensional (3D) reconstructions of samples with BioCaP and newly formed

bone were shown in Fig. 8. New bone was only found in the sample containing BioCaP

with BMP-2. Bone and BioCaP were separated by the analysis software (Fig. 9).

Micro-CT analysis revealed that the volume and the volume density of newly formed

bone for BioCaP with internally- or a coating-incorporated BMP-2 was significantly

higher than around an adsorbed depot (Table 1), but no significant differences were found

between the internally- and the coating- incorporated depots. 5 weeks after implantation,

the volume of BioCaP of all the four groups decreased significantly, compared with the

volume before implantation (298.48±8.87 mm3) (p<0.05). The volume of BioCaP with

internally- or a coating-incorporated BMP-2 was significantly higher than those with

adsorbed or no BMP-2 (p<0.05). No significant difference was found in the bone mineral

density among the three BMP-2 groups.

Chapter 3

40

Figure 8. Three-dimensional (3D) reconstructions of BioCaP (left column) and bone (right column)

by micro-CT. Group (1), BioCaP granules bearing an internally-incorporated depot of BMP-2 (A,

B); Group (2), BioCaP granules bearing a coating-incorporated depot of BMP-2 (C, D); Group (3),

BioCaP granules bearing an adsorbed depot of BMP-2 (E, F); and Group (4), BioCaP granules

bearing no BMP-2 (G, H). In the Group (4), no bone was found (H).


41

Figure 9. Two-dimensional (2D) image of BioCaP and bone by Micro-CT (A). Bone (red) and

BioCaP (white) were separated by the analysis software (B).

Table 1

Micro-CT Evaluation on BioCaP and bone 5 weeks after implantation

Groups BV (mm3) BV/TV MV (mm3) BMD

(mg HA/cm3)

(1) BioCaP + internally-incorporated

BMP-2 11.78±2.67* 0.05±0.01* 229.69±16.13*† 649.10±26.69

(2) BioCaP + coating-incorporated

BMP-2 13.76±4.92# 0.06±0.03# 236.43±29.82#§ 605.84±28.77

(3) BioCaP + adsorbed BMP-2 1.22±1.87 0.01±0.01 124.97±66.93 648.10±41.43

(4) BioCaP - - 118.18±35.29 -

BV, bone volume; BV/TV, bone volume/total volume; MV, material volume; BMD, bone mineral density *Significant difference (p < 0.05) between Group (1) and Group (3). #Significant difference (p < 0.05) between Group (2) and Group (3). †Significant difference (p < 0.05) between Group (1) and Group (4). §Significant difference (p < 0.05) between Group (2) and Group (4).

DISCUSSION

In this study, the mechanical strength of BioCaP granules is comparable with that of

trabecular bone. These granules have a series of characteristics that make them attractive

for bone regeneration purposes. First, BioCaP granules as a bone-defect filling material

are easy to handle. Second, both delivery modes showed a slow release of protein in vitro.

Third, the BioCaP granules can be resorbed as shown by the human osteoclasts which

Chapter 3

42

confirmed the previous results by using mouse osteoclasts [15]. Forth, micro-CT analysis

showed 3D construction and 2D image of newly formed bone around the granules

containing BMP-2 after the subcutaneous implantation. This micro-CT analysis with an

“onion-peeling” algorithm successfully distinguished BioCaP and bone. Micro-CT results

showed that BioCaP granules were biodegradable and bone formation proved to be

higher with the granules with the two delivery modes of BMP-2 compared with the

adsorbed mode, which confirmed the histological results in the previous study [15]. Our

findings demonstrate that the BioCaP granules in which BMP-2 had been incorporated

are osteoinductive and we propose the use of this implant material if efficient bone

formation is needed.

Compressive strength is most often used to characterize the mechanical behaviour of

bone substitutes [25]. The compressive strength of BioCaP is about 4.58 MPa, thus being

similar to that of human trabecular bone which ranges from 0.22 to 10.44 MPa, with a

mean value of 3.9 MPa [26]. The XRD and EDX analysis showed that BioCaP had the

characteristics of calcium-deficient apatite with a low crystallinity [27], also indicating

similarities to native bone [28, 29]. The bone-like characteristics of BioCaP could be

caused by the mixture of the two phases of precipitations under a biomimetic

environment. In addition, the biomimetic environment used in this study can retain

BMP-2 activity [30]. The hardness of BioCaP granules makes it easy of handling for

filling bone defects.

A slow and sustained delivering mode is critical to maximize the functional efficiency

of a cytokine [12, 31]. The protein release from the internally- and the coating-

incorporated modes is highly depending on the degradation rate of BioCaP or the coating.

In the present study, these two modes exhibited a low burst release within the first 24

hours and subsequently a slow release period in vitro. On the other hand, the resorption

assay revealed that human osteoclasts can resorb BioCaP and the coating in vitro.

Resorbing cells such as osteoclasts may increase the degradation rate of material to speed

up the agent's rate of delivery [32], and consequently to affect the functional efficiency of

the agent. The cell-mediated protein release from BioCaP has been investigated in

another study [15]. In the present study, micro-CT analysis revealed that the two delivery

modes induced more bone formation compared to the adsorption mode. The findings

suggest that the two delivery modes of BMP-2 have the capacity to maintain the slow

release in vivo, whereas the adsorbed mode resulted in a burst release.

In the internally-incorporated mode, protein distributes throughout the whole volume

of BioCaP granules, which means that the protein can be gradually liberated as long as

the granules are degraded. After 5-week implantation BioCaP granules with the

internally-incorporated BMP-2 had not been completely degraded. This probably implies

that a certain amount of BMP-2 is still present in the BioCaP granules, thus increasing

the duration of BMP-2 activity. In contrast, about 80-100% of the coating (about 20-µm

thickness) can be degraded within 5 weeks in vivo [12, 33]. However, micro-CT could

not distinguish BioCaP and its coating. Histological observation indicated the coating of

BioCaP was degraded [15].

Moreover, the in vitro Ca2+

release represents the self-dissolubility of BioCaP and its

coating, and indicates that the solubility of BioCaP is higher than the coating. The

degradation of BioCaP with coating-incorporated BMP-2 would be delayed because of

the coating. The micro-CT analysis revealed that the degradation of BioCaP with


43

internally- or coating-incorporated BMP-2 was slower than that of BioCaP with adsorbed

BMP-2 or without BMP-2. These results are is consistent with the previous study [15].

As a dual protein-delivery model, BioCaP can be used for different clinical

applications. Growth factors, anticancer drugs and antibiotics can be candidates for this

model. For example, BioCaP granules with internally-incorporated BMP-2 and in

addition antibiotics incorporated into the coating may be considered to treat bone defects

in peri-implantitis. However, the potential applications of this dual protein-delivery

model need to be evaluated further.

CONCLUSION

The biomimetic mineralization approach confers BioCaP bone-like mechanical strength.

BioCaP showed the characteristics of calcium-deficient apatite. The two delivery modes

of BioCaP showed slow release of protein in vitro in 35 days. However, the in vivo

protein release may more complicated, which needs to be investigated further. Human

osteoclasts assay indicated the good biodegradability of BioCaP which is also confirmed

in vivo by micro-CT analysis. Micro-CT results showed more bone formation by the two

protein-delivery modes, suggesting these two modes can achieve a highly efficient

delivery of BMP-2. This results confirmed the histological result in the previous study

[15]. This micro-CT method provides an alternative for the evaluation of bone substitute

and bone formation.

ACKNOWLEDGEMENTS

The authors acknowledge Cees Kleverlaan, Arie Werner, Ben Norder, Teun J. de Vries,

and Ineke Jansen for their assistance with the operation of SEM, XRD, and cell culture.

Chapter 3

44

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14. Kempen DH, Lu L, Heijink A, Hefferan TE, Creemers LB, Maran A, et al. Effect of local

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BMP-2/BMP-7 delivery system into chitosan-based scaffolds for bone tissue engineering.


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BMP-2/BMP-7 delivery on in vitro bone regeneration. J Mater Sci Mater Med

2010;21:2999-3008.



21. Olivier BJ, Schoenmaker T, Mebius RE, Everts V, Mulder CJ, van Nieuwkerk KM, et al.


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Increased osteoclast formation and activity by peripheral blood mononuclear cells in chronic

liver disease patients with osteopenia. Hepatology 2008;47:259-67.

22. Faust J, Lacey DL, Hunt P, Burgess TL, Scully S, Van G, et al. Osteoclast markers

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24. Schulten EA, Prins HJ, Overman JR, Helder MN, ten Bruggenkate CM, Klein-Nulend J. A

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Chapter 3

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47

Chapter 4

Osteoinductive biomimetic bone substitute for the

repair of critical-sized bone defects in sheep

Tie Liu, Gang Wu, Daniel Wismeijer, and Yuelian Liu.

Submitted, 2013.

Chapter 4

48

ABSTRACT

As a synthetic alternative to autologous bone grafting, a biomimetic calcium phosphate

bone substitute (BioCaP) was developed with two protein delivery modes: 1) protein in

the interior of BioCaP (internally-incorporated mode), 2) protein in the coating on the

surface of BioCaP (coating-incorporated mode). The aim of this study is to investigate

the therapeutic effectiveness of BioCaP with each delivery mode of BMP-2 in the repair

of a large cylindrical bone defect (8mm in diameter and 13mm in depth) in sheep. Six

groups were estabilshed: (i) BioCaP only; (ii) BioCaP with coating-incorporated BMP-2;

(ii) BioCaP with internally-incorporated BMP-2; (iv) no graft material; (v) autologous

bone; (vi) deproteinized bovine bone (DBB, a commercial product). 4 and 8 weeks after

implantation, samples were withdrawn for histological and histomorphometric analysis.

BioCaP with BMP-2 showed equal efficacy as autologous bone in the bone defect repair

at 8 weeks post-implantation. Both delivery modes of BMP-2 accelerated the bone

formation in an early period of 4 weeks. The internally-incorporated mode enhanced

bone formation after 8 weeks, showing more efficient than DBB. Within 8 weeks, about

half of BioCaP with either internally-incorporated BMP-2 or without BMP-2 was

degraded, which was significantly higher than that of BioCaP with coating-incorporated

BMP-2. In conclusion, both two delivery modes of BMP-2 enhance bone formation.

Benefiting from these two delivery modes, BioCaP can be a promising alternative to the

autografts.

Keywords: Biomimetic calcium phosphate; Protein delivery; Bone repair; Critical-sized

bone defect; BMP-2

INTRODUCTION

The treatment of bone defects requires adequate volume of bone tissue, which is of

paramount importance to achieve an excellent aesthetic and functional restoration. When

the bone defects are too large to be self-healed, it requires bone grafting in order to fill

the defect [1, 2]. Autografts (gold standard), allografts, xenografts, and synthetic

materials are available for the repair of bone defects in the fields of dentistry, orthopedics

and traumatology [3]. Synthetic calcium phosphate (CaP) biomaterials are widely used in

the regeneration of bone defects because of their chemical similarity to native bone tissue.

Currently, there is an increased interest in biomimetic calcium-phosphate materials

because of their capacity to carry (delivery) bioactive agents without compromising their

bioactivity [4-8]. For example, the delivery of bone morphogenetic protein 2 (BMP-2)

can enhance bone regeneration [9, 10].

Biomimetic materials are capable of eliciting specific cellular responses and directing

new tissue formation [11]. Biomimetic CaP coating has been developed to deliver growth

factors for bone regeneration [4]. However, the increase of the thickness of this coating is

highly dependent on underlying substrates as a scaffold such as dental titanium implants,

polymers, and deproteinized bovine bone [4]. To overcome this limit, we recently

developed a biomimetic calcium-phosphate bone substitute (BioCaP) using a refined

biomimetic coating approach [12]. The BioCaP granule can be used as a dual

protein-delivery model which possesses two delivery modes: 1) an

Osteoinductive BioCaP for the repair of bone defects in sheep

49

internally-incorporated mode, protein can be incorporated into the interior of BioCaP; 2)

a coating-incorporated mode, protein can be incorporated into the coating on the surface

of BioCaP [13].

Our previous study has demonstrated that BioCaP with the two delivery modes

resulted in a slow release of protein in vitro, and an efficient osteoinduction in rats when

BMP-2 was delivered. The aim of this study was to evaluate the therapeutic effects of

BioCaP bone substitute with two delivery modes of BMP-2 in the repair of the

critical-sized bone defect in sheep. New bone formation and the degradation of BioCaP

were evaluated histologically and histomorphometrically after a 4- and 8-week

implantation.


Fabrication of biomimetic calcium phosphate (BioCaP) bone substitute

BioCaP was fabricated by refining a well-established biomimetic mineralization

approach [14]. A CaP solution (200 mM HCl, 20 mM CaCl2·2H2O, 680 mM NaCl, and

10 mM Na2HPO4) buffered by TRIS (250 mM) to a pH of 7.4. The whole solution was

incubated in a shaking water bath (50 agitations/min) at 37°C for 24 hours. Thereafter all

precipitations were retrieved and gently washed by Milli-Q water, strongly filtered and

compressed to a block using a vacuum exhaust filtering method with a vacuum filter

(0.22-μm pore, Corning, NY, USA) and an air pump. After drying in air circulation at

room temperature for 2 hours, the hardened block can be ground and filtered to obtain

granules with a size of 0.25-1.0mm using metallic mesh filters. For sterilization, the CaP

solution was filtered with the vacuum filter (0.22-μm pore) before buffering. All the

following procedures were performed under aseptic conditions.

BMP-2 (INFUSE® Bone Graft, Medtronic, USA) can be introduced into the CaP

solution at a final concentration of 0.2μg/ml before buffering as mentioned above and

thereafter was co-precipitated into the interior of BioCaP, viz., the internally-incorporated

mode.

Biomimetic coating procedure

The superficial coating was deposited on BioCaP according to the well-established

biomimetic mineralization approach [14]. Briefly, 0.58g of BioCaP with a size of

0.25-1mm was incubated in the coating solution [40 mM HCl, 4 mM CaCl2·2H2O, 136

mM NaCl, 2 mM Na2HPO4, and 50 mM TRIS (pH 7.4); total volume of 150 ml] in a

shaking water bath (50 agitations/min) at 37°C for 24 hours.

BMP-2 present in the coating solution at a final concentration of 0.2μg/ml can be

co-precipitated into the coating on BioCaP, viz., the coating-incorporated mode. The

granules were then freeze-dried. The entire procedure was conducted under sterile

conditions.

Quantification of the amount of the incorporated BMP-2

The amount of incorporated BMP-2 was determined by a commercially available

enzyme-linked immunosorbent assay (ELISA) kit (PeproTech, London, UK). 0.05g of

BioCaP with the two carry (delivery) modes of BMP-2 (n=6) was dissolved in 1ml 0.5M

EDTA (pH 8.0). The ELISA assay was performed according to the manufacturer's

Chapter 4

50

instructions. The BioCaP without BMP-2 was evaluated as a control. According to the

ELISA result, each sample of BioCaP granule that bore internally-incorporated or

coating-incorporated BMP-2 contained 10.5±1.27μg and 18.56±0.88μg of BMP-2,

respectively.

The morphology of BioCaP

The morphology of BioCaP granules with or without BMP-2 was visualised using a

scanning electron microscope (SEM, XL20, FEI Company, the Netherlands), under an

accelerating voltage of 10 kV after being sputter-coated with gold.

Surgical procedure

Twelve sheep were anesthetized by administering Sumianxin II (0.3 ml/kg, purchased

from the Military Veterinary Institute, Quartermaster University of PLA, Chang Chun,

China) with the addition of Penicillium (5 × 104 U/kg) and atropine (0.03 mg/kg) at 30

min before surgery. After applying local anaesthesia (1% lidocaine with 1:100,000

adrenaline) and skin disinfection (0.5% iodophor solution) to the implantation sites, the

surgery and animal care was performed and the cylinder-shaped defects were created (8

mm in diameter and 13 mm in depth) as described in a previous study [15]. The

implantation sites were the proximal part of the diaphysis and distal epiphysis of humerus

and femur of 12 adult female sheep. Each sheep provided 8 standardized implantation

sites. Six implantation sites were randomly chosen. These implantation sites were

assigned to the experiment groups according to a randomization protocol [16].

Membranes (Bio-Gide®, Geistlich Biomaterials, Wolhuser, Switzerland) were used to

cover the defects. Samples with surrounding tissues were retrieved at 4 weeks and 8

weeks post-operation.

Experimental groups

Six groups were established to treat CSBD (n=6 animals per group per time point, Table

1):

(i) BioCaP bearing neither a coating nor BMP-2 (experimental);

(ii) BioCaP bearing a coating-incorporated BMP-2(experimental);

(iii) BioCaP bearing an internally-incorporated BMP-2(experimental);

(iv) No graft material (negative control);

(v) Autologous bone (positive control); and

(vi) Deproteinized bovine bone (DBB, Bio-Oss®, control, a bovine xenograft, is one of

the most widely used commercial bone substitutes used in bone repair and

augmentation in clinical dentistry).

In the case of autograft, the bone was harvested at the same time as the creation of the

defect and reduced to 0.25-1mm particles using a rongeur.

Histological procedures

Samples with surrounding tissues chemically were fixed and embedded into a block as

previously reported [17, 18]. Applying a systematic random-sampling strategy [19], the

samples were sawed vertical to the long axis of the cylindrical defect, into 10-12 slices of

600-μm thickness, 1 mm apart (interval). All slices of each sample were separately


51

mounted on plexiglass holders and polished. The slices were surface-stained with

McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue [18] and examined with a light

microscope with a digital camera (Leica, Wetzlar, Germany).

Table 1

Experimental groups

Groups Abbreviation Granule

size

Graft

materials

BMP-2

Total

Loading

Volume

(amount) of graft

material

per

sample

Absence (−)

Presence

(+)

Dose of BMP-2

(per

sample)

(i) BioCaP bearing neither

a coating nor BMP-2 BioCaP 0.25-1.0mm

0.65cm3

(0.58g) - -

(ii)

BioCaP bearing

coating-incorporated

BMP-2

BioCaP BMP inc.

0.25-1.0mm 0.65cm3 (0.58g)

+ 10.5μg

(iii)

BioCaP bearing

internally-incorporated BMP-2

BioCaP

BMP int. 0.25-1.0mm

0.65cm3

(0.58g) + 18.56μg

(iv) No graft material

(negative control) NGM - - - -

(v) Autologous bone

(positive control) AB 0.25-1.0mm 0.65cm3 - -

(vi) Deproteinized bovine

bone (Bio-Oss®) DBB 0.25-1.0mm

0.65cm3 (0.35g)

- -


In addition to a subjective histological description, 10 slices of each sample was used for

quantitative histomorphometric analysis including the volume of newly formed bone,

bone marrow (fat), BioCaP and the volume density of multinucleated giant cells (MGCs)

on BioCaP or DBB. Using the point-counting technique [20], the surface area (S) of a

component per slice was obtained. The interval between two slices was 1mm. Therefore,

the volume (V) of a component is defined as: V ∑ ( )

The volume density of MGCs was normalized to the volume of BioCaP or DBB. The

volume density of MGCs (Va) is defined as its volume (Vb) per unit volume of graft

materials (Vc): Va= Vb/Vc. To evaluate the degradation of BioCaP granules, the volume

of BioCaP before implantation (time 0, as control) were evaluated by the same

Chapter 4

52

histological method. Six chemically fixed and plastic-embedded samples (0.58g of

BioCaP granules per samples) were specifically reserved for this purpose.


All data are presented as mean values together with the standard deviation (SD). Data

were compared using a one way analysis of variance (ANOVA), and post-hoc

comparisons were made using Tukey's corrections. The significance level was set at p

< .05.

RESULTS

In-vitro characterization

A scanning electron microscopy (SEM) micrograph of BioCaP granules is depicted in

Fig. 1A. BioCaP granule that bore internally-incorporated BMP-2 showed rough surface

(Fig. 1B and C). BioCaP granule with a layer of coating was shown in Fig. 1D. BioCaP

granule that bore coating-incorporated BMP-2 showed crystalline surface (Fig. 1E and F).

The incorporation of BMP-2 did not affect the surface morphology of BioCaP or the

coating [13].

Clinical observations

After 4- and 8-week implantation, a total of 72 implants were harvested (36 implants at 4

weeks and 36 implants at 8 weeks). All the sheep exhibited good health and all the

surgical implant sites healed well without any significant wound complication. No visual

signs of inflammatory or adverse tissue reaction were observed.

Figure 1. SEM micrographs of the BioCaP granules (A). BioCaP with internally-incorporated

BMP-2 showed a rough surface (B) with calcium phosphate microspheres (C). A cross section of

BioCaP granules with coating-incorporated BMP-2 showed the coating layer on the surface of

BioCaP (D). This coating displayed a crystalline rough surface (E) with tiny crystalline plates (F).


53

Descriptive light microscopy

Histological images of each group at 4 and 8 weeks of implantation are depicted in Fig. 2.

At 4 week, newly formed bone was observed in close contact with the BioCaP granules

in the three BioCaP groups (see Figs 2A-C). BioCP with BMP-2 showed more bone

formation than BioCaP without BMP-2. The new bone started to form a network in some

areas. The empty group (negative control) confirmed that the defect was critically sized

(Fig. 2D and Fig. 2d). Bone defect was not healed after 8 weeks. New bone only

appeared sporadically near the defect boundary and fibrous tissues were constantly

appeared in the center part of the defect. The DBB group (Fig. 2F) showed significantly

less bone formation compared with the BioCaP with incorporated BMP-2 or autologous

groups (Fig. 2E). Images at higher magnification of the three BioCaP groups showed a

thin layer of bone which capsulated BioCaP granules (Fig. 3A and C), and new bone was

also observed between the granules (Fig. 3E).

At 8 weeks, all the graft groups showed significant bone formation (see Figs 2a-f).

BioCaP with internally-incorporated BMP-2 (Fig. 2c) displayed significantly more bone

formation compared with BioCaP without BMP-2 (Fig.2a). In areas where BioCaP with

or without BMP-2 (Figs 2a-c) was still present, a complete interconnected bone network

could be observed. The deposited bone was constantly in close contact with these BioCaP

granules, most of which were entirely encapsulated in the new bone. The newly formed

bone had a woven appearance including the presence of remodeling lacunae. DBB also

showed a similar trabecular bone structure (Fig. 2f). The autologous bone group showed

uneven woven bone formation (Fig. 2e). Images at higher magnification of the three

BioCaP groups showed that the areas mainly consisting of bone, bone marrow and

BioCaP with or without BMP-2 can be observed indicating a healthy bone environment

(Fig. 3B, D, and F). The group containing BioCaP with internally-incorporated BMP-2

showed that BioCaP had been replaced by newly formed bone in some areas with a

trabecular bone appearance (Fig. 4A). It was displayed that the woven bone was

undergoing remodeling to be the lamellar bone due to the osteoblasts and multinucleated

osteoclasts (Fig. 4B).

At 4 weeks, a few BioCaP granules were surrounded by multinucleated giant cells,

which were apparently osteoclastic cells (Fig. 5A). No bone formation occurred in this

area. Multinucleated giant cells were observed sporadically on BioCaP at 8 weeks (Fig.

5B). In both implantation time points, it was difficult to recognize or find the coating on

BioCaP, which indicated the coating to be degraded completely.

Chapter 4

54

Figure 2. Representative histological micrographs at higher magnification of bone defect of each

group at 4 and 8 weeks after implantation. Groups: (i) BioCaP (asterisk) bearing neither a coating

nor BMP-2 (A; a); (ii) BioCaP bearing a coating-incorporated BMP-2 (B; b); (iii) BioCaP bearing

an internally-incorporated BMP-2 (C; c); (iv) No graft material (D; d); (v) Autologous bone (E; e);

and (vi) Deproteinized bovine bone (#) (F; f). At 4 weeks, the newly formed bone (unmineralized)

was dark purple (arrow). At 8 weeks, the newly formed bone (mineralized) was reddish (arrow).

The newly formed bone and the autologous (+) bone can be separated in the group (v). The slices

were surface-stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue. Scale bar = 1

mm.


55

Figure 3. Representative histological micrographs at higher magnification of bone defect of the

three BioCaP groups at 4 and 8 weeks after implantation. At 4 weeks, BioCaP (A), BioCaP with

coating-incorporated BMP-2 (C), BioCaP with internally-incorporated BMP-2 (E). New bone

(white arrow) was observed in close contact with BioCaP (asterisk) or encapsulating BioCaP. At 8

weeks, BioCaP (B), BioCaP with coating-incorporated BMP-2 (D), BioCaP with

internally-incorporated BMP-2 (F). Most areas of the bone defect were filled with bone, bone

marrow (M) and BioCaP. Most residual BioCaP granules were entirely encapsulated in the new

bone. Bone marrow was in close contact with BioCaP. The slices were surface-stained with

McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue. Scale bar = 200 µm.

Chapter 4

56

Figure 4. Representative histological micrographs of trabecular bone structure with bone

remodeling in the group containing BioCaP (asterisk) with internally-incorporated BMP-2 (A). At

higher magnification (B), osteoblasts (white arrow) and osteoclasts (black arrow) were observed.

The slices were surface-stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue.

Scale bar = 200µm in A. Scale bar = 50µm in B.

Histomorphometric results

Quantitative evaluation of the amount of bone formation after 4 and 8 weeks of

implantation (Fig. 6) revealed that bone formation significantly increased with increasing

implantation time (p<0.05). At 4 weeks post-implantation, the bone formation in the

samples containing autologous bone was the highest. Significantly more bone formation

was observed in the groups containing BioCaP with internally- or coating-incorporated

BMP-2 than the group containing BioCaP without BMP-2 or containing DBB. No

significant difference was found between the two groups containing BioCaP with

BMP-2.

At 8 weeks implantation time, no significant difference in bone formation was found

between autologous bone and BioCaP with internally- or coating-incorporated BMP-2.

Significantly more bone formation was observed in the group containing BioCaP with

internally-incorporated BMP-2 compared with the group containing DBB. Significantly

more bone formation was observed in the group containing BioCaP with

internally-incorporated BMP-2 than the group containing BioCaP without BMP-2.

Quantitative evaluation of the amount of bone marrow (Fig. 7) revealed that at 4

weeks bone marrow only appeared in the groups containing BioCaP with or without

BMP-2 and autologous bone. Significantly more bone marrow was observed in the group

containing BioCaP with coating-incorporated BMP-2 compared with the group

containing BioCaP with internally-incorporated BMP-2 or without BMP-2. At 8 weeks,

no significant difference was found between the groups containing BioCaP with or

without BMP-2 and autologous bone. Significant more bone marrow formation was

observed in the groups containing BioCaP with or without BMP-2 compared with the

group containing DBB.

BioCaP degradation significantly increased with increasing implantation time (p<0.05)

(Fig. 8). BioCaP with internally-incorporated BMP-2 or without BMP-2 showed

significantly more BioCaP degradation after 4 and 8 weeks of implantation than the

BioCaP with coating-incorporated BMP-2.


57

The volume density of multinucleated giant cells (MGCs) at 4 weeks was lowest in

association with samples containing BioCaP with internally or coating-incorporated

BMP-2 (Fig. 9). At 8 weeks, no significant difference in the volume density of MGCs

was found among the four groups contain BioCaP or DBB.

Figure 5. Representative histological micrographs of multinucleated giant cell (black arrow) on the

surface of BioCaP at 4 weeks (A) and 8 weeks (B) from the group containing BioCaP with

internally-incorporated BMP-2. At 4 weeks, multinucleated giant cells were easily observed on a

few BioCaP granules in all the three BioCaP groups, whereas at 8 weeks, multinucleated giant cells

were occasionally found, since most BioCaP granules had been covered by new bone. Osteocytes

(white arrow). The slices were surface-stained with McNeal's Tetrachrome, basic Fuchsine and

Toluidine Blue. Scale bar = 50 µm.

Chapter 4

58

Figure 6. Graph depicting the volume of newly formed bone within the bone defect at 4 and 8

weeks after implantation for each group (see Table 1 for an explanation of the abbreviations). Mean

values (n=6 samples per group) are represented together with the standard deviation. *p<0.05.

Figure 7. Graph depicting the volume of bone marrow within the bone defect at 8 weeks after

implantation for each group (see Table 1 for an explanation of the abbreviations). Mean values

(n=6 samples per group) are represented together with the standard deviation. *p<0.05.


59

Figure 8. Graph depicting the percentage degradation of BioCaP within the bone defect at 4 and 8

weeks after implantation for each of the BioCaP groups. See Table 1 for an explanation of the

abbreviations. Mean values (n=6 samples per group) are represented together with the standard

deviation. *p<0.05.

Figure 9. Graph depicting the volume density of multinucleated giant cells on the surface of

BioCaP or DBB at 4 and 8 weeks after implantation. See Table 1 for an explanation of the

abbreviations. Mean values (n=6 samples per group) are represented together with the standard

deviation. *p<0.05; +p<0.01.

DISCUSSION

Recently, a biomimetic calcium phosphate bone substitute (BioCaP) was developed as a

dual delivery model with two delivery modes, viz., an internally-incorporated mode and a

coating-incorporated mode. In current study, BioCaP with the two delivery modes of

BMP-2 proved to be equally efficient as autologous bone in the repair of critical-sized

bone defects at 8 weeks after implantation. The findings indicate that BioCaP with each

delivery mode of BMP-2 can be a promising alternative to the autografts.

Chapter 4

60

Critical-sized bone defect is defined as the intraosseous wound with the smallest size,

which cannot spontaneously heal completely without intervention [21]. It is usually used

as an experimental model to test bone repair materials [22]. The critical-sized bone defect

(CSBD) model in this study was created by drilling holes in the humerus and femur of

sheep according to a widely published protocol by Nuss et al. [15]. This drill hole model

in sheep has proved to be an excellent animal model for testing biomaterials for use in

orthopedics, maxillofacial and dental surgery [23]. It allowed the intraosseous

implantation of up to 8 different test materials within one animal due to the

standardization of the bone defect, while at the same time it can reduce the overall

suffering of animals and give the necessary numbers to satisfy statistical requirements

[15, 24]. Because of the similarities with humans in weight, bone and joint structure and

bone regeneration, the results from sheep are more valid than those obtained from small

laboratory animals [25]. Although rodents may be less expensive, they have a different

bone morphology and they are often are too small for testing bone substitute. Positive

results in rodents may have to be repeated and verified in larger species before human

clinical trials can be initiated.

In the current study, we showed that the coating-incorporated mode of BMP-2

significantly induced more bone formation for BioCaP at 4 weeks but not 8 weeks. The

accelerated bone formation is attributed to the gradual degradation of the coating with the

slow release of BMP-2 [26]. However, the coating could be totally degraded within 5

weeks [17]. The coating on BioCaP cannot be recognized or distinguished from BioCaP

using the histological staining. The reason could be the similar chemical property of the

coating and BioCaP, since both of which are obtained by precipitating calcium phosphate.

The new bone observed wrapping the BioCaP granules may cover the coating and

thereby delay its degradation. The internally-incorporated mode of BMP-2 significantly

accelerated more bone formation for BioCaP at both implantation time points (4 and 8

weeks), which indicated that the release of BMP-2 may be sustain during the 8 weeks.

The internally-incorporatedBMP-2 could be continually released as long as BioCaP has

not been totally degraded. The coating has been demonstrated as a three-dimensional

reservoir from which BMP-2 can be gradually liberated [4], while BioCaP using the

granular form can offer a larger reservoir than the coating.

Moreover, BioCaP resulted in more bone marrow compared with DBB. The

abundance of bone marrow bodes well for the health and the endurance of the newly

formed bone, since it is an important source of nutriments and pluripotent progenitor

cells for osseous tissue [27]. Both osteoblasts and osteoclasts are derived from

progenitors that reside in the bone marrow. Therefore, bone marrow is critical for bone

remodeling. The further degradation of BioCaP may be dependent on the bone

remodeling.

Histological and histomorphological analysis revealed the good biodegradability of

BioCaP. Ideal biodegradability refers to that bone substitute can be degraded in short

time, enabling bone remodeling, and concomitantly replaced by bone tissue [28]. The

degradation of calcium phosphate materials depended on the self-dissolubility and

cell-based resorption [29]. The use of coating can delay the degradation of BioCaP.

When the induced bone covered or even encapsulated BioCaP, the degradation of

BioCaP could also be delayed. In this case, the encapsulated residual BioCaP can be

degraded in the following bone remodeling process. The observed degradation of BioCaP


61

was associated with the multinucleated osteoclast-like cells. There cells were not only

observed on bone but also on BioCaP. However, the two modes of BMP-2 resulted in a

lower volume density of these osteoclast-like cells on material surface. This might also

prevent the degradation of BioCaP. Our previous study has proven that the slow release

of BMP-2 from the biomimetic coating suppress the formation of multinucleated giant

cells [30]. In other studies, BMP-2 has been shown to stimulates the recruitment,

proliferation and differentiation of osteoprogenitor cells at low doses, whereas it induces

the recruitment, formation and activation of osteoclasts at high doses [31, 32].

In this study, BioCaP bone substitute was produced by the precipitation of calcium

phosphate in a biomimetic environment which can retain the protein biological activity

[7]. Both of BioCaP and the biomimetic calcium phosphate coating are produced in this

environment using similar calcium phosphate solutions. The biomimetic coating has been

demonstrated to be highly biocompatible and osteoconductive [4]. Good biocompatibility

refers to the ability of a biomaterial to perform its desired function with respect to a

medical therapy, without eliciting any undesirable local or systemic effects in the

recipient or beneficiary of that therapy, but generating the most appropriate beneficial

cellular or tissue response in that specific situation, and optimizing the clinically relevant

performance of that therapy [33]. Osteoconductivity is the ability of the graft to function

as a scaffold to permit bone growth on its surface or for ingrowth of new bone [34]. In

the present study, BioCaP granules were observed in close contact with bone or bone

marrow. These findings indicated the good biocompatibility and osteoconductivity of

BioCaP, This may be attributed to its biomimetic chemical property.

It has been known that the slow delivery of BMP-2 enhances osteoinduction [9], and

the slow delivery of vascular endothelial growth factor (VEGF) promotes vascularization

[35]. Furthermore, since bone regeneration is a coordinated cascade of events regulated

by several growth factors, the local sequential delivery of VEGF and BMP-2 could

enhance bone formation compared to BMP-2 alone [36]. Therefore, BioCaP can just turn

to this application for the local, sequential and slow delivery of VEGF and BMP-2. Other

candidates could be antibiotics and anti-cancer drugs. There is a need for further

investigation into this dual release model.

CONCLUSION

In conclusion, the findings indicate that BioCaP with the two delivery modes of BMP-2

can be a promising alternative to the autografts. The coating-incorporated mode of

BMP-2 accelerated the bone formation for BioCaP in an early period (4 weeks); the

internally-incorporated mode enhanced bone formation in a longer period (8 weeks),

which is more efficient for the large bone defect repair. BioCaP with the two delivery

modes of BMP-2 give better bone formation compared with the deproteinized bovine

bone (a commercial product). BioCaP showed good biodegradability. It might not be too

earlier to conclude that BioCaP with the two delivery modes of BMP-2 would be the

substitute for autologouse bone for clinic applications. Benefiting from the two delivery

modes, BioCaP could be used as a dual delivery vehicle for the sequential delivery of

different protein/drugs.

Chapter 4

62

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reconstruct peri-implant bone defects: a micro-CT evaluation. J Clin Periodontol

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22. Hollinger JO, Kleinschmidt JC. The critical size defect as an experimental model to test bone

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23. Theiss F, Apelt D, Brand B, Kutter A, Zlinszky K, Bohner M, et al. Biocompatibility and

resorption of a brushite calcium phosphate cement. Biomaterials 2005;26:4383-94.

24. Apelt D, Theiss F, El-Warrak AO, Zlinszky K, Bettschart-Wolfisberger R, Bohner M, et al. In

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morphogenetic protein 2 signaling in osteoclasts is negatively regulated by the BMP

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Chapter 4

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65

Chapter 5

A novel BMP2-coprecipitated, layer-by-layer

assembled biomimetic calcium phosphate particle: a

biodegradable and highly-efficient osteoinducer

Yuanna Zheng, Gang Wu, Tie Liu, Daniel Wismeijer, and Yuelian Liu.

Clinical Implant Dentistry and Related Research,

2013 Mar 4. doi: 10.1111/cid.12050. [Published online]

Chapter 5

66

ABSTRACT

Statements of the problem: To repair large-size bone defects, most bone-defect-filling

materials in clinic need to obtain osteoinductivity either by mixing them with particulate

autologous bone or adsorbing bone morphogenetic protein-2 (BMP-2). However, both

approaches encounter various limitations. In this study, we hypothesized that our novel

particles of biomimetic BMP-2-coprecipitated calcium phosphate (BMP2-cop.BioCaP)

could serve as an independent and biodegradable osteoinducer to induce bone formation

efficiently for these bone-defect-filling materials, e.g. deproteinized bovine bone (DBB).

Method of study: We alternately layer-by-layer assembled amorphous and crystalline

CaP triply to enable a “bamboo-like” growth of the particles. We functionalized BioCaP

by coprecipitating BMP2 into the most outer layer of BioCaP. We monitored the

degradation, osteoinductivity and foreign-body reaction of either BMP2-cop.BioCaP or

its combination with DBB in an ectopic site in rats.

Results: After 5 weeks, the BMP2-cop.BioCaP significantly induced new bone

formation not only alone but also when mixed with DBB. Its osteoinductive efficiency

was 10-fold higher than the adsorbed BMP2. Furthermore, BMP2-cop.BioCaP also

reduced significantly the host foreign body reaction to DBB in comparison with the

adsorbed BMP2. After a 5-week implantation, more than 90% of BMP2-cop.BioCaP

degraded.

Conclusions: These findings indicate a promising clinical potential for

BMP2-cop.BioCaP in the repair of large-size bone defects.

Keywords: Biomimetic; Bone morphogenetic protein; Bone regeneration; Calcium

phosphate; Layer-by-layer; Osteoinducer

INTRODUCTION

Large-size bone defects exceed the self-healing capacity of bone tissue and often a

pro-fibrotic microenvironment is formed in the defects [1]. To realize their osseous

restoration, bone-defect-filling materials are indispensible. Although autografts are still

regarded as the “gold-standard” bone-defect-filling materials, their application is still

limited because of the low available quantity as well as donor-site pain and morbidity [2].

Consequently, allografted, xenografted and synthetic calcium phosphate (CaP)-based

materials (e.g. deproteinized bone and biphasic CaP) are widely adopted clinically for the

treatment of large-size bone defects. These materials are also highly osteoconductive

which enhances the migration of osteogenic cells. However, such an enhancement is still

too limited to realize osseous restoration. They intrinsically lack osteoinductivity for

inducing bone regeneration in a pro-fibrotic environment. One common approach used

clinically is to combine bone-defect-filling materials with ground autografts [3] — which

supplies the necessary osteogenic elements — for repairing large-size bone defects. In

this case, the limitations above mentioned of autografts ensue.

One promising approach to this problem is to confer osteoinductivity to these

CaP-based materials by using an osteogenic agent, such as bone morphogenetic protein 2

(BMP2). BMP2 is a dimeric disulfide-linked polypeptide growth factor under

transforming growth factors- superfamily. BMP2 has been approved by FDA and shown

A highly-efficient osteoinducer

67

to induce bone formation in animal studies and clinical trials [4-7]. A consensus has been

reached that the in-vivo osteoinductive efficiency of BMP2 is highly dependent on its

release kinetics. The present mode of delivery in clinic ─ superficial adsorption of BMP2

onto bone-defect-filling materials [8] ─ is associated with a rapid and burst release [9,

10]. Most of such delivered BMP2 is released too rapidly to induce a sustained

osteogenic response at the site of the implantation. This difficulty cannot be overcome

satisfactorily merely by increasing the loading dose of BMP2. Apart from the tremendous

expense that would be incurred, the transient high local concentration of BMP2, which

would be generated, could induce deleterious side effects, such as an excessive

stimulation of local bone resorption and the induction of bone formation at unintended

sites [11-13].

To be optimally osteoinductive, BMP2 needs to be delivered to target sites at low

level concentrations in a sustained manner. One such approach is to coprecipitate BMP2

into a thin layer of biomimetic CaP coating that is prepared on the surfaces of

biomaterials [4]. We have recently shown that coating-coprecipitated BMP2 induced a

significantly higher volume of new bone surrounding the biomaterials than the

superficially adsorbed BMP2 [14]. In addition, the coating-coprecipitated BMP2 could

also suppress significantly the host foreign-body reaction to the biomaterials, while the

superficially adsorbed BMP2 could not [15]. On the other hand, although the biomimetic

coating technique is broadly applicable to a series of bone-defect-filling materials [16],

its application is not unlimited because of the dependence of coating growth on the

physicochemical properties of the underlying biomaterials as well as the need to prepare

the coatings on these materials.

Recently, we made a breakthrough in modifying the biomimetic coating procedure.

Thereby, we have for the first time alternately layer-by-layer assembled

BMP2-coprecipitated biomimetic CaP particles (BMP2-cop.BioCaP) that could serve as

an independent “osteoinducer”. This novel BMP2-cop.BioCaP was designed to be mixed

directly with clinically-used bone-defect-filling materials to induce bone formation. In

this study, we monitored the biological properties of BMP2-cop.BioCaP such as

degradation, osteoinductivity and foreign-body reaction. We also ascertained whether

BMP2-cop.BioCaP could efficiently induce bone formation surrounding, and suppress

the host foreign-body reaction to a clinically-used bone-defect-filling material ─

deproteinized bovine bone (DBB).


In-vitro investigation

Preparation of Layer-by-layer assembled biomimetic calcium phosphate (BioCaP)

particles with or without coprecipitated BMP2

The protocol (Fig. 1) to produce the Layer-by-layer assembled BioCaP particles was

derived from our well established biphasic biomimetic coating protocols [4, 17, 18].

Briefly, micro-particles of amorphous CaP were obtained in 2000ml of a

five-fold-concentrated simulated body fluid [684mM NaCl; 12.5mM CaCl2·2H2O; 21mM

NaHCO3; 5mM Na2HPO4·2H2O and 7.5mM MgCl2·2H2O (Sigma, St. Louis, USA)] for

24 hours at 37C. Thereafter, the amorphous CaP micro-particles were immersed in

1000ml of a supersaturated calcium phosphate solution [40mM HCl; 2mM

Chapter 5

68

Na2HPO4·2H2O; 4mM CaCl2·2H2O; 50mM TRIS base (Sigma, St. Louis, USA) (pH 7.4)]

for 48 hours at 37C. Thereby, a thick layer of crystalline CaP was deposited on

amorphous CaP micro-particles. After drying at room temperature, these particles were

then immersed in the 5-fold simulated body fluid (24 hours) and the supersaturated

calcium phosphate solution (48 hours) alternately for a total of three cycles. During the

preparation of the final crystalline CaP layer, BMP2 (INFUSE® Bone Graft, Medtronic,

USA) was introduced into this supersaturated calcium phosphate solution at a final

concentration of 2µg/ml and coprecipitated with the crystalline CaP layer. The samples

were then air-dried. The entire procedure was conducted under sterile conditions.

Figure 1. Schematic graphs demonstrate the layer-by-layer assembling process of biomimetic

(CaP). Micro-particles of amorphous CaP that were initially obtained from a 5-fold simulated body

fluid were immersed into supersaturated CaP solution for 48 hours and 5-fold simulated body fluid

for 24 hours alternately. Thereby, amorphous CaP and crystalline CaP were layer-by-layer

assembled. Then, the particles were immersed into a supersaturated CaP solution with 2µg/ml

BMP2. After 48 hours, the particles were air-dried and ready for use. The increase of particle size

was attributed to both the layer-by-layer growth of coatings and the aggregation of particles by the

growing coatings.

Surface characterization of the BioCaP

The surface characteristics of BioCaP were evaluated in a scanning electron microscope

(XL 30, Philips, the Netherlands). For this purpose, samples of the material were

mounted on aluminium stubs and sputtered with gold particles to a thickness of 10-15nm.

Determination of the amount of the coprecipitated BMP2

The amount of coprecipitated BMP2 was determined using a commercially available



69

BMP2-cop.BioCaP was dissolved in 1ml 0.5M EDTA (pH 8.0). The ELISA assay was

performed according to the manufacturer's instructions. Three samples were used for this

purpose.

Confirmation of the homogeneous distribution of the coprecipitated protein by

fluorescence microscopy

To investigate the distribution of the coprecipitated protein within BioCaP, BMP2 was

substituted by a model protein ─ bovine serum albumin that had been conjugated with

fluorescein isothiocyanate [19] [FITC-BSA (Sigma, St. Louis, USA)]. FITC-BSA was

introduced into the supersaturated calcium phosphate solution at a final concentration of

2µg/ml. After freeze-drying, the coated samples were embedded in methylmethacrylate.

600m-thick sections were prepared and affixed to Plexiglas holders. These sections

were then ground down to a thickness of 80 m for an inspection in a fluorescence

microscope.

In-vitro monitoring of the release kinetics of the coprecipitated protein in BioCaP

To monitor the release kinetics of the coprecipitated protein in BioCaP, FITC-BSA

(2µg/ml) was introduced into supersaturated calcium phosphate solution for the final

immersion. Six samples were used to determine the total amount of coprecipitated

FITC-BSA. These samples were immersed in 1ml of 0.5% EDTA (pH 8.0) and vortexed

twice for 5 minutes to ensure complete dissolution of coatings. The supernatants were

withdrawn for analysis of total loading of FITC-BSA.

To monitor the release kinetics, six samples of DBB mixed with

FITC-BSA-cop.BioCaP at a volume ratio of 4:1 and six samples of DBB bearing an

equivalent amount of adsorbed FITC-BSA (included for the purpose of comparison and

prepared likewise as the adsorption of BMP2) were incubated in sealed 10-ml glass tubes

containing 2ml of phosphate-buffered 0.9% saline (pH 7.4). The tubes were incubated for

up to 35 days in a shaking waterbath (60 agitations/min), which was maintained at 37C.

The sampling and measurement with spectrophometer were performed following the

protocol as previously published [15]. Fluorescence readings were converted to amounts

of protein using a standard curve, which was generated by preparing a dilution series of

FITC-BSA in 5ml of phosphate-buffered 0.9% saline. The temporal release of FITC-BSA

was expressed as a percentage of the total amount that had been coprecipitated into the

crystalline layer of the BioCaP or that had been adsorbed directly onto the DBB particles.


We adopted a subcutaneous bone induction model in rats to further evaluate the

BMP2-cop.BioCaP in vivo in aspects of degradation, osteoinductivity and foreign-body

reactivity. We measured the following parameters: 1) volume density of newly formed

bone; 2) volume density of foreign-body giant cells; 3) volume density of BioCaP and 4)

osteoinductive efficiency of BMP2.

Grouping

As an experimental animal model, we used adult male Wistar rats (200-220g). Six groups

were established (n=6 animals per group): (1) BioCaP; (2) BMP2-cop.BioCaP; (3) DBB

alone (4) DBB bearing adsorbed BMP2; (5) DBB mixed with 0.07cc BioCaP; (6) DBB

Chapter 5

70

mixed with 0.07cc BMP2-cop.BioCaP. The amount of BMP2-cop.BioCaP (0.07) was

determined according to our previous study [15]. It showed that about 10-15µg of the

coating-coprecipitated BMP2 could sufficiently induce bone formation. 0.07cc

BMP2-cop.BioCaP contains 10.29±1.94µg BMP2 according to the ELISA result.

0.15g of DBB (about 0.35cc) per sample was used. The samples of DBB bearing

adsorbed BMP2 (about 13.5µg) were prepared as described previously [15]. The loading

process was achieved by introducing a 75-µl aliquot of stock solution (0.18µg/µl) into

1-ml Eppendorf tubes containing 0.15g of DBB particles.

Surgery and histology

Animal experiments were conducted with the permission of and in accordance with the

regulations laid down by the Animal Protection Commission of the State of Bern

(Switzerland). 18 rats were used in this study. Each rat received two samples from two

different groups: they are either non-BMP-2-containing discs (group 1, 3, 5) or

BMP-2-containing discs (group 2, 4, 6). To the end, 6 animals were used for each group.

(n=6 animals per group).

The rats were acclimatized to their new surroundings for 5 days. Housing is in

compliance with the national guidelines for animal experimentation. Surgery was

performed under conditions of general anesthesia [using Vetalar® (ketamine

hydrochloride) (Boehringer Ingelheim Vetmedica, Inc., St. Joseph, USA] [4]. Two

samples per rat were surgically implanted within lateral dorsal subcutaneous pockets (one

on the left side and one on the right), and were trapped therein by suturing the incision

site. After surgery, the rats were kept in cages of Animal facility of Bern University for 5

weeks. The animals were fed ad libitum with hay, granulated food and water.

Five weeks after surgery, the samples were retrieved, chemically fixed and embedded

in methylmethacrylate as previously reported [4, 18]. By applying a systematic

random-sampling strategy [20], the samples were sawed vertical to the short axis, into

10-12 slices of 600µm-thickness with 1mm apart. Odd- or even-numbered slices of each

sample were separately mounted on Plexiglas holders and polished. The odd-numbered

slices were surface-stained with McNeal’s Tetrachrome, basic Fuchsine and Toluidine

Blue O [21] for the histomorphometric analysis of various parameters (see below). The

even-numbered slices were subjected to the tartrate-resistant acid phosphatase (TRAP)

reaction [4, 22] and counterstained with Methyl Green. They were used to estimate the

volume density of multinucleated osteoclasts. Applying a two-step systematic

random-sampling strategy, 25-30 images at a final magnification of 320 were recorded

in a Nikon-Eclipse light microscope and printed in color for the histomorphometric

analysis.


In the present study, the space under the fibrous capsule that embraced the whole block of

implants (subcapsular space) was taken as the reference space. The reference space was

estimated using Cavalieri’s methodology [23]. This involves measuring the

cross-sectional area of a defined number of tissue sections at a fixed distance apart

through the reference volume. The cross-sectional area of each section was estimated

using the point-counting technique [24].

The volume densities of bone, of the multinucleated cells and of the remaining


71

materials were determined stereologically from its area density on tissue sections by the

point-counting technique [24]. The volume density of foreign-body giant cells (FBGCs)

was obtained by subtracting that of TRAP-positive osteoclasts from that of

multinucleated cells [4]. To compare the foreign-body reaction to either

BMP2-cop.BioCaP or BioCaP or DBB in different groups, the volume dentisity of

FBGCs was normalized to the corresponding volume density of BMP2-cop.BioCaP or

BioCaP or DBB.

The total volume of bone and the remaining BioCaP material were estimated by

multiplying the volume densities of each parameter by the corresponding subcapsular

reference volume. The osteoinductive efficiency of BMP2 was estimated by dividing the

total volume of bone by the amount of BMP2. Since more than 90% of

BMP2-cop.BioCaP was degraded, we assumed all the coprecipitated BMP2 in its outer

layer was completely used. The BMP2 that was adsorbed onto DBB should also be

exhausted after 5 weeks since it exhibited a burst release. Therefore, we use the total

loading of BMP2 to estimate the osteoinductive efficiency.


All data are presented as mean values together with the standard deviation (Mean±SD).

Data were compared using a one-way analysis of variance (ANOVA) with the

significance level being set at p<0.05. Post-hoc comparisons were made using

Bonferroni’s corrections.

RESULTS

In-vitro characterization:

In this study, we assembled 3-dimensional particles using this novel biomimetic

layer-by-layer assembling technique. Under scanning electron microscopy, the

amorphous CaP microparticles that were derived from the 5-fold simulated body fluid in

the first cycle showed morphology of irregular clusters of microspheres with a diameter

of 1.5-3µm (Fig. 2A). After immersing these amorphous CaP microparticles in

supersaturated calcium phosphate solution for 48 hours, a crystalline CaP deposited on

their surfaces and showed plate or needle-like crystals (Fig. 2B). After three cycles of

alternate immersion, the particle size increased from the initial 5-20 µm up to 100-1000

µm (Fig. 2D) with a crystalline outer layer (Fig. 2C). The coprecipitated protein is

located within the whole outer crystalline CaP layer (Fig. 3A). As anticipated, the

FITC-BSA that was adsorbed onto DBB was released rapidly, being completely

exhausted after 13 days (Fig. 3B). In contrast, protein that was coprecipitated into

BioCaP was released gradually and at a steady rate after the 3rd

day until the 35th

day, at

which juncture the initial depot had been depleted by no more than 50.1% (Fig. 3B). The

total loading of BMP2 in 0.35cc BMP2-cop.BioCaP is 51.13±9.68 µg with a

coprecipitation rate of 30.1±5.7%.

Chapter 5

72

Figure 2. Scanning electron micrographs depicting the morphologies of the initial amorphous CaP

particles (A), the initial layer of crystalline CaP (B), and the final BMP2-cop.BioCaP (C&D).

Bars=5m in A, B, and C. Bar=200m in D.

Figure 3. (A) Fluorescence micrographs depicting the distribution of coprecipitated protein in the

outer layer of BioCaP. FITC-BSA (green signal) was used to as a substitute for bone morphogenetic

protein-2. Bar=100m. (B) Graph depicting the in-vitro release kinetics of BMP2 from DBB with

BMP2-cop.BioCaP and DBB with adsorbed BMP2.


73

In-vivo study:

Histological results

Five weeks after the subcutaneous implantation, BioCaP distributed compactly and did

not induce new bone formation (Fig. 4A). Connective tissue infiltrated into the BioCaP

particles, on the surfaces of which multinucleated FBGCs was frequently found (Fig.

4A1). In contrast, BMP2-cop.BioCaP distributed loosely and induced a large volume of

new bone (Fig. 4B). BMP2-cop.BioCaP was tightly integrated into the new bone (Fig.

4B1).

Figure 4. Light micrographs of the cross-sections through BioCaP (A&A1) and BMP2-cop.BioCaP

(B&B1) after a 5-week implantation in subcutaneous site in rats. The sections were stained with

McNeal’s Tetrachrome, basic Fuchsine and Toluidine Blue O. Yellow arrows points to the

foreign-body giant cells (FBGCs) lying on BioCaP. Black arrows points to the remaining BioCaP.

Asterisks indicates the newly formed bone. Bars=200m in A and B. Bars=30m in A1 and B1.

Five weeks after implantation, new bone was only found surrounding the DBB either

with adsorbed BMP2 (Fig. 5B) or mixed with BMP2-cop.BioCaP (Fig. 5D). No new

bone formation was found surrounding the DBB either alone (Fig. 5A) or mixed with

BioCaP (Fig. 5B). Bone was deposited abundantly surrounding the DBB with

BMP2-cop.BioCaP (Fig. 5D), but only sporadically surrounding the DBB with an

adsorbed BMP2 (Fig. 5B). The remaining BioCaP showed clusters of round or ellipse or

irregular microparticles (Fig. 5C1). They distributed among the DBB particles (Fig. 5C)

Chapter 5

74

with connective tissue filling in between (Fig. 5C1). For the DBB with

BMP2-cop.BioCaP, bone tissue formed with BMP2-cop.BioCaP as centre and deposited

on the surfaces of both BMP2-cop.BioCaP and DBB (Fig. 5D). Bone tissue was found

tightly integrated with BMP2-cop.BioCaP and DBB without intervening tissue (Fig.

5D1).


Volume density of bone surrounding BMP2-cop.BioCaP was [0.36±0.07 (mm3/mm

3)]

(Fig. 6A). The remaining percentage of BMP2-cop.BioCaP (5.6±2.1%) was significantly

lower than that of BioCaP (34.2±6.4%) (Fig. 6B). The volume ratio of FBGCs to

BMP2-cop.BioCaP [0.063±0.0198 (mm3/mm

3)] was also significantly lower than that to

BioCaP [0.110±0.0188 (mm3/mm

3)] (Fig. 6C).

Volume density of bone surrounding DBB mixed with BMP2-cop.BioCaP [0.06±0.03

(mm3/mm

3)] was significantly higher than that surrounding DBB with adsorbed BMP2

[0.007±0.009 (mm3/mm

3)] (Fig. 7A). The osteoinductive efficiency of BMP2 in the

group of DBB mixed with BMP2-cop.BioCaP was 10-fold higher than that in the group

of DBB with adsorbed BMP2 (Fig. 7B). The remaining percentage of BioCaP in the

group of DBB with BMP2-cop.BioCaP (8.4±5.5%) was significantly lower than that in

the group of DBB with BioCaP (24.9±6.1%) (Fig. 6B). The mixture with DBB did not

significantly influence the remaining percentage of BioCaP regardless of the

coprecipitation of BMP2 (Fig. 6). The volume ratio of FBGCs to BMP2-cop.BioCaP

[0.013±0.018 (mm3/mm

3)] was also significantly lower than that to BioCaP [0.155±0.019

(mm3/mm

3)] at the presence of DBB (Fig. 6C). The volume ratio of FBGCs to the DBB

mixed with BMP2-cop.BioCaP [0.009±0.005 (mm3/mm

3)] was significantly lower than

that to the DBB either alone [0.039±0.012 (mm3/mm

3)] or with adsorbed BMP2

[0.038±0.006 (mm3/mm

3)] or with BioCaP [0.043±0.004 (mm

3/mm

3)] (Fig. 7C).


75

Figure 5. Light micrographs of the cross-sections through DBB alone (A), DBB with adsorbed

BMP2 (B), DBB with BioCaP (C&C1) and DBB with BMP2-cop.BioCaP (D&D1) after a 5-week

implantation in subcutaneous site in rats. The sections were stained with McNeal’s Tetrachrome,

basic Fuchsine and Toluidine Blue O. Asterisks indicates the newly formed bone. Bars=200m in A,

B, C, and D. Bars=30m in C1 and D1.

Chapter 5

76

Figure 6. Graph depicting the volume density of new bone (A), percentage of remaining BioCaP

(B) and volume ratio of foreign-body giant cells (FBGCs) to BioCaP that were associated with

BioCaP within the subcapsular space (reference volume) for the four groups, 5 weeks after

subcutaneous implantation in rats. Mean values (n=6 animals per group) are represented together

with the standard deviation. *: p< 0.05; **: p< 0.01; ***: p< 0.001.

Figure 7. Graph depicting the volume density of new bone (A), osteoinductive efficiency of BMP2

(B) and volume ratio of foreign-body giant cells (FBGCs) to BioCaP that were associated with

BioCaP within the subcapsular space (reference volume) for the four groups, 5 weeks after

subcutaneous implantation in rats. Mean values (n=6 animals per group) are represented together

with the standard deviation. *: p< 0.05; **: p< 0.01; ***: p< 0.001.

DISCUSSION

In this study, we have for the first time developed 3-dimensional biomimetic CaP

(BioCaP) particles (100-1000µm) by modifying the principle for preparing the thin

(10-50µm), and substrate-dependent biomimetic CaP coatings. In this novel particle, the

advantage of the coatings in coprecipitating and slowly releasing proteinaceous cytokines

was maintained. We showed that this novel BMP2-cop.BioCaP, serving as an

independent “osteoinducer”, could induce bone formation efficiently and suppress the

host foreign-body reaction when it was mixed with DBB ─ a clinically-used

bone-defect-filling material. In addition, BMP2-cop.BioCaP also exhibited a proper

degradation rate in vivo.

In our previous studies, we have already shown that the BMP2-coprecipitated

biomimetic coating is very broadly applicable to bone-defect-filling materials and dental

implants. This was proven by the success in the preparation of this coating on a broad

range of biomaterials (e.g. metallic [4, 25], inorganic [15], polymeric materials [26]) that


77

have completely different geometries, topographies and surface chemistries [16]. Albeit

so, this type of biomimetic coating on bone-defect-filling materials has the limitation that

their growth relies still highly on the proper surface roughness and/or active surface

chemistry of the bone-defect-filling materials [16]. In this study, we modified the

biomimetic coating technique and developed this BMP2-cop.BioCaP with an aim of

completely breaking through these limitations. The BMP2-cop.BioCaP exhibited no

dependence on the physiochemical properties of bone-defect-filling materials and thus

can possibly be applied with any kind of granular bone-defect-filling materials used

clinically. Meanwhile, this BMP2-cop.BioCaP is also easily handled clinically, which

will significantly favor its clinical application.

The alternate assembling of the amorphous and crystalline layer was indispensible to

increase significantly the volume of BioCaP particles. This is because the amorphous CaP

layer is very thin (1.5-10 µm) and the crystalline CaP is hardly beyond 100 µm. By this

alternate layer-by-layer approach, we use the amorphous CaP layer as a connection and

seeding layer for the growth of another layer of crystalline CaP. The BioCaP grows in a

“bamboo-like” pattern with the amorphous CaP as the nodes and the crystalline CaP as

the internodes. After three cycles of alternate soaking in 5-fold simulated body fluid and

supersaturated calcium phosphate solution alternately (Fig. 1), the size of the BioCaP

significantly increased from the initial 5-20µm to 100µm-1mm (Fig. 2). The increase in

size was attributed both to the “bamboo-like” layer-by-layer growth of coatings and to

the aggregation of underlying particles by the growing coatings (Fig. 1). The current size

of BMP2-cop.BioCaP seemed correct for sustaining the osteoinductive effect of

coprecipitated BMP2, since a large amount of new bone was induced with high

efficiency (Fig. 7B).

Besides the size, the degradability of a CaP-based biomaterial is very important for

the in-vivo longevity and efficacy of its biological effects [27]. After 5 weeks, 60-82% of

BioCaP degraded (Fig. 6B), which indicated a significantly higher degradability of

BioCaP than most of the clinically-used, CaP-based bone-filling materials. Such a rapid

degradation is associated with its high dissolubility of BioCaP. This is because BioCaP

was prepared in biomimetic principle without the involvement of non-physiological

conditions (e.g. high temperature) and was composed of both amorphous CaP and

crystalline calcium-deficient hydroxiapatite with a low crystallity [26]. In contrast, most

of the clinically-used bone-defect-filling materials are sintered, which leads to the

significantly increased crystallinity and thus decreased dissolubility [28].

Apart from the spontaneous dissolution, the degradation of a material is also

accelerated by many types of cells (e.g. fibroblasts, monocytes/macrophages) through

phagocytotic mechanisms [29]. When their phagocytic capacity is exceeded,

macrophages can also fuse to form FBGCs. In contrast, these multinucleated FBGCs had

a significantly higher resorptive efficiency [30] and played a major role in the

degradation of BioCaP. Interestingly, although the volume ratio of FBGCs to

BMP2-cop.BioCaP was significantly decreased (Fig. 6C), the degradation rate of

BMP2-cop.BioCaP was significantly increased in comparison with BioCaP (Fig. 6B). In

fact, the suppression of FBGCs to CaP coatings in the presence of coprecipitated BMP2

could be found from 2-3 weeks [4]. These findings suggested that other resorption

mechanisms played key roles in the degradation of BMP2-cop.BioCaP. The activities of

osteoblasts and osteoclasts during the osteogenesis may account for this phenomenon.

Chapter 5

78

Besides, except phagocytic activity [29], osteoblasts-mediated mineralization can

generate many protons [31] that may promote the degradation of BMP2-cop.BioCaP.

Conventionally, these protons have to be neutralized by an extracellular buffering system

to prevent their accumulation [32]. CaP materials with a high dissolubility may directly

neutralize the protons, which promotes the activities of osteoblasts. The calcium and

phosphate ions generated in this way can greatly support the process of osteogenesis.

Consequently, a CaP material that bears the greater solubility shows the higher

osteoconductivity [33]. In this study, the osteogenesis was significantly promoted by the

coprecipitated BMP2, which also increased significantly the osteoblast-mediated

degradation and reuse of BioCaP. On the other hand, the mixture with DBB did not

significantly influence the degradation rate of either BioCaP or BMP2-cop.BioCaP (Fig.

6B), which indicated that the degradation property of BMP2-cop.BioCaP was not

influenced by the targeting bone-defect-filling materials.

The release kinetics is a crucial factor for the osteoinductive efficiency of BMP2. In a

clinical application, BMP2 is simply adsorbed superficially onto the bone-defect-filling

materials, which is associated with a high-dose burst release and thus low osteoinductive

efficiency [8]. In contrast, the coating-coprecipitated BMP2 showed a slow and sustained

release and thus a significantly higher osteoinductive efficiency than the adsorbed BMP2

[14, 15]. In line with this principle, DBB with BMP2-cop.BioCaP induced significantly

higher volume density of bone than the DBB with adsorbed BMP2 (Fig. 7A).

Accordingly, the osteoinductive efficiency of BMP2 in the group of DBB with

BMP2-cop.BioCaP was 10-fold higher than that in the group of DBB with adsorbed

BMP2 (Fig. 7B). These findings indicated that BMP2-cop.BioCaP could act as a

powerful “osteoinducer” to induce efficiently new bone formation for other granular

clinically-used bone-defect-filling materials. Although the newly formed bone originated

from the BMP2-cop.BioCaP, it did not stay unattached but integrated tightly onto the

DBB (Fig. 5D1) without the intervening of connective tissues. Thereby, DBB,

BMP2-cop.BioCaP and the new bone form an interconnected bony network (Fig. 5D). In

contrast, for the BioCaP without the coprecipitation of BMP2, BioCaP and DBB were

isolated by fibrous connective tissues (Fig. 5C1) and no bone tissue was detected (Fig.

5C).

One concern associated with the use of DBB is its biocompatibility. Although DBB

can integrate with bone in a pro-osteogenic environment such as in non-critical-sized

bone defects and/or in the presence of a sufficiency of autologous bone chips [34], it can

provoke significant foreign-body reactions in a pro-fibrotic environment such as at a

subcutaneous site [35] or in critical-sized bony defects [1]. Foreign-body reactivity is

histologically characterized by the local accumulation of macrophages, their fusion to

form FBGCs, and the deposition of dense fibrous connective tissue [30]. FBGCs begin to

appear between the 2nd and the 10th day after implantation [36]. They often persist for

the whole lifetime of the implant [37] and their presence is known to be associated with

the failure of biomaterials [30]. The foreign-body reaction may significantly hinder the

regeneration of bone and the osseointegration of DBB. In this study, we found that the

volume ratio of FBGCs to DBB was significantly lower in the group of DBB with

BMP2-cop.BioCaP than that in the group of either DBB alone, or DBB with adsorbed

BMP2, or DBB with BioCaP (Fig. 7C). This finding indicated that BMP2-cop.BioCaP

could not only induce bone formation efficiently but also significantly suppress the host


79

foreign-body reaction to DBB. Such suppression was most probably attributed to the

extensive osteogenesis [15]. The suppression of osteogenic activity on the formation of

FBGCs may be partially mediated by the elevated levels of osteopontin, that is enriched

during bone regeneration. Osteopontin was previously shown to suppress the fusion of

macrophages into FBGCs both in vitro and in vivo [38].

The volume ratio of FBGCs to DBB in the group of DBB with adsorbed BMP2 is

similar with that in the group of DBB alone. This finding indicated that the transient high

local concentration of BMP2 that was generated by its burst release did not influence the

formation and accumulation of FBGCs at the 5-week juncture. Since bone-formation

activity cannot be sustained when BMP2 was liberated in a single high-dose burst, the

volume density of osseous tissue that was laid down was low (Fig. 7A) and insufficient to

hinder the formation of FBGCs (Fig. 7C).

CONCLUSION

In this study, we developed a novel BMP2-cop.BioCaP as an independent slow delivery

system for BMP2. BMP2-cop.BioCaP can serve as “osteoinducer” to induce bone

formation efficiently and to suppress the foreign-body reaction to a clinically-used

bone-defect-filling material. In addition, this material also exhibited proper degradability.

All these properties confer this BMP2-cop.BioCaP a very promising potential for the

application clinically for the repair of large-size bone defects.

AUTHOR DISCLOSURE STATEMENT

All authors have no conflicts of interest. We sincerely thank Prof. Dr. Tony Hearn for

editing the English.

Chapter 5

80

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83

Chapter 6

A biomimetic osteoinducer enhances the therapeutic

effects of deproteinized bovine bone in a sheep

critical-sized bone defect (Ø8×13mm) model

Tie Liu, Gang Wu, Yuanna Zheng, Daniel Wismeijer, and Yuelian Liu.

Submitted, 2013

Chapter 6

84

ABSTRACT

Purpose: Most materials used clinically for filling bone defects [e.g. deproteinized

bovine bone (DBB)] lack osteoinductivity so that their therapeutic effects are far from

satisfactory. We have recently developed a novel biomimetic “osteoinducer” to provide

an alternative viable approach. We hypothesize that this osteoinducer could enhance the

therapeutic effect of DBB significantly in the repair of critical-sized bone defects

(CSBD).

Materials and Methods: The osteoinducer as an alternative of autograft was synthesised

by assembling a triple layer of amorphous and crystalline calcium phosphate into which

BMP2 was co-precipitated (BMP2-cop.BioCaP). DBB mixed with BMP2-cop.BioCaP

was tested. These samples and proper positive (autologous bone and DBB mixed with

autologous bone) and negative (DBB alone and DBB mixed with BioCaP without BMP2)

controls were implanted in the critical-sized bone defects in sheep for 4 and 8 weeks. We

assessed the degradability, foreign body reaction and osteoinductivity of the materials to

evaluate the efficacy of BMP2-cop.BioCaP of changing the therapeutic effects of DBB.

Results: The volume of newly formed bone associated with the test group

(BMP2-cop.BioCaP/DBB) was significantly higher than the negative controls and the

positive control which is DBB mixed with autologous bone after 4 and 8 weeks; The

newly formed bone of the test group was comparable with the autologous bone group

after 8 weeks. About 95% BMP2-cop.BioCaP had been degraded and replaced by newly

formed bone after 8 weeks. A significantly lower foreign-body reaction was found with

BMP2-cop.BioCaP/DBB than the other groups.

Conclusions: BMP2-cop.BioCaP significantly induced bone formation and thus

enhanced the therapeutic effect of DBB. DBB mixed with this osteoinducer may reduce

the use of autograft in the repair of critical-sized bone defects.

Key words: Osteoinducer, Deproteinized bovine bone, Biomimetic calcium phosphate,

BMP, Osteoinductive, Critical-sized bone defect, Bone repair

INTRODUCTION

The essence for treating bone fractures and defects is to achieve an adequate volume of

bone tissue. When the bone defects are too large to heal by themselves, bone grafting is

needed to fill the defect [1, 2]. An autograft is still regarded as the gold standard in

treatment since it provides an osteoconductive 3-dimensional scaffold for bone growth,

osteogenic cells and osteoinductive growth factors [3]. However, autogenous bone is

often associated with limitations such as the need for additional surgical intervention,

pain in the donor site, morbidity and a high and unpredictable resorption [4, 5]. These

limitations have led to a continual search for alternatives [6, 7]. A better understanding of

the biology of the healing of bones and technological development have resulted in the

development of numerous alternative materials for filling bone defects, such as allografts,

xenografts, and synthetic materials. Most of these materials that are used clinically are

highly osteoconductive. This enhances the migration of osteogenic cells. However, most

of them lack intrinsic osteoinductivity so that their therapeutic effects on large bone

defects are far from satisfactory [8].

A highly-efficient osteoinducer for the repair of bone defects

85

One of the materials is deproteinized bovine bone (DBB) and it is widely used

clinically. It is a bovine xenograft [9]. The use of DBB has the potential of reducing

morbidity since taking an autograft is unnecessary. Because DBB shows a physical

chemical structure similar to that of natural bone [10], it has excellent osteoconductive

properties for serving as a scaffold for bone formation [11]. Previous studies have

demonstrated its efficiency in the repair of critical-sized bone defect (CSBD) and the

augmentation of the maxillary sinus compared with other materials [12, 13]. However, in

some cases DBB delays early bone formation which probably results from the lack of

osteoinductivity [14, 15]. On the other hand, both surgeons and patients would like to see

a shortening of the recovery phase. The use of DBB in combination with a particulate

autograft has been suggested for inducing an adequate volume of bone tissue for an

excellent restoration. This can provide osteogenic elements [11, 16], but the limitations

mentioned above follow.

The application of osteogenic growth factors such as bone morphogenetic protein 2

(BMP2) by superficially adsorbing them onto DBB did not promote new bone formation

in CSBD [17, 18], since such a delivery mode only gives a short term burst release of

BMP2. Consequently, DBB had been considered as an unfeasible carrier for BMP [19].

We have shown previously that the biomimetic calcium phosphate (CaP) coating with

incorporated BMP2 can functionalize DBB and render the material efficiently

osteoinductive in an ectopic rat mode [20]. The sustained release of BMP2 from the

carrier coating enhances osteoinductivity [21]. However, the application of such a

coating is also limited since the whole procedure needs several days before the operation

[20].

To provide a viable alternative, we recently developed a novel “osteoinducer” by

biomimetically assembling calcium phosphate layer by layer and co-precipitating BMP2

into it (BMP2-cop.BioCaP). In the previous study, we have shown that this novel

osteoinducer can be directly mixed with DBB and it enhances the bone formation highly

efficiently in a rat ectopic model (subcutaneously) [22]. Whether this osteoinducer can

work in an osseous environment is still unknown. In this study, we hypothesized that the

osteoinducer could enhance the therapeutic effect of DBB significantly in a sheep

critical-sized bone defect model, the bone defect size is 8 mm in diameter and 13 mm

depth (Ø8×13mm). DBB mixed with BMP2-cop.BioCaP was tested. These samples and

proper controls were implanted in the critical-sized bone defects in sheep for 4 and 8

weeks. The degradability, foreign body reaction and osteoinductivity of the materials

were evaluated.


In-vitro preparation and characterization

Preparation of layer-by-layer assembled biomimetic calcium phosphate (BioCaP)

particles with or without incorporated BMP2

The layer-by-layer assembled BioCaP particles were produced according to our recent

publication [22]. Briefly, micro-particles of amorphous CaP were formed and deposited

by incubating in a beaker containing 2000ml of a five-fold concentrated simulated body

fluid (684mM NaCl; 12.5mM CaCl2·2H2O; 21mM NaHCO3; 5mM Na2HPO4·2H2O and

7.5mM MgCl2·2H2O) for 24 hours at 37°C. These particles served as the cores for the

Chapter 6

86

subsequent layer-by-layer assembly. They were immersed in 1000ml of a supersaturated

calcium phosphate solution [40mM HCl; 2mM Na2HPO4·2H2O; 4mM CaCl2·2H2O;

50mM TRIS base (pH 7.4)] for 48 hours at 37°C. Thereby, the first layer of crystalline

CaP coating deposited/grew on the amorphous CaP micro-particles. Thereafter, as

mentioned above, these particles were immersed in the five-fold concentrated simulated

body fluid for 24 hours and subsequently the supersaturated calcium phosphate solution

for 48 hours for the second layer of coating. Consequently, the size of BioCaP particles

was enlarged by assembling layer-by-layer.

In this study, the BioCaP particle was assembled in three cycles. During the

preparation of the outer layer, BMP2 (INFUSE® Bone Graft, Medtronic, USA) was

introduced into this supersaturated calcium phosphate solution at a final concentration of

2μg/ml and co-precipitated into the outermost crystalline CaP layer (BMP2-cop.BioCaP).

The BMP2-cop.BioCaP particles were then freeze-dried and retrieved with the size of

0.25-1mm. The entire procedure was conducted under sterile conditions.

Quantification of the amount of the incorporated BMP2

The amount of incorporated BMP2 was determined by a commercially available


BMP2-cop.BioCaP (n=6) was dissolved in 1ml 0.5M EDTA (pH 8.0). The ELISA assay

was performed according to the manufacturer's instructions.


Experimental groups

We used an experimental animal model in sheep with drill holes with 8mm in diameter

and 13mm in depth within the proximal and distal humerus and femur. Deproteinized

bovine bone granules (DBB, size: 0.25–1mm, Bio-Oss®, Geistlich, Switzerland) was

used in this study. One experimental and four control groups were established (n=6 sheep

per group, Table 1):

(1) Autologous bone particles (positive control, taken from the same sheep);

(2) DBB granules mixed with autologous bone particles (positive control);

(3) DBB granules mixed with BMP2-cop.BioCaP particles (experimental group);

(4) DBB granules mixed with BioCaP particles (negative control for the effects of

BMP2);

(5) DBB granules alone (negative control for the effects of BioCaP and of BMP2).

In Group 2, a 1 : 1 ratio of DBB and autologous bone particles was determined

according to previous studies [11, 23-25]. Autologous bone was harvested from the

cylindrical bone defects during the surgery and ground to 0.25-1mm particles under

sterile conditions. These bone chips were reserved for Group 1 and 2. In Group 3 and 4,

0.59cm3 of DBB (size: 0.25-1.0mm) and 0.07cm

3 of BMP2-cop.BioCaP or BioCaP (size:

0.25-1.0mm) per sample were placed into 1-ml Eppendorf tubes and homogeneously

mixed by manually shaking. The amount of BMP2-cop.BioCaP was determined

according to our previous study [22].


87

Table 1 Experimental groups

Groups Abbreviation

Graft materials

Total

Loading

Volume of graft

material per sample

Dose of

BMP2 (per

sample)

(1) Autologous bone AB 0.66cm3 -

(2) DBB granules mixed with

autologous bone AB DBB

AB 0.33cm3

DBB 0.33cm3 -

(3)

DBB granules mixed with

BMP2-cop.BioCaP particles

(experimental)

DBB BioCaP

BMP

BMP2-cop.BioCaP

0.07cm3;

DBB 0.59cm3

10.3μg

(4) DBB granules mixed with

BioCaP particles DBB BioCaP

BioCaP 0.07cm3;

DBB 0.59cm3 -

(5) DBB granules DBB 0.66cm3 -

Experimental animal model

A total of 12 adult (2- to 4-year-old) female Australia sheep (40-50 kg in weight) were

used in the present study, which was approved by Ethical Committee of School of

Stomatology in Zhejiang University. All the animal experiments were carried out

according to the ethics laws and regulations of China. Throughout the study, the sheep

were treated following the guidelines of animal care established by Zhejiang University.

The sheep were subjected to anaesthesia by administering Sumianxin II (0.3 ml/kg,

purchased from the Military Veterinary Institute, Quartermaster University of PLA,

Chang Chun, China) with the addition of Penicillium (5 × 104 U/kg) and atropine (0.03

mg/kg) at 30 min before surgery. A local anaesthesia (1% lidocaine with 1:100,000

adrenaline) and skin disinfection (0.5% iodophor solution) were applied to the

implantation sites. The implantation sites were the proximal part of the diaphysis and

distal epiphysis of humerus and femur of sheep.[26] Each sheep can provide 8 totally

standardized implantation sites. 5 implantation sites were randomly chosen, and these

sites were assigned to the five groups (n=6 sheep per group) according to a

randomization protocol [27]. The surgery procedures are shown in Fig. 1. The surgery

and animal care were performed and the cylinder-shaped defects (8mm in diameter and

13mm in depth) were created as described in a previous study [26]. Membranes

(Bio-Gide®, Geistlich, Switzerland) were used to cover the defects. Samples with

surrounding tissues were retrieved at 4 weeks and 8 weeks post-operation.

Chapter 6

88

Figure 1. Surgical procedures: perforation of the cylinder-shaped defects (8mm in diameter and

13mm in depth) (A), and the cylinder bone was shown in the pane; filling materials (B); cover the

defects with membranes (C); suture of inner soft tissue (D), and suture of outside soft tissue (E).


Samples were chemically fixed and embedded into a block as previously reported.[20, 28]

Applying a systematic random sampling strategy,[29] the samples were sawn vertically to

the long axis, into 10 slices of 600-μm thickness, 1 mm apart (interval). All the slices of

each sample were mounted separately on plexiglass holders and polished. The slices were

surface stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue [20] and

examined with a light microscope (Leica).


In addition to a subjective histological description, 10 slices of each sample was used for

quantitative histomorphometric analysis. The volume of newly formed bone, BioCaP and

DBB and the volume density of multinucleated giant cells (MGC) on DBB were

measured using the point-counting technique.[22, 30]

The volume density of MGC on DBB was normalized to the volume of DBB. The

volume density of MGC (D) is defined as its volume (Va) per unit volume of DBB (Vb):

D = Va / Vb.

To evaluate the degradation of BioCaP, the volume of BioCaP before implantation

(Vtime0) and after 5 weeks of implantation (V5weeks) was evaluated by using the same

histological method. Six samples containing BioCaP (0.07cm3) were reserved for ‘time

0’. Therefore, the percentage of non-degraded BioCaP (P) is defined as: P= V5weeks /

Vtime0 × 100%.


All data are presented as mean values with the standard deviation (SD). Data were

compared using a one way analysis of variance (ANOVA), and post hoc comparisons

were made using Bonferroni’s corrections. The significance level was set at p< .05.

RESULTS

In-vitro characterization:

After three cycles of alternate immersion, the particle size increased up to 100-1000μm

with a crystalline outer layer. According to our previous results [22], BMP2 has been

successfully incorporated in the outermost coating layer of BioCaP, and an efficient

ectopic bone formation was induced by about 10 μg of BMP2 in BMP2-cop.BioCaP. In


89

the current study, each sample with BMP2-cop.BioCaP contains 10.3±1.9μg BMP2 with

an incorporation rate of 30.1±5.7% according to the ELISA result.


At the end of the two-implantation periods (6 sheep per time point), a total of 60 implants

were harvested (30 implants at 4 weeks and 30 implants at 8 weeks). All the sheep

exhibited good health and all the surgical implant sites were healed well without any

complications. No visual signs of inflammation or adverse tissue reaction were observed.

Histological results

Representative histological images of each group are shown in Fig. 2. In the positive

controls, plenty of autografts were found at 4 weeks (Fig. 2 A, B). After 8 weeks, most

autografts had been replaced by newly formed bone (Fig. 2 A1, B1).

In the samples containing DBB (Fig. 2 C-E, C1-E1), the whole bone defect was filled

uniformly with DBB granules. The newly formed bone was always in close contact with

the DBB surface and more mature bone presented at 8 weeks than at 4 weeks. At 4 weeks

of implantation, in the samples containing BMP2-cop.BioCaP or BioCaP, newly formed

bone with a woven appearance was observed between DBB granules or deposited on

DBB (Fig. 3A and B), while in the samples with only DBB, most DBB did not have

bone deposition (Fig. 3C). At 8 weeks, an interconnected bone DBB network was

observed (Fig. 3D-F). DBB granules were always encapsulated in bone. More bone

growth was observed throughout the space between DBB granules in the samples

containing BMP2-cop.BioCaP (Fig. 3D) compared with those containing BioCaP (Fig.

3E) and DBB only (Fig. 3F).

At a higher magnification, BMP2- cop.BioCaP and BioCaP particles were observed

constantly in close contact with new bone or completely encapsulated in the new bone at

both time points (Fig. 4). BMP2-cop.BioCaP and BioCaP particle consisted of many

calcium phosphate microspheres. Representative images at high magnification of

BMP2-cop.BioCaP are shown in Fig. 5A and B. Mononuclear cells were observed in

close contact with BMP2-cop.BioCaP particle or in the interior of the particle between

the small calcium-phosphate spheres at both time points (Fig. 5A, B). Moreover,

multinucleated giant cells (MGCs) were occasionally observed on the surface of

BMP2-cop.BioCaP particles at 4 weeks (Fig. 5A), whereas no MGC was found in contact

with BMP2-cop.BioCaP at 8 weeks (Fig. 5B). At 4 weeks, MGCs were observed on the

surface of DBB granules (Fig. 5C), while MGCs were sporadically found on DBB

granules at 8 weeks (Fig. 5D). A light zone of DBB was often observed beneath these

MGCs (Fig. 5C and D). The light zone in Fig. 5D seemed to be a sign of resorption.

Chapter 6

90

Figure 2. Representative histological micrographs of the whole bone defect (in the white circle) of

each group at 4 weeks (A-E) and 8 weeks (A1-E1) after material placement. Group 1 (A, A1);

Group 2 (B, B1); Group 3 (C, C1); Group 4 (D, D1); and Group 5 (E, E1) (see Table 1 for an

explanation of the groups). Autograft (#); Newly formed bone (arrow); DBB granules (asterisk).

The slices were surface-stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue.

Figure 3. Representative histological sections of DBB granules and newly formed bone (arrow) in

Group 3, 4 and 5 after 4 and 8 weeks of implantation at higher magnification (see Table 1 for an

explanation of the groups). After 4 weeks of implantation, newly formed bone was observed in

close contact with DBB granules in Group 3 (A) and Group 4 (B), but not in Group 5 (C). After 8

weeks of implantation, more bone growth was observed throughout the space between DBB

granules in Group 3 (D) compared to Group 4 (E) and Group 5 (F). The slices were surface-stained

with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue. Bar=500µm.


91

Figure 4. Representative histological sections of BioCaP (asterisk), DBB, and bone (arrow) in

Group 3 and 4 after 4 and 8 weeks of implantation at higher magnification (see Table 1 for an

explanation of the groups). Group 3 at 4 weeks (A); Group 4 at 4 weeks (B); Group 3 at 8 weeks

(C); Group 4 at 8 weeks (D). Most BMP2-cop.BioCaP particles and BioCaP particles without

BMP2 were in close contact in newly formed bone at both time points. The slices were

surface-stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue. Bar=100µm.

Chapter 6

92

Figure 5. Mononuclear cells (yellow arrow) appeared in the interior of BMP2-cop.BioCaP particles

at 4 weeks (A) and 8 weeks (B). Osteocytes (white arrow). Multinucleated giant cells (MGC; arrow

head) were occasionally observed on BMP2-cop.BioCaP at 4 weeks (A), while no MGCs were

found on BMP2-cop.BioCaP at 8 weeks. MGCs were observed on DBB granules at 4 weeks (C),

while MGCs were sporadically observed on DBB at 8 weeks (D). A light zone (black arrow)

commonly observed when MGCs could be observed adjacent to DBB. The slices were

surface-stained with McNeal's Tetrachrome, basic Fuchsine and Toluidine Blue. Bar=50µm.


For each treatment, the volume of new bone at 8 weeks was significantly higher (p<0.05)

than that at 4 weeks (Fig. 6). At 4 weeks, the volume of new bone in the group of BioCaP

(no BMP2)/DBB was significantly higher than with DBB alone. The volume of new bone

associated with BMP2-cop.BioCaP/DBB was comparable with an autograft at 8 weeks,

and it was significantly higher than autograft/DBB, BioCaP (no BMP2)/DBB, and DBB

alone at both 4 and 8 weeks.

The degradation of BMP2-cop.BioCaP and BioCaP increased with increasing

implantation time (p<0.05). BMP2-cop.BioCaP showed significantly more degradation

than BioCaP at both time points (Fig. 7A). After 8 weeks, about 95% of

BMP2-cop.BioCaP was degraded. The volume of DBB at 4 and 8 weeks after

implantation revealed that there was no significant difference between the two time

points in all the four groups (Fig. 7B).

The volume density of multinucleated giant cells (MGC) on the surface of DBB

revealed that bone formation decreased significantly with increasing time after


93

implantation (p<0.05). At 4 weeks after implantation, the volume density of MGC on

DBB was the lowest in samples containing BMP2-cop.BioCaP (Fig. 8), while at 8 weeks

there were no significant differences among the four groups with DBB.


weeks after implantation for each of the 5 groups (see Table 1 for an explanation of the

abbreviations). Mean values (n=6 samples per group) are represented together with the standard

deviation. *p<0.05.

Figure 7. Graph depicting the percentage of non-degraded BioCaP (A) and the volume of DBB (B)

within the bone defect at 4 and 8 weeks after implantation (see Table 1 for an explanation of the


deviation. *p<0.05.

Chapter 6

94


deproteinized bovine bone at 4 and 8 weeks after implantation (see Table 1 for an explanation of

the abbreviations). Mean values (n=6 samples per group) are represented together with the standard

deviation. *p<0.05.

DISCUSSION

The purpose of the present study is to test our hypothesis that our novel osteoinducer ─

BMP2-cop.BioCaP could significantly enhance the therapeutic effects of DBB on the

repair of critical-sized bone defects. BMP2-cop.BioCaP is a biodegradable and highly

efficient osteoinducer. It resulted in more bone formation than the single use of DBB and

it significantly suppressed foreign-body reaction not only in an orthotopic environment,

but also in an ectopic environment [22]. The volume of new bone associated with

BMP2-cop.BioCaP/DBB was significantly higher than autograft/DBB. These findings

indicate that BMP2-cop.BioCaP could significantly enhance the therapeutic effects of

DBB on critical-sized bone defects.

An ectopic model (subcutaneous) of ossification is useful for testing the principle of

an osteoinductive system [28]. However, the osseous environment is different from a

non-osseous environment. The critical-sized bone defect (CSBD) model in this study was

created by drilling holes in the humerus and femur of sheep according to the well

published protocol by Nuss et al [26]. This drill hole model in sheep has proved to be an

excellent animal model for testing biomaterials for use in orthopedics, maxillofacial and

dental surgery [31]. It allowed the intraosseous implantation of up to 8 different test

materials within one animal due to the standardization of the bone defect, while at the

same time it can reduce the overall suffering of animals and give the necessary numbers

to satisfy statistical requirements [26, 32]. Our previous study has confirmed that these

critical-sized bone defects cannot heal by themselves [33]. In addition, the results from

sheep are more convincing than those obtained with small laboratory animals because of

the similarities in the bone structure of humans [34].

In the current study, all BioCaP particles (with or without BMP2) were observed to be

in close contact with bone or entirely encapsulated in the bone, and mononuclear cells

(osteocyte-like cells) were observed to be in close contact with BioCaP. This finding


95

suggests that BioCaP is highly biocompatible, which has been proved in our previous

study, since each layer of BioCaP is produced using the biomimetic coating technique

[22]. During the preparation of BioCaP, the five-fold concentrated simulated body fluid

provided the CaP microspheres which are formed under the nucleation inhibitory

influence of Mg2+

and HCO32-

[35, 36]. These microspheres then serve as seed for the

subsequent growth of a crystalline latticework of octacalcium phosphate under conditions

that are conducive to nucleation [37]. Therefore, the physicochemical property of BioCaP

might be similar to the biomimetic coating. A number of studies showed that the coating

has excellent biocompatibility, biodegradability, osteoconductivity and the capability of

slow delivery of growth factors such as BMP2 and vascular endothelial growth factor

(VEGF) [20, 21, 28, 38].

The slow delivery of BMP2 plays a very important role in bone formation [20, 21]. It

has been shown that the protein incorporated in BioCaP resulted in a sustained release of

protein in vitro and BMP2 delivered by BioCaP led to a highly osteoinductive efficiency

in vivo in our previous study [22]. The results in the current study have confirmed the

high efficiency of BMP2-cop.BioCaP with 10.3ug of BMP2, which is significantly less

than the clinic applications [39]. Moreover, the results revealed that BioCaP without

BMP2 is also conducive to bone formation at 4 weeks after implantation. One possible

mechanism for this might be related to the degradation of BioCaP which provides

calcium for the process of osteogenesis. Moreover, mononuclear cells were observed in

the interior of BioCaP particle between the small CaP spheres. This suggests that BioCaP

has a porous structure. The porosity also contributes to the degradability of the material

[40].

Histomorphometric analysis revealed that BMP2-cop.BioCaP had high degradability

and resulted in a significant increase in bone formation. A proper degradability is an

essential property for biomaterials [41]. The degradation mechanism of

BMP2-cop.BioCaP is associated with its dissolubility (the spontaneous dissolution), and

the cell based resorption [21, 22, 28]. There are two possible mechanisms for the cell

based degradation of material and they are a phagocytosis mechanism and an acidic

mechanism [42]. The phagocytosis mechanism is associated with fibroblasts and

monocytes/macrophages and the acidic mechanism is related to multinucleated giant cells

(FBGCs, osteoclasts) and osteoblasts. FBGCs and osteoclasts can produce at the surfaces

of biomaterials an acidic microenvironment that exists between the cell membrane and

the surface of the biomaterial [43, 44]. The mineralization mediated by osteoblasts

(osteogenesis) can generate many protons [45] which have to be neutralized

conventionally by an extracellular buffering system to prevent their accumulation [46].

Therefore, these protons may promote the degradation of BMP2-cop.BioCaP, since the

osteogenesis was significantly promoted in this study by BMP2-cop.BioCaP.

Moreover, MGCs were shown to be in close contact with DBB in this study.

Histomorphometric analysis revealed that BMP2-cop.BioCaP significantly decreased the

formation of MGCs on DBB. This finding coincides with a previous study which showed

that BMP2-cop.BioCaP suppressed these cells in an ectopic environment [22]. We

assume that these MGCs could be regarded as foreign-body reaction in agreement with

our previous studies [20, 22]. In addition, a light zone of DBB appeared beneath the

MGCs. This finding is in line with a previous study [16], indicating a sign of resorption

of DBB. It should also be noted that MGCs play a critical role in the surface treatment

Chapter 6

96

and the degradation of material.[44]

In the present study, DBB served as an osteoconductive scaffold for bone formation

[11]. However, the low degradability of DBB is one of its drawbacks [10, 16]. Although

the presence of cell based demineralization of DBB was observed, the histomorphometric

analysis of the DBB volume demonstrated the low degradability of DBB. The ideal bone

regeneration requires that the material can be gradually replaced by new bone in a short

period of time [41]. Therefore, we propose that the combination of BMP2-cop.BioCaP

and a biodegradable material for filling a bone defect may result in a better regeneration

of the bone in a shorter period. Our on-going study is developing a biodegradable

biomimetic calcium phosphate bone substitute for filling bone defects.

CONCLUSION

It was shown that BMP2-cop.BioCaP can serve as a highly efficient osteoinducer for

inducing bone formation with DBB and for suppressing the foreign-body reaction in a

critical-sized bone defect. BMP2-cop.BioCaP also showed good degradability. This

novel material has a very promising clinical potential as an osteoinducer which can be a

substitute for an autograft and which can enhance significantly the therapeutic effects of

materials for filling bone defects.

ACKNOWLEDGMENT

Authors would like to thank Prof. Dr. Tony Hearn for editing the grammar.


97

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101

Chapter 7

Deproteinized bovine bone functionalized with the

slow delivery of BMP-2 for the repair of

critical-sized bone defects in sheep

Tie Liu, Gang Wu, Daniel Wismeijer, Zhiyuan Gu and Yuelian Liu.

Bone. 2013, 56: 110–118.

Chapter 7

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ABSTRACT

As an alternative to an autologous bone graft, deproteinized bovine bone (DBB) is widely

used in the clinical dentistry. Although DBB provides an osteoconductive scaffold, it is

not capable of enhancing bone regeneration because it is not osteoinductive. In order to

render DBB osteoinductive, bone morphogenetic protein 2 (BMP-2) has previously been

incorporated into a three dimensional reservoir (a biomimetic calcium phosphate coating)

on DBB, which effectively promoted the osteogenic response by the slow delivery of

BMP-2. The aim of this study was to investigate the therapeutic effectiveness of such

coating on the DBB granules in repairing a large cylindrical bone defect (8mm diameter,

13mm depth) in sheep. Eight groups were randomly assigned to the bone defects: (i) no

graft material; (ii) autologous bone; (iii) DBB only; (iv) DBB mixed with autologous

bone; (v) DBB bearing adsorbed BMP-2; (vi) DBB bearing a coating but no BMP-2; (vii)

DBB bearing a coating with adsorbed BMP-2; and (viii) DBB bearing a

coating-incorporated depot of BMP-2. 4 and 8 weeks after implantation, samples were

withdrawn for a histological and a histomorphometric analysis. Histological results

confirmed the excellent biocompatibility and osteoconductivity of all the grafts tested. At

4 weeks, DBB mixed with autologous bone or functionalized with coating-incorporated

BMP-2 showed more newly-formed bone than the other groups with DBB. At 8 weeks,

the volume of newly-formed bone around DBB that bore a coating-incorporated depot of

BMP-2 was greatest among the groups with DBB, and was comparable to the autologous

bone group. The use of autologous bone and BMP-2 resulted in more bone marrow

formation. Multinucleated giant cells were observed in the resorption process around

DBB, whereas histomorphometric analysis revealed no significant degradation of DBB.

In conclusion, it was shown that incorporating BMP-2 into the calcium phosphate coating

of DBB induced strong bone formation around DBB for repairing a bone defect.

Keywords: Deproteinized bovine bone, Biomimetic calcium phosphate coating, BMP-2,

Critical-sized bone defect, Bone repair, Drug delivery

INTRODUCTION

In recent years, a critical-sized bone defect (CSBD) is defined as an intraosseous wound

that will not spontaneously heal completely without intervention [1, 2]. Autograft is

regarded as the ‘gold standard’ because of its excellent combination of osteoconduction

and osteoinduction. However, it is always associated with irregular rates of resorption,

pain and morbidity of the donor site, and requires additional surgical procedures. These

limitations have already led to the pursuit of alternatives including allografts, xenografts

and synthetic alloplasts [3, 4]. Most of them are osteoconductive, while the lack of an

intrinsic property of osteoinductivity is always the main problem [5]. To solve this

problem, one of the strategies in bone tissue engineering has been the introduction of

bone growth factors into a suitable scaffold [2, 6].

The scaffold that acts as a template for cell interactions and provides a structural

support for the newly formed tissue is a key component for bone regeneration [7].

Deproteinized bovine bone (DBB), a bovine xenograft of which there is an unlimited

supply, is one of the most widely used scaffolds used in bone repair and augmentation in

Deproteinized bovine bone functionalized with the slow delivery of BMP-2

103

clinical dentistry. DBB is derived from a bovine source and is treated by a chemical

extraction process to remove all the organic components and pathogens [4]. In terms of

its inorganic composition and its isomeric crystalline dimensions, DBB has a physical

and chemical structure similar to that of natural bone [8]. It shows osteoconductive

properties when it is in close contact with the newly formed bone [9]. However, it was

reported that DBB delays the early bone formation [10] and lacks intrinsic

osteoinductivity [11]. This suggested using DBB in combination with autogenous bone

chips which can then provide osteogenic elements [9, 12]. But this still has the limitations

of autografts mentioned above. The addition of platelet-rich plasma which contains

various growth factors on DBB did not enhance early and late healing of the bone [13].

The local delivery of mesenchymal stem cells (MSC) by DBB offers the promising

potential of augmenting the healing of CSBD [14, 15], but it needs harvesting a cell from

a secondary site, which is then expanded in vitro and seeded onto DBB directly prior to

implantation. Another simple option is to use osteogenic agents such as bone

morphogenetic protein 2 (BMP-2) adsorbed on DBB [16]. However, due to the high burst

release of BMP-2, the adsorption mode has not promoted de novo bone formation [17].

To be effective, this mode usually needs very high doses (in the milligram range) [18, 19],

and is neither efficient nor cost effective [20, 21]. Therefore, DBB was once considered

not to be a feasible carrier for BMPs [22].

It is well known that a slow delivery of BMP-2 plays a crucial role in bone formation

[23, 24]. For the slow delivery of BMP-2, technique of a biomimetic calcium phosphate

(CaP) coating has been developed and applied on different materials such as titanium

implants [25], polymers [26] and DBB [27]. This biomimetic coating deposited on the

surface of carrier materials can serve as a three-dimensional reservoir for growth factors.

BMP-2 incorporated into the crystalline latticework of this biomimetic coating during its

growth (deposition) can retain its biological activity [28]. In the previous studies, the

coating incorporated with BMP-2 has been applied on dental titanium implants to

improve osteoconductivity and osteoinductivty, especially for the early bone formation

(1-3 weeks) in an orthotopic site [29]. Recently, we found that the functionalization of

DBB granules with the BMP-2-incorporated biomimetic coating induced efficient bone

formation at an ectopic (subcutaneous) site in rats [27]. Moreover, the

BMP-2-incorporated biomimetic coating can suppress foreign body reaction which may

significantly hinder the regeneration of bone and the osseointegration of DBB. However,

Whether this BMP-2-incorporated coating on DBB can facilitate bone formation for the

repair of CSBD is not clear.

The aim of the current investigation was to study the therapeutic effectiveness of

DBB functionalized with coating-incorporated BMP-2 for the repair of critical-sized

bone defect. To this end, autologous bone was used as a positive control; DBB granules

that bore either a directly or a coating-adsorbed depot of BMP-2, or a

coating-incorporated depot of this agent, were filled into the bone defects as testing

groups in the adult sheep. There were 8 experimental groups in total. The volumes of

newly formed bone within the bone defect 4 and 8 weeks after surgery were estimated

histomorphometrically.


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Biomimetic calcium phosphate coating procedure

The DBB granules (size: 0.25–1mm, Bio-Oss®, Geistlich, Switzerland) were

biomimetically coated, according to a well-established protocol [25, 28, 30, 31], with a

layer of crystalline calcium phosphate in the absence or presence of BMP-2 [27]. Briefly,

0.35g of the DBB granules were immersed in 300ml of five-fold-concentrated simulated

body fluid (684 mM NaCl; 12.5 mM CaCl2·2H2O; 21 mM NaHCO3; 5 mM

Na2HPO4·2H2O; 7.5 mM MgCl2·2H2O) for 24 h at 37 °C and afterwards in 130 ml of a

supersaturated solution of calcium phosphate [40 mM HCl; 2 mM Na2HPO4·2H2O; 4

mM CaCl2·2H2O; 50 mM TRIS base (pH 7.4)] for 48 h at 37 °C described as the

precious study [27]. The entire procedure was conducted under sterile conditions.

Incorporation of BMP-2 into the calcium phosphate coating

BMP-2 (INFUSE® Bone Graft, Medtronic, USA) was present in the supersaturated

solution of calcium phosphate at a final concentration of 1μg/ml, and was subsequently

coprecipitated into the biomimetic calcium phosphate coating of the DBB granules. The

samples were then freeze-dried. The entire procedure was conducted under sterile

conditions. The quantification of the amount of BMP-2 encapsulated in the coating was

determined using an enzyme linked immunosorbent assay (ELISA) kit (PeproTech EC,

London, UK), as described the previous studies [26, 31].

Adsorption of BMP-2 onto DBB granules with or without the coating

According to the results of ELISA, 35.0 ± 0.62 μg (mean ± SD) of BMP-2 were

incorporated into the coating of each sample. Hence, 35.0 μg of BMP-2 was likewise

adsorbed onto each 0.35 g sample of DBB granules with or without the coating as

described in the previous study [27]. Briefly, the loading process was achieved by

introducing a 200-μl aliquot of a stock solution with 175 μg/ml of BMP-2 into 1-ml

Eppendorf tubes containing 0.35g of DBB granules. Finally the DBB granules were

homogeneously mixed and wetted. Afterwards, the samples were freeze dried for 24 h.

The entire procedure was conducted under sterile conditions

Surface characterization of the coating on DBB

The surface characteristic of the calcium phosphate coating with or without incorporated

BMP-2 on DBB was evaluated with a scanning electron microscope (SEM, XL 30,

Philips, The Netherlands). For this purpose, samples of the material were mounted on

aluminum stubs and sputtered with gold particles to a thickness of 10–15 nm.

Confirmation of the homogeneous distribution of a coating-incorporated depot of

proteins

To confirm the homogeneous distribution of the protein in the crystalline latticework of

the coating on DBB granules, BMP-2 was substituted by the model protein bovine serum

albumin labeled with fluorescein-isothiocyanate (FITC-BSA, Sigma, St. Louis, MO,

USA). FITC-BSA (green signal) was introduced into the supersaturated calcium

phosphate solution at a final concentration of 1 μg/ml. The coated samples were

embedded in methylmethacrylate, sectioned, and ground [27]. A series of 50-µm-thick

sections were prepared for analysis by fluorescence microscopy. Micrographs were taken

with a digital camera (Leica, Wetzlar, Germany) mounted on an inverted light


105

microscope (Leica) equipped with a fluorescence lamp.

Experimental animal model

A total of 12 adult (2- to 4-year-old) female Australia sheep (40-50 kg in weight) were

used in the present study, which was approved by Ethical Committee of School of

Stomatology, Zhejiang University. All animal experiments were carried out according to

the ethic laws and regulations of China. Throughout the study, the sheep were treated

following the guidelines of animal care established by Zhejiang University.

The sheep were anesthetized by administering Sumianxin II (0.3 ml/kg, purchased

from the Military Veterinary Institute, Quartermaster University of PLA, Chang Chun,

China) with the addition of Penicillium (5 × 104 U/kg) and atropine (0.03 mg/kg) 30 min

before surgery. After applying local anesthesia (1% lidocaine with 1:100,000 adrenaline)

and skin disinfection (0.5% iodophor solution) to the implantation sites, the surgery and

animal care was performed and the cylinder shaped defects were created (8mm in

diameter and 13mm in depth) as described in a previous study [32]. The implantation

sites were the proximal part of the diaphysis and distal epiphysis of humerus and femur

of 12 adult female sheep. Eight implantation sites per animal were assigned to the eight

groups according to a randomization protocol [33]. Membranes (Bio-Gide®, Geistlich

Biomaterials, Wolhuser, Switzerland) were used to cover the defects after filling

materials. The sheep were sacrificed at 4 weeks and 8 weeks post-operation, and samples

with surrounding tissues were retrieved.

Experimental groups

Eight groups were established to treat CSBD (n=6 animals per group per time point,

Table 1):

(i) No graft material;

(ii) Autologous bone;

(iii) Deproteinized bovine bone (DBB, Bio-Oss®) bearing neither a calcium phosphate

coating nor a depot of BMP-2;

(iv) DBB bearing neither a calcium phosphate coating nor a depot of BMP-2, but

mixed with autologous bone (1:1);

(v) DBB bearing no coating but a superficially adsorbed depot of BMP-2;

(vi) DBB bearing a calcium phosphate coating but no BMP-2;

(vii) DBB bearing a calcium phosphate coating upon which BMP-2 was superficially

adsorbed; and

(viii) DBB bearing a calcium phosphate coating into which BMP-2 was incorporated.

Autogenous bone was harvested from the cylindrical bone defects and ground to

chips under sterile conditions. These bone chips were reserved for groups (ii) and (iv).


Samples with surrounding tissues were fixed chemically and embedded into a block as

previously reported [27, 31]. Applying a systematic random sampling strategy [34], the

samples were sawn vertically to the long axis into 10-12 slices of 600-μm thickness, 1

mm apart (interval). All slices of each sample were separately mounted on plexiglass

holders and polished. The surfaces of the slices were stained with McNeal's

Chapter 7

106

Tetrachrome, basic Fuchsine and Toluidine Blue [27] and examined with a light

microscope (Leica).

Table 1

Experimental groups

Groups Abbreviation

DBB

Coating

BMP-2

Total

Loading

Amount of

DBB (per sample)

Absence (−)

Presence

(+)

Absence (−)

Presence

(+)

Dose of BMP-2

(per

sample)

(i) No graft material (negative control)

NGM _ _ _ _

(ii) Autologous bone

(positive control) AB _ _ _ _

(iii)

Deproteinized bovine bone

(Bio-Oss®) bearing

neither a coating nor a depot of

BMP-2

DBB 0.35g

(0.65cm3) _ _ _

(iv) DBB mixed with autologous bone

(1:1)

DBB+AB 0.175g (0.325

cm3)

_ _ _

(v)

DBB bearing no coating but a

superficially

adsorbed depot of

BMP-2

DBB+BMP ads. 0.35g

(0.65 cm3) _ + 35µg

(vi) DBB bearing a

coating but no BMP-2

DBB+CaP 0.35g

(0.65 cm3) + _ _

(vii)

DBB bearing a

coating upon which BMP-2 was

superficially

adsorbed

DBB+CaP+BMP

ads.

0.35g

(0.65 cm3) + + 35µg

(viii)

DBB bearing a coating into which

BMP-2 was incorporated

DBB+BMP inc. 0.35g

(0.65 cm3) + + 35µg


In addition to a subjective histological description, 10 slices of each sample were used for

quantitative histomorphometric analysis. The volume of newly formed bone, bone

marrow, DBB and the volume density of multinucleated giant cells (MGC) on DBB were

measured using the point counting methodology [35]. During analysis, the evaluator was

always blinded for the groups.


107

The volume density of MGC was normalized to the volume of DBB. The volume

density of MGC (Va) is defined as its volume (Vb) per unit volume of DBB (Vc): Va=

Vb/Vc. To evaluate the degradation of DBB granules, the volume of DBB before

implantation (time 0, as control) was evaluated by the same histological method. Six

samples (0.35g of DBB granules per sample) which had been chemically fixed and

embedded in plastic were specifically reserved for this purpose.


All data are presented as mean values together with the standard deviation (SD). Data

were compared using a one way analysis of variance (ANOVA), and post-hoc

comparisons were made using Tukey's corrections. The significance level was set at p

< .05.

RESULTS

In-vitro investigation

In the scanning electron microscope, the biomimetic coating of calcium phosphate with

incorporated BMP-2 on DBB displayed a uniform crystalline surface (Fig .1A). The

incorporation of BMP-2 did not affect the coating morphology. Fluorescence microscopy

revealed that protein (green signal) was homogeneously distributed in the crystalline

latticework of the coating (Fig. 1B). The thickness of the coating was 21.2±13.8μm.

When the coating has no fluorescently tagged protein, it was not visible in the

fluorescence micrograph (Fig. 1C). There was 35µg of BMP-2 incorporated and/or added

per sample of 0.35 g of DBB (Table 1).

Figure 1. SEM micrographs of the crystalline calcium phosphate coating with

incorporated BMP-2 on deproteinized bovine bone (A). Fluorescence micrographs

illustrating the even distribution of a depot of protein (green signal) in the coating (B).

The coating without fluorescently tagged protein was not visible in the fluorescence

micrograph (C).


All 12 sheep exhibited good health and all the surgical implant sites healed well without

any complications in the wound. At the end of the two implantation periods, a total of 96

implants were harvested (48 implants at 4 weeks and 48 implants at 8 weeks). No visual

sign of inflammation or adverse tissue reaction was observed.

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Descriptive light microscopy

Representative histological images of each group at a low magnification are depicted in

Fig. 2. In the negative control group (i) without graft material, newly formed bone was

found sporadically close to the defect border at 4 and 8 weeks. There was no new bone

formation in the center part of bone defect. In group (ii) containing autologous bone,

plenty of autologous bone chips were observed at 4 weeks, while at 8 weeks most

autologous bone chips had been replaced by newly formed bone which had encapsulated

the residual autologous bone chips. Bone formation was irregular in most sections due to

the irregular resorption rate of autologous bone. Large regions filled with bone marrow or

fibrous tissues were usually observed in the bone defect.

In group (iii) which contained only DBB, the bone defect was mainly filled with four

components: newly formed bone, bone marrow, fibrous tissues, and DBB granules. In

general, DBB granules were distributed uniformly in the bone defect. Bone formation

always started from the borders of the bone defect. The newly formed bone was always

in close contact with the DBB surface and presented a more mature appearance at 8

weeks than at 4 weeks. At 4 weeks, unmineralized bone was observed but not uniformly

between the DBB granules. At 8 weeks, mineralized trabecular structures were observed

uniformly within the bone defect and most DBB granules were encapsulated in the bone.

The trabecular appearance can be also observed in the other groups with DBB.

In group (iv) which contained DBB mixed with autologous bone, newly formed bone

was observed at 4 weeks between autologous bone chips and DBB. At 8 weeks, residual

autologous bone chips were observed sporadically and encapsulated in the new bone.

Group (v), containing DBB with adsorbed BMP-2, group (vi), containing DBB with

coating but without BMP-2, and group (vii) containing DBB with coating-adsorbed

BMP-2 did not show a significantly histological difference compared with group (iii)

containing only DBB at both implantation times.

Group (viii), which contains DBB with coating-incorporated BMP-2, showed a

different behavior compared to the other groups with DBB. An interconnected bone

network, which had a woven appearance, can be easily observed at 4 weeks. At 8 weeks,

the bone growth was observed throughout the space between DDB granules in group

(viii), and thus formed compact bone areas within the defects. This kind of area was

rarely found in other DBB groups (iii-vi).

A representative image at higher magnification of the unmineralized bone at 4 weeks

from group (v) containing DBB with adsorbed BMP-2 is shown in Fig. 3A. The

unmineralized new bone with deep purple can be observed in all the groups at 4 weeks.

At this time point, new bone in group (viii) containing DBB with coating-incorporated

BMP-2 appeared more mature (Fig. 3B) compared with other DBB groups such as group

(v) (Fig. 3A). Moreover, a development stage of bone marrow was observed in groups

(ii), (iv) and (viii) containing autograft or the coating-incorporated BMP-2 (Fig. 3B),

while bone marrow was rarely found in other DBB groups. Multinucleated giant cells

were found on the bone or DBB surface in each group with DBB at both times. A

representative multinucleated giant cell enveloping a very small DBB granule at 4 weeks

is shown in Fig. 3A. Light demineralized regions of DBB were always observed under

these cells (Fig. 3B). With a further resorbing process, the resorption lacunae were

clearly created by the multinucleated giant cells (Fig. 4A). However, these cells were

found sporadically at 8 weeks because most of the DBB granules had been encapsulated


109

in the new bone. The calcium phosphate coating can be found at 4 weeks but not at 8

weeks (Fig. 4B).

Representative images at higher magnification showed different appearances of bone

tissues in group (viii) containing DBB with coating-incorporated BMP-2 at 8 weeks of

implantation (Fig. 5). A compact bone structure with the presence of small porous

structure was observed (Fig. 5 A and B). In this compact bone area, bone marrow was

observed in some porous structure (Fig. 5C). Also, trabecular-like bone was visible as

characterized by the presence of an open porous structure with bone marrow formation

(Fig. 5D).

Figure 2. Representative histological sections of bone defect of each group at 4 and 8 weeks after

material placement. (i) No graft material; (ii) Autologous bone (#); (iii) Deproteinized bovine bone

(DBB, asterisk) bearing neither a coating nor BMP-2; (iv) DBB mixed with autologous bone (1:1);

(v) DBB bearing an adsorbed depot of BMP-2; (vi) DBB bearing a calcium phosphate coating but

no BMP-2; (vii) DBB bearing a calcium phosphate coating upon which BMP-2 was superficially

adsorbed; and (viii) DBB bearing a calcium phosphate coating into which BMP-2 was incorporated.

At 4 weeks, the newly formed bone (unmineralized) was purple (black arrow). At 8 weeks, the

newly formed bone (mineralized) was reddish (black arrow). The newly formed bone and the

autologous bone can be separated in groups (ii and iv). Scale bar = 500 µm.

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110

Figure 3. Representative histological sections at 4 weeks of DBB granules that bore an adsorbed

depot of BMP-2 (A) in group (vi) and those that bore a coating-incorporated depot of this agent (B)

in group (viii). The newly formed bone was more in contact with the DBB surface and presented

more mature in group (viii) than those in group (vi). At this juncture, a development stage of bone

marrow was found in group (viii) (M). Multinucleated giant cells presented on the surface of DBB

(arrow) and the underlying DBB presented demineralized region (light region). Capsular fibrous

tissues (F) were also found around DBB. Scale bar = 100 µm.

Figure 4. Representative histological sections at 4 weeks of multinucleated giant cells (black arrow)

and the resorption lacunae on DBB granules that bore an adsorbed depot of BMP-2 from group (v)

(A) and the calcium phosphate coating (white arrow) from group (vii) (B). Scale bar = 100 µm.


111

Figure 5. Representative histological sections at 8 weeks of DBB granules that bore a

coating-incorporated depot of BMP-2 with different bone tissues in group (viii). A stable compact

bone (CB) area (A); a compact bone area in an active phase with osteoblasts (arrow, B); Bone

marrow in close contact with DBB (C); and trabecular bone appearance (D). Scale bar = 100 µm

for (A-C); Scale bar = 200 µm for (D).

Histomorphometry

Bone formation

Quantitative evaluation of the amount of bone formation 4 and 8 weeks after

implantation (Fig. 6) revealed that bone formation increased significantly with increasing

time after implantation (p < 0.05). The statistical data revealed that 4 weeks or 8 weeks

after implantation group (viii) with coating-incorporated BMP-2 especially had a

significant effect on new bone formation among the groups with DBB. At 4 weeks, the

volume of newly formed bone was significantly the highest in group (ii) with only

autologous bone, and the lowest in group (i) without treatment. Significantly more bone

formation was found in group (iv) containing DBB mixed with autologous bone and

group (viii) compared with other groups with DBB. No significant difference was found

in bone formation between groups (iv) and (viii). Moreover, significantly more bone

formation was found in group (v) with directly adsorbed BMP-2 compared with group

(iii) with only DBB at 4 weeks.

At 8 weeks, the volume of newly formed bone was significantly the highest in groups

(ii) and (viii), and significantly the lowest in group (i). There were no significant

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112

differences in bone formation between groups (ii) and (viii) and there were no significant

differences among the other five groups with DBB. There was significantly more bone

formation in group (iii DBB only) compared with group (i).


weeks after implantation for each of the 8 groups (see Table 1 for an explanation of the


deviation. *p<0.05; **p<0.01; ***p<0.001.

Bone marrow

Quantitative evaluation of the amount of bone marrow revealed that there were no

significant differences among groups (ii), (iv) and (viii) containing autologous bone or

the coating-incorporated BMP-2 at 4 weeks, while bone marrow was not found in the

other five groups (Fig. 7). At 8 weeks, the volume of bone marrow was significantly the

highest in group (ii) with autologous bone, while bone marrow was still not found in

group (i) with no graft materials. Significantly more bone marrow was observed in

groups (vii) and (viii) with coating-adsorbed or coating-incorporated BMP-2 compared

with groups (iii) and (vi) without BMP-2. Furthermore, a trend was observed: the groups

containing autologous bone or BMP-2 resulted in more bone marrow than the other

groups.

DBB volume

Quantitative evaluation of the DBB volume after 4 and 8 weeks of implantation (Fig. 8)

revealed that no significances were found in the DBB volume among groups (iii), (v),

(vi), (vii), and (viii). As anticipated, the DBB volume of group (iv) containing DBB

mixed with autologous bone was about half that compared with other groups. Compared

with the start (time 0), no significant decrease of the DBB volume in each group was

found.


113

Figure 7. Graph depicting the volume of bone marrow within the bone defect at 8 weeks after

implantation for each of the 8 groups (see Table 1 for an explanation of the abbreviations). Mean

values (n=6 samples per group) are represented together with the standard deviation. *p<0.05;

**p<0.01; ***p<0.001.

Figure 8. Graph depicting the volume of DBB within the bone defect at 4 and 8 weeks after

implantation for each of the DBB-relevant groups (see Table 1 for an explanation of the


deviation.

Volume density of multinucleated giant cells on DBB

The multinucleated giant cells at 8 weeks were too few to count. Therefore, only data at 4

weeks were obtained (Fig. 9). At 4 weeks, the volume density of multinucleated giant

cells was the highest in DBB that bore an adsorbed depot of BMP-2, and the lowest in

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114

those that bore a coating-incorporated depot of the agent. No significant differences were

found among the other 4 DBB groups.


deproteinized bovine bone at 4 weeks after implantation (see Table 1 for an explanation of the


deviation. *p<0.05; **p<0.01; ***p<0.001.

DISCUSSION

In the current study, the histomorphometric analysis of bone formation 4 and 8 weeks

after implantation revealed that the functionalization of DBB with coating-incorporated

BMP-2 in group (viii) induced significant bone formation in the treatment of

critical-sized bone defects in sheep. This mode of drug delivery was more efficient

osteoinductively compared with the adsorption modes (directly adsorbed depot of BMP-2;

coating-adsorbed depot of BMP-2). After 8 weeks of implantation, group (viii) exhibited

a volume of induced new bone similar to the positive control group (ii) containing

autologous bone. Group (viii) also showed significantly more bone formation compared

with group (iv) containing DBB mixed with autologous bone. This study provided

evidence in support of the functionalization of DBB with the coating-incorporated

BMP-2 for optimizing the osteoinductivity for treatment of critical-sized bone defect.

The critical-sized bone defect (CSBD) model in this study was created by drilling

holes in the humerus and femur of sheep according to a widely published protocol by

Nuss et al. [32]. This drill hole model in sheep has proved to be an excellent animal

model for testing biomaterials for use in orthopedics, maxillofacial and dental surgery

[36]. It allowed the intraosseous implantation of up to 8 different test materials within

one animal due to the standardization of the bone defect, while at the same time it can

reduce the overall suffering of animals and give the necessary numbers to satisfy

statistical requirements [32, 37]. Because of the similarities with humans in weight, bone

and joint structure and bone regeneration, the results from sheep are more valid than

those obtained from small laboratory animals [38]. Although rodents may be less


115

expensive, they have a different bone morphology and they are often are too small for

testing bone substitute. Positive results in rodents may have to be repeated and verified in

larger species before human clinical trials can be initiated. Our previous study has

demonstrated that DBB with coating-incorporated BMP-2 can induce bone formation

efficiently in rats subcutaneously [27]. Whilst the ectopic model of ossification is useful

for testing the osteoinduction of materials, it is not suitable for its optimization [29].

To improve the osteoinductive effect substantially, BMPs need to be delivered to

target sites gradually at a sustained and low level [29, 39]. Biomimetic calcium

phosphate coating has been proven as a simple and effective tool for delivering growth

factors slowly, such as BMP-2 and vascular endothelial growth factor (VEGF) [23, 40].

In the present study, the histomorphometric analysis of bone formation revealed a

coating-incorporated depot of BMP-2 to be more efficient compared with the

adsorption mode. This is reflected in the volume of bone that had been deposited by the

end of both the sampling times: the value was higher in group (viii) with slow delivery of

BMP-2 compared with groups (v) and (vii) with a rapid delivery of BMP-2. Moreover,

no significant difference was found between groups (v) and (vii). The value in group (v)

with directly adsorbed BMP-2 was significantly higher compared with that in group (iii)

with DBB at 4 weeks only, while no significant differences were found between these

two groups at 8 weeks. These findings were anticipated and can be readily accounted for

by the relatively rapidly release of BMP-2 [41], which was highly water-soluble within a

biological milieu, and speedily borne away from DBB, despite the fact that BMP-2 has a

strong affinity for DBB [42]. Hence, the osteoinductive effect of the agent is exerted

within a short time span. On the contrary, group (viii) with coating-incorporated BMP-2

resulted in a sustained BMP-2 release at a low level [2, 27], and thereby led to more bone

formation.

The way of BMP-2 release of the coating-incorporated mode includes: 1) controlled

low burst release (initial diffusion), 2) release controlled by dissolution based on the

solubility of the coating itself, and 3) cell (osteoclast) mediated release based on the

digestive activity of cells [12]. It was demonstrated that the degradation of calcium

phosphate coating was enhanced by osteoclasts and thus resulted in an elevated protein

release, but it was still maintained in a sustained manner in vitro [43]. The efficiently

induced bone formation at an ectopic site confirmed in turn the gradual, sustained and

cell-mediated release of BMP-2 from the calcium phosphate coating in vivo [44].

Therefore, the lifetime of the coating determined the duration of the protein release. In

the present study, the BMP-2-incorporated coating can be observed at 4 weeks but not at

8 weeks, which indicates that its BMP-2 release can last at least 4 weeks. A previous

study showed that the coating (thickness: about 20μm) had not been completely degraded

during a 5 week period at an ectopic site [31]. The incorporation of BMP-2 could

increase the degradation of the coating on titanium implants and it resulted in the

complete degradation within 3 weeks in an orthotopic (maxillary) site [29]. The coating

may result in a faster degradation in the orthotopic site compared with the ectopic site,

because any material placed in the soft tissue may stimulate the formation of a soft tissue

capsule in an attempt to wall it off. Even the local cellular mechanisms of degradation

may be different in soft and osseous tissue. Therefore, different environments could

affect the degradation of the coating.

The abundance of bone marrow bodes well for the health and the endurance of the

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newly formed bone, since it is an important source of nutriments and pluripotent

progenitor cells for osseous tissue [45]. Both osteoblasts and osteoclasts are derived from

progenitors that reside in the bone marrow. DBB granules were observed in close contact

with bone marrow, which indicates the excellent biocompatibility of DBB. The

histomorphometric data revealed a trend indicating that the groups containing autologous

bone or BMP-2 resulted in more bone marrow than the rest groups. These findings

indicate that autologous bone and BMP-2 may be conducive in developing a healthy

bone environment [27], since autologous bone also provides osteogenic cells and growth

factors for osteoinduction. Moreover, no significant differences were found in bone

marrow among the groups (v, vii, and viii) with slow or rapid delivery of BMP-2 after 8

weeks of implantation, whereas in a previous study the slow delivery of BMP-2

significantly induced more bone marrow than the rapid delivery mode at an ectopic site

[27].

In all the groups with DBB, most DBB granules had become an integral part of the

bone network 8 weeks after the implantation. DBB can continually serve as a scaffold

because it degrades slowly. There is a controversy about the degradation of DBB. It was

reported that the resorption of DBB was very minor within 11 years after grafting with no

significant changes in the DBB particle size [12]. However, a severe resorption of DBB

was observed recently in a porcine calvaria augmentation model under a certain

experimental condition [46]. On the basis of current knowledge, the biodegradation of

biomaterials is based on the dissolubility of the material itself and the cell (osteoclast)

based resorption [47]. It was reported that an average of 4.7% (± 1.61) for DDB was

dissolved in Tris-HCl (120 h, pH 7.3, 37°C) [8]. Osteoclasts can be found on the surface

of DBB within 2 weeks at an orthotopic site [48]. In the present study, although a certain

amount of DBB was observed in a very small size and the resorption lacunae by cells

were clearly observed, the quantitative evaluation of the DBB volume in each group

supports the notion of the low degradability of DBB. The formation of the capsular

fibrous tissue and the bone deposited on the surface of DBB could prevent the dissolution

and resorption of DBB. Moreover, it was shown that the coating with incorporated

BMP-2 might increase the degradation of underlying materials [26]. The adsorbed mode

of BMP-2 was shown to increase the degradation of ceramic [49]. However, in the

current study, the different delivery modes of BMP-2 did not significantly influence the

degradation of DBB.

When DBB was implanted into an extraction wound healing model (not a

critical-sized bone defect model), a series of different processes were involved: 1) innate

inflammation; 2) formation of granulation tissue and provisional matrix; 3) surface

cleaning and resorption; and 4) de novo bone formation and hard tissue integration of the

material [48]. In the present study, the release of coating-incorporated BMP-2 from DBB

resulted in the lowest volume density of multinucleated giant cells on DBB, whereas the

adsorbed BMP-2 led to the highest one. These multinucleated cells could be osteoclasts

or foreign body giant cells (FBGCs), both of which can degrade biomaterials. It was

reported that the coating-incorporated BMP-2 with slow release can surpress the foreign

body reaction [27]. The foreign body reaction producing mcrophages and FBGCs is the

end stage of the inflammatory responses following the implantation of biomaterial.

FBGCs can release mediators of degradation such as reactive oxygen intermediates

(ROIs, oxygen free radicals), degradative enzymes, and acid into this privileged zone


117

between the cell membrane and biomaterial [50]. FBGCs can also release inflammatory

cytokines which stimulate circulating stem cells to become osteoprogenitors [50, 51]. On

the other hand, BMP-2 (endogenous or released from the carrier biomaterial) has been

shown to regulate both osteoblasts and osteoclasts [52, 53]. At low doses, BMP-2

stimulates the recruitment, proliferation and differentiation of osteoprogenitor cells,

whereas at high doses, it induces the recruitment, formation and activation of osteoclasts.

Osteoclast has become the common term to denote any cell that has a unique function to

break down mineralized matrices [54]. Osteoclasts exhibited tartrate resistant acid

phosphatase (TRAP) positivity and a well defined ruffled border, and they were observed

at the surface of both newly formed bone and biomaterials [55]. The precise

identification of the osteoclasts and the FBGCs seems to be difficult in vivo, since both

can be TRAP-positive [56]. It has also been shown that osteoblasts could be positively

stained with TRAP [57]. Therefore, staining with TRAP would not be specific for

osteoclast [58]. However, it should be noted that all these multinucleated giant cells play

a critical role in the surface treatment and the degradation of material and the bone

formation and remodeling. The precise distinction of them in vivo needs to be

investigated further.

Clinically it is necessary to accelerate bone formation for a curtailment of the

recovery phase in the bone defect repair or bone augmentation, since the expectations of

surgeons and patients alike are continually rising [59]. The calcium phosphate coating

incorporated with growth factors has been widely studied in animal models in an ectopic

or an orthotopic site. More studies need to devote to exploring its clinical performance

with titanium implants or bone substitutes. Meanwhile, the coating technique has been

continually modified and developed. By adjusting the ratio of the coating solution

volume to the surface area of the substrate, the coating thickness and the incorporation

rate of BMP-2 can be customized for a more precisely controlled release [60]. All in all,

we are well on the way to developing a simple and effective tool to optimize the

commercial products of bone substitute by giving them osteoinduction, and ultimately to

achieve a more satisfactory therapeutic effectiveness.

CONCLUSION

Our findings show the excellent biocompatibility and osteoconductivity of DBB. This

material can undergo cell-mediated resorption, but still showed slow degradation. The

capacity of BMP-2 to induce and sustain local bone formation in critical-sized bone

defects can be influenced by its mode of delivery. The osteogenic response can be more

efficiently promoted by its sustained release from a three dimensional reservoir, which

is a calcium phosphate coating with incorporated BMP-2, than by its rapid release from

an adsorption way. At the same time, the coating-incorporated BMP-2 on DBB led to an

excellent therapeutic effect which is comparable with that of autograft. This

functionalization approach could greatly enhance the clinical potential of DBB to be an

alternative to bone autografts in the repair of large or critical-sized bone defects.

ACKNOWLEDGMENTS

We would like to thank Prof. Dr. Tony Hearn for his scientific input and English editing

Chapter 7

118

as a native speaker for this publication. This project was supported by Osteology grant

(2008-015 / Dr. Liu) and KNAW grants (08CDP043, 09CDP036 and 11CDP011).


119

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123

Chapter 8

Low-dose rhBMP2/7 heterodimer to reconstruct

peri-implant bone defects: a micro-CT evaluation

Jingxiao Wang, Yuanna Zheng, Juan Zhao, Tie Liu, Lixia Gao, Zhiyuan Gu,

and Gang Wu

Journal of Clinical Periodontology, 2012 Jan;39(1): 98-105.

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ABSTRACT

Objectives: To delineate the dynamic micro-architectures of bone induced by low-dose

bone morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects

compared to BMP2 and BMP7 homodimer.

Material and Methods: Peri-implant bone defects (8mm-in-diameter, 4mm-in-depth)

were created surrounding SLA-treated titanium implants (3.1mm-in-diameter,

10mm-in-length) in minipig’s calvaria. We administrated collagen sponges with adsorbed

low-dose (30ng/mm3) BMP2/7 to treat the defects using BMP2, BMP7 or no BMP as

controls. 2, 3, and 6 weeks after implantation, we adopted micro-computer tomography to

evaluate the micro-architectures of new bone using the following parameters: relative

bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th),

trabecular separation (Tb.Sp), connectivity density (Conn.D), and structure mode index

(SMI). Bone implant contact (BIC) was also revealed histologically.

Results: Consistent with 2 and 3 weeks, after 6 weeks post-operation, BMP2/7 resulted

in significantly higher BV/TV (63.033±2.055%) and significantly lower SMI

(-4.405±0.500) than BMP2 (BV/TV: 43.133±2.001%; SMI: -0.086±0.041) and BMP7

(BV/TV: 41.467±1.850%; SMI: -0.044±0.016) respectively. Significant differences were

also found in Tb.N, Tb.Th and Tb.Sp at all the time points. At 2 weeks, BMP2/7 resulted

in significantly higher BIC than the controls.

Conclusions: Low-dose BMP2/7 heterodimer facilitated more rapid bone regeneration in

better quality in peri-implant bone defects than BMP2 and BMP7 homodimers.

Keywords: Bone morphogenetic protein, Heterodimer, Bone regeneration, MicroCT,

Bone defect

INTRODUCTION

More rapid repair of peri-implant bone defects has been pursued for years in the field of

dental implantology. The treatment with autograft is not satisfactory due to its high

resorption rate and limited availability [1, 2]. Such a situation has engendered vigorous

efforts to develop alternative materials. Albeit so, most of the commercially available

bone-defect-filling materials, such as collagen and deproteinized bovine bone, are not

intrinsically osteoinductive. Consequently, they have to be premixed with particulate

autologous bone to obtain osteogenicity when they are applied to repair the large-volume

bone defects. In this process, the limitations of autografts ensue.

As a viable option, homodimeric bone morphogenetic proteins (BMPs) can confer

osteoinductivity and expedite osteogenesis [3]. Absorbable collagen sponges with

adsorbed BMP2 or BMP7 homodimers have been approved by FDA for clinical use [1].

However, the effective doses of BMP homodimers are very high (e.g. 12 milligrams) [4],

which results in not only a substantial economic burden, but also a series of potential

side-effects, such as the overstimulation of osteoclastic activity [5].

One alternative approach to overcome this dilemma is to adopt more potent BMPs [6].

Heterodimeric BMPs exhibited several- or dozens-fold more effects than the respective

homodimers in inducing in-vitro osteoblastogenesis [7]. Most of previous studies were

based on the BMP heterodimers through the technology of combined BMP2 and BMP7

Low-dose rhBMP2/7 heterodimer

125

gene transfer, which is still far away from clinical applications [8]. However, hitherto, as

a promising cytokine therapy, the effects of purified recombinant human BMP

heterodimers on in-vivo osteogenesis were merely reported. We hereby designed this

experiment using a peri-implant bone defect model in minipigs in order to identify the

dynamic changes in micro-architectures of BMP2/7-induced bone. We hypothesized that

BMP2/7 could facilitate more rapid bone regeneration in better quality than BMP2 and

BMP7.

In the field of bone tissue engineering, a suitable carrier is important to optimize

osteoinductive effects of BMPs. Although many controlled-release carriers have been

well developed recently, collagen sponge is the only FDA-approved BMP-delivery

carrier for clinical use [9]. Collagen sponge has been proved to be a acceptable carrier of

BMPs in numerous clinical trials [10]. In the present study, we selected collagen sponge

as the carrier of BMP2/7 heterodimer with a view to giving a direct relevance and

significance for clinical practice.

The minimal dose of BMP homodimers to induce bone regeneration in minipig’s

calvaria was not documented. A previous study showed that BMP-2 of 30 to 240ng/mm3

could induce bone regeneration in a dose-dependent-increasing manner in the critical-size

defects [11]. Consequently, we adopted 5.0ug (equivalent to 30ng/mm3 in bone defects)

as test concentration.

Although histological analyses provide unique information on cellularity and dynamic

indices of bone remodeling, they have limitations in assessing bone micro-architectures.

Histological analyses are derived from stereological analysis of a few 2D sections,

usually assuming that the underlying structure is plate-like [12]. The inclusion of dental

implants may further lessen the histomorphometric information of bone because only one

central section of each implant can be used for analysis [13]. In contrast, micro-computed

tomography (microCT) can directly measure bone micro-architectures independent of

stereological models [14].

In this study, we adopted microCT in order to clarify 1) the dynamic 3D

micro-architectures of newly regenerated bone induced by BMP2/7 heterodimer

compared to BMP2 and BMP7 homodimers, and 2) the efficacy of low-dose BMP2/7 to

repair peri-implant bone defects.


Preparation of collagen sponges containing BMP2/7, BMP2 or BMP7

According to the manufacturer’s instruction, recombinant human BMP2/7 heterodimer,

BMP2 homodimer or BMP7 homodimer (R&D System Inc., Minneapolis, USA) was

reconstituted to a final concentration of 0.05μg/μl in a sterile 4mM HCl solution

containing 0.1% bovine serum albumin (BSA). The sterile 4mM HCl solution containing

0.1% BSA without BMP was used as control (non-BMP-containing suspension). The

collagen sponges (collagen type I, Helistat®, Integra, USA) were adapted into uniform

small pieces (15mm×4mm×2.5mm) under sterile condition. 100μl of either

BMP-containing or non-BMP-containing suspension was then homogeneously adsorbed

onto each collagen sponge piece. The final loading of BMP was 5μg per collagen sponge

piece. The sponge pieces were then dried under sterile condition.

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126

Animal models

We chose mini-pigs as experimental animals because they bear a comparable rate of bone

regeneration to human [15]. The calvaria bone defects were selected by the following

considerations: 1) calvaria bone does not depend on central blood supply [16]; 2) calvaria

has a more stable mechanical and chemical environment than in mouth, which will

significantly increase the successful rate and decrease analysis complexity.

Eighteen 9-month-old Guangxi Bama minipigs (9 male, 9 female, weighing from

16.50 to 19.80kg), purchased from and kept in Animal Research Centre of Zhejiang

University. Throughout the study, the minipigs were treated following the guidelines of

animal care established by Zhejiang University.

Group set-up

Four groups were set up (n=6 animals per group per time point): 1) Collagen with

BMP2/7 heterodimer (experimental group); 2) Collagen with BMP2 homodimer; 3)

Collagen with BMP7 homodimer; 4) Collagen without BMPs. To balance the influence

from the gender of animals and defect sites (Fig. 1c, d), the samples were subjected to

different defects of different animals following a randomization protocol (Table 1).

Table 1. A randomization protocol for the distribution of collagen sponges with adsorbed

BMP2/7, BMP2 or BMP7 or control (no BMP) to the different defects of different

animals at one of the three time points.


127

Figure 1. Images depicting the schematic model (a, b) and surgical images (c, d) of peri-implant

bone defects that were surgically created in minipig’s calvariae and were treated using collagen

with adsorbed BMPs. (a) A defect (8mm-in-diameter, 4mm-in-depth) was created using a trephine

drill. An implant (8mm-long fixture) was implanted in the center defects with 4mm fixture within

the bone defect. (b) The peri-implant bone defect was fulfilled with collagen sponge with or

without BMPs, and covered with Bio-Gide® membrane before suture. (c) The borders of four

defects (8mm-in-diameter and 4mm-in-depth) were created using trephine drills in the calvarial

bone of minipigs. (d) Four implants were centrally implanted into the four defects with interval

spaces filling with collagen sponges. From the left upper, the defects were numbered as 1, 2, 3 and

4 respectively in the anticlockwise direction.

Surgeries

The pigs were subjected to anesthesia by administrating Sumianxin II (0.3ml/kg,

purchased from the Military Veterinary Institute, Quartermaster University of PLA,

Chang Chun, China) with the addition of Penicillium (5×104

Unit/kg) and Atropine

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128

(0.03mg/kg) at 30 minutes before surgery. After applying a local anesthesia (1%

Lidocaine with 1:100000 Adrenaline) to the frontal calvariae of the minipig, a

10-cm-long sagittal incision was made on the forehead region. The calvarial bone was

exposed after lifting a full thickness flap. Four bone defects (8mm-in-diameter,

4mm-in-depth) with a 1cm interval distance were prepared on minipig’s calvaria using a

trephine drill. The cylinder-shaped bone tissue in the centre of each circular defect was

thereafter removed (Fig. 1a).

We adopted the titanium implants (Zhejiang Guangci Medical Appliance co., ltd.,

Cixi, China) with large-grit sand-blasted, acid-etched (SLA) fixtures (3.1mm-in-diameter

and 8mm-in-length) in this experiment. They were implanted in the center of the bone

defects with 4mm-long fixture within the defect (Fig. 1a). The total volume of each bone

defect after implantation was 166mm3. The remaining circular bone defect around the

implant was filled with collagen sponges with or without adsorbed BMPs

(5000ng/166mm3≈30ng/mm

3) (Fig. 1b).

The bone defects and the implants were covered with a piece of Bio-Gide® membrane

(40mm×50mm, Geistlich PhamaAG, Switzerland, Fig. 1b). The soft tissue was sutured

layer by layer. In addition, Penicillium (50000U/kg) was administered for 3 days

postoperatively to protect the minipig from any inflammation. The suture was removed

on the 7th day after surgery.

Sample retrievement and preparation

At 2, 3 and 6 weeks post-operation, minipigs were sacrificed by intramuscular injection

of overdose of Sumianxin II. All the calvarial blocks of the sacrificed animals were

harvested and immediately immersed into the 10% neutrally buffered formalin for

fixation. After a 7-day fixation, each individual bone sample including the implant and

the bone defect around it was separated using a gypsum saw from each calvarial block.

The specimens and uninjured bone (obtained in the same location as the defect region)

were dehydrated with alcohol and embedded in methyl methacrylate (MMA).

MicroCT evaluation

Embedded specimens and uninjured bone were scanned by micro-CT (micro-CT80,

ScancoMedical, Bassersdorf, Switzerland) with a resolution of 10µm followed by

off-line reconstruction. After image acquisition, the titanium and mineralized tissue were

segmented from each other by applying a multilevel thresholding procedure [17, 18].

We evaluated the micro-architectures of bone within the defects using following

parameters: 1) Relative bone volume (bone volume/tissue volume, BV/TV, %); 2)

Trabecular number (Tb.N, 1/mm); 3) Trabecular thickness (Tb.Th, mm); 4) Trabecular

separation (Tb.Sp, mm); 5) Connectivity density (Conn.D, 1/mm3) and 6) Structure mode

index (SMI).


The histomorphometric analysis was subsequently performed to measure bone implant

contacts (BIC) in histological sections. Details can be seen in supplementary data.


All data were presented as mean values together with the standard deviations (Mean±SD).

http://www.all-acronyms.com/SLA/sand-blasted,+large-grit,+acid-etched/962522


129

Data were analyzed by one-way analysis of variance (ANOVA). SPSS software (version

18 for windows, SPSS Inc., Chicago, IL, USA) was employed for the statistical analysis.

Post Hoc comparisons were made using Bonferroni’s corrections with the level of

significance at p<0.05.

RESULTS

All implants achieved good primary stability. The healing period was uneventful and the

surgical sites healed well during the 6 weeks.

Significant increases in BV/TV, Tb.N and Tb.Th as well as significant decreases in

Tb.Sp were found for each group at a later time point than at an earlier time point (Table

2), except for Tb.Th in the non-BMP-treated group at 3 weeks and in the BMP2/7 group

at 6 weeks. At each time point, significantly higher BV/TV, Tb.N and Tb.Th as well as

significant lower Tb.Sp were found in the three BMP-treated groups than that in the

non-BMP-treated group. Significantly higher BV/TV, Tb.N and Tb.Th as well as

significant lower Tb.Sp were also found in the BMP2/7-treated group than those in the

BMP2- and BMP7-treated group. However, no significant difference in BV/TV, Tb.N,

Tb.Th and Tb.Sp was detected between the BMP2- and BMP7-treated groups at each

time point.

BMP2/7-treatment for 6 weeks restored 87.3% BV/TV of the uninjured bone, which

was significantly superior to BMP2- (59.8%), BMP7- (57.5%), and non-BMP-treatment

(31.1%). Tb.N and Tb.Sp reached the equivalent level to that of the uninjured bone only

in the BMP2/7-treated group at 6 weeks. On the two-dimensional images that were

perpendicular to the implants, the Tb.N after 6-week BMP2/7-treatment was significantly

higher than those after 6-week BMP2-, BMP7- and non-BMP-treatments (Fig. 2). The

border between the defects and the surrounding uninjured bone was also less distinct in

the BMP2/7-treated groups than those in the other three groups (Fig. 2). For the

non-BMP-treated group, significant increases in Tb.Th were only found at 6 weeks. For

the BMP2/7-treated group, Tb.Th reached the equivalent level to that of the uninjured

bone at 3 weeks and maintained in this level in the monitoring span.

For each time point, significantly lower SMI was found in the BMP2/7-treated groups

than that in the either BMP2- or BMP7- or non-BMP-treated group (Table 2). No

significant differences in SMI were found among BMP2-, BMP7- and non-BMP-treated

groups at each time point. At 6 weeks, BMP2/7-treatment resulted in the least difference

(2.45) in SMI from the uninjured bone than the treatments of BMP2 (6.76), BMP7 (6.80)

and non-BMP (7.47).

Conn.D in non-BMP-treated group significantly increased at 3 weeks and thereafter

maintained in the equivalent level to that in the uninjured bone (Table 2). Conn.D in the

three BMP-treated groups significantly increased at 3 weeks and then significantly

decreased at 6 weeks. Conn.D in the three BMP-treated groups was significantly higher

than the non-BMP-treated group for each time point. Conn.D in BMP2/7-treated group

was significantly higher at 2 weeks but significantly lower at 3 weeks than the BMP2-

and BMP7-treated groups. At 6 weeks, Conn.D in BMP2/7-treated group reached the

equivalent level to that in the uninjured bone. In contrast, Conn.D in BMP2- and

BMP7-treated group was significantly higher than that in the uninjured bone.

At as early as 2 weeks post-operation, BMP2/7 resulted in a significantly higher BIC

Chapter 8

130

than the groups of BMP2, BMP7 and non-BMPs. And BIC in BMP2/7 group remained in

this level in the following monitoring span. (Details can be seen in supplementary data.)

Figure 2. Graph depicting 2-dimensional microCT images of peri-implant bone defects in the

minipigs’ calvaria at 6 weeks post-operation. (a) collagen without BMPs (non-BMP-treated); (b)

collagen with BMP2 homodimer; (c) collagen with BMP7 homodimer; (d) collagen with BMP2/7

heterodimer. White arrows pointed to the borders of the bone defects.


131

DISCUSSION

Grafts of collagen sponge with adsorbed BMP homodimers, in particular of BMP2 and

BMP7, were shown to accelerate bone formation [19]. However, the use of the BMP

Chapter 8

132

homodimers are associated with a high cost [4, 20] and potential side-effects [5]. BMP

heterodimers can be a promising approach to solve this dilemma. The study, for the first

time, showed that purified recombinant human BMP2/7 heterodimer in a low dose could

induce in-vivo bone regeneration more rapidly and in a significantly higher

dose-efficiency than rhBMP2 and rhBMP7 homodimers.

The quality of newly formed bone is crucial to the long-term stability of implants. It

depends on both its volumetric properties such as bone mineral density, and its geometric

properties such as bone structure and micro-architecture [21, 22]. In contrast to

conventional histological evaluation, microCT is superior to show not only bone mineral

density but also bone micro-architectures [21, 23].

Among the six parameters, BV/TV, Tb.N and Tb.Th directly reflected the amount of

new bone. This study confirmed that BMP homodimers could significantly promote bone

regeneration in such a low dose (30ng/mm3). We also found that the BV/TV in

BMP2/7-treated group was significantly higher than that in BMP2- or BMP7-treated

group for all time points. This finding indicated that BMP heterodimer induced bone

regeneration in a significantly higher dose-efficiency than the homodimers. This

specificity of BMP heterodimers was previously attributed to their higher osteoinductive

potency than the respective homodimers [24]. Recently, we systematically delineated the

functional characteristics of BMP2/7 heterodimer in inducing bone regeneration in a

time-course and dose-dependent study [25]. We found that the maximum effect of

BMP2/7 heterodimer on promoting in-vitro osteoblastogenesis was not superior to BMP2

or BMP7 homodimers; instead, the effective concentration of BMP2/7 heterodimer for

each osteoblastogenetic event was significantly lower than that of the two homodimers.

These findings suggested that the advantages of BMP2/7 over BMP2 and BMP7 were

significant when they were applied in and probably only in, relatively lower doses.

However, this hypothesis needs to be clarified in in-vivo dose-dependent studies.

In the present study, the volume density of newly formed bone tissue (BV/TV)

induced by BMP2/7 was 1.163- or 1.379-fold of those induced by BMP2 or BMP7 at 3

weeks, and 1.489- or 1.512-fold of those at 6 weeks, which were not as high as

previously reported (2-3-fold) [8, 24, 26]. We supposed that this inconsistency might be

due to the different animal models and different BMP concentrations, etc.

The time-course and BMP-dependent patterns of BV/TV that were obtained from

microCT were identical to those of area percentage of bone (APB) that was obtained

from histological sections (Supplementary data). The high positive correlation (Pearson

coefficient=0.992, p<0.001) between these two parameters validated the reliability of

microCT. This finding is consistent with previous studies in animal [27] and human

specimens [28]. Albeit so, APB was relatively lower than the corresponding BV/TV.

This may be attributable to several factors: inadequate resolution of microCT images

relative to the trabecular size and the use of a plate model to estimate trabecular size, etc

[28].

After 6 weeks of BMP2/7-treatment, the Tb.N reached the equivalent level to that in

the uninjured bone, while either BMP2 or BMP7 or non-BMP treatment failed to do so.

Tb.Th in BMP2/7-treated group reached the same level as the uninjured bone at as early

as 3 weeks (Fig. 2). In contrast, it was obtained in neither BMP2- nor BMP7- nor

non-BMP-treated groups even after 6 weeks. All together, these findings indicated that

BMP2/7 heterodimer could more rapidly increase Tb.Th than the respective homodimers.


133

The effect of BMP2/7 in increasing Tb.Th was also more rapid than its effect on

increasing Tb.N. Consequently, the significant increase of BV/TV in BMP2/7-treated

group from 3 to 6 weeks should be attributed to the increase of not Tb.N but to Tb.Th.

These findings may add new knowledge to the specificity of BMP2/7 heterodimer’s

function.

BIC in each group increased gradually. At 6 weeks post-operation, no significant

difference could be found among the four groups (Supplementary data). The difference in

BIC among different groups was not as significant as other parameters. This may be due

to the short vertical depth (4mm) of the bone defects and the high conductivity of the

dental implants. Bone tissue could easily grow along the surface of dental implants. In

BMP2, BMP7 and non-BMP groups, a thin layer bone could be seen on the surface of

dental implants with few contacts at 2 weeks, while the surrounding space remained

unfilled with bone. On the other hand, BMP2/7 could significantly enhance the BIC at as

early as 2 weeks with surrounding space fulfilled with newly regenerated bone. This

finding suggested that BMP2/7 in the selected dose could facilitate significantly earlier

functioning of implants than BMP2 and BMP7.

Conn.D and SMI reflect the network and structure of trabecular bone tissue [29] and

they cannot be obtained from 2-D histological sections. Conn.D in non-BMP-treated

group significantly increased 1.6-fold at 3 weeks than at 2 weeks, and thereafter

maintained at about 8.2 which was equivalent to that in the uninjured bone till the end of

the monitoring span. In contrast, Conn.D increased 3.60-fold for BMP2 and 4.08-fold for

BMP7 at 3 weeks than at 2 weeks. Conn.D in the two BMP homodimer-treated groups

decreased at 6 weeks to 14.1 which was higher than the uninjured bone. These results

indicated BMP2 and BMP7 heterodimers could significantly induce de novo bone

regeneration at about 3 weeks post-operation. The Conn.D in the two BMP

homodimer-treated groups were about 4.2- to 4.5-fold of that in non-BMP treated group

at 3 weeks and reached 34.9-37.4. Such a high Conn.D suggested that the BMP

homodimers resulted in a dense stellate-reticulum network of newly formed trabeculae.

The thereafter decrease of Conn.D may be due to the re-organization of trabecular by

osteoclastic activity [30]. Unexpectedly, although a significant increase was also detected

for BMP2/7 heterodimer at 3 weeks, Conn.D in this group was significantly lower than

those of BMP2 or BMP7 homodimers. Two possible mechanisms might account for this

phenomenon: 1) the peak of BMP2/7-induced de novo bone regeneration appeared earlier

and was not detected in the selected time point; 2) BMP2/7 may facilitate a rapid

osteoclastic activity to simultaneously re-organizing the trabeculae [30]. Further studies

need to be performed to clarify this mechanism. At 6 weeks post-operation, Conn.D in

BMP2/7-treated group was significantly lower than those of BMP2 or BMP7 group and it

was equivalent to that in the uninjured bone. This finding indicated that BMP2/7 can

favor more rapid bone maturation than BMP2 and BMP7 homodimers.

At 6 weeks, SMI significantly decreased to -4.4, which was the nearest to that in the

uninjured bone (-6.8). In contrast, SMI were at about 0 in the two BMP

homodimer-treated groups and was 0.6 in the non-BMP-treated group. Negative values

are the result of pores within bones that have high bone volume fractions, which

presented a type of Swiss cheese-like structure with a concave surface [21, 31]. In

consistent with other parameters, SMI indicated BMP2/7 heterodimer resulted in the

more rapid maturation of bone than BMP2 and BMP7 homodimers. This finding was also

Chapter 8

134

confirmed by the indistinct border between the new and the old bone in BMP2/7 group at

6 weeks (Fig. 2).

One limitation in this study was that only one concentration of BMPs was adopted in

this study. Further studies with serial concentrations could provide more complete

delineation of the functional characteristics of BMP heterodimer in inducing in-vivo bone

regeneration. Caution should be taken when extrapolating the findings to alveolar bone

defects due to the different biological properties between the calvarial bone and alveolar

bone. Although we adopted a protocol to enable the homogeneous distribution of BMPs

within collagen sponges, the direct adsorption of BMP2 onto collagen sponges can be

still clinician-dependent and the potential influence of incubation protocol could,

therefore, totally excluded. Release profile of BMPs was also a key determinant of

osteoinductive efficacy of BMPs [32]. This study was based on a delivery system of

collagen sponge, which may present a different release profile of BMPs and different

results from other delivery systems such as biomimetic coatings [33, 34] and polymers

[35].

In summary, this study indicated that collagen sponge-delivered rhBMP2/7

heterodimer could repair peri-implant bone defects more rapidly and in a significantly

higher dose-efficiency than rhBMP2 and rhBMP7 homodimers when they were applied

in the same low dose (30ng/mm3).

ACKNOWLEDGEMENTS

We thank Dr. Yuelian Liu from Academic Center for Dentistry Amsterdam, VU

University, the Netherlands for giving technical assistance during the experiment and we

also thank Geistlich Phama AG Company for providing Bio-Gide® membrane.


135

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137

Chapter 9

Icariin: does it have an osteoinductive potential for

bone tissue engineering?

Xin Zhang&, Tie Liu

&, Yuanliang Huang, Daniel Wismeijer, and Yuelian Liu.

&Xin Zhang and Tie Liu share first authorship.

Phytotherapy Research, 2013 Jul 4. doi: 10.1002/ptr.5027.

Chapter 9

138

ABSTRACT

Traditional Chinese Medicines (TCMs) have been recommended for bone regeneration

and repair for thousands of years. Currently, the Herba Epimedii and its multi-component

formulation are the attractive native herbs for the treatment of osteoporosis. Icariin, a

typical flavonol glycoside, is considered to be the main active ingredient of the Herba

Epimedii from which icariin has been successfully extracted. Most interestingly, it has

been reported that icariin can be delivered locally by biomaterials and that it has an

osteoinductive potential for bone tissue engineering. This review focuses on the

performance of icariin in bone tissue engineering and on blending the information from

icariin with the current knowledge relevant to molecular mechanisms and signal

pathways. The osteoinductive potential of icariin could be attributed to its multiple

functions in the musculoskeletal system which is involved in the regulation of multiple

signaling pathways in anti-osteoporosis, osteogenesis, anti-osteoclastogenesis,

chondrogenesis, angiogenesis, and anti-inflammation. The osteoinductive potential and

the low price of icariin make it a very attractive candidate as a substitute of

osteoinductive protein − bone morphogenetic proteins (BMPs), or as a promoter for

enhancing the therapeutic effects of BMPs. However, the effectiveness of the local

delivery of icariin needs to be investigated further.

Keywords: icariin, osteoinductive, BMPs, bone regeneration, bone tissue engineering

Abbreviations: ALP, alkaline phosphatase; BMD, bone mineral density; BMP, bone

morphogenetic protein; BR, bone resorption; BSP, bone sialoprotein; BMSCs, bone

marrow mesenchymal stem cells; Cbfa1, core-binding factor alpha 1; CD14/TLR4,

cluster of differentiation 14/toll-like receptor 4; CPC, calcium phosphate cement;

EGF-EGFR, epidermal growth factor-epidermal growth factor receptor; ERK,

extracellular regulated protein kinases; GAGs, glycosaminoglycans; LPS,

lipopolysaccharide; MEK, mitogen-activated protein kinase; OCG, osteoclastogensis;

OCN, osteocalcin; OPG, osteoprotegerin; PA, proliferative activity; PGE2, prostaglandin

E2; RANKL, receptor activator of nuclear factor-kB ligand; Runx2, runt-related

transcription factor 2; Smad, drosophila mothers against decapentaplegic protein; Sox9,

SRY (sex determining region Y)-box 9; TBA, trabecular bone area; TCP, tricalcium

phosphate

Icariin: does it have an osteoinductive potential

139

Contents

1. Introduction

1.1 Osteoinduction and osteoconduction in bone regeneration

1.2 Clinical and economic backgrounds

1.3 Traditional Chinese Medicine in bone regeneration

2. What is icariin?

3. Icariin-based multi- or single- component formulation

3.1 Xian Ling Gu Bao

3.2 Herba Epimedii

4. Icariin applications in bone tissue engineering

5. Underlying mechanisms of icariin for bone regeneration

5.1 Anti-osteoporosis

5.2 Osteogenesis

5.3 Anti-osteoclastogenesis

5.4 Chondrogenesis

5.5 Angiogenesis

5.6 Anti-inflammation

6. Toxicity of icariin

7. Concluding remarks and perspectives

Acknowledgment

References

1. Introduction

1.1. Osteoinduction and osteoconduction in bone regeneration

A satisfactory bone regeneration of bone defects which are so large that they cannot heal

by themselves remains a big problem for surgeons (Otto and Rao 2004). An ideal

osteoinductive bone graft is still desired. The osteoinductive property of such a graft has

become the most important issue for bone substitutes. Autografts are the gold standard

due to their osteoconductive and osteoinductive properties, while they are unfortunately

associated with a limited availability as well as with pain and morbidity at the donor site

(Ahlmann et al., 2002). The use of allografts or xenografts can overcome these problems

but they are associated with possible infections and immune responses (Bauer and

Muschler 2000; Stevenson 1998; Donos et al., 2004). At present, synthetic bone

substitutes such as calcium phosphate based biomaterials have become widely used in

clinics because of their high osteoconductivity (Dorozhkin 2010), but most of them lack

an intrinsic osteoinductivity. Consequently, bone growth factors and mesenchymal stem

cells are usually introduced into the system to render these synthetic biomaterials

osteoinductive followed by protein or gene delivery (Cowan et al., 2004; Franceschi et

al., 2004; Byers et al., 2004; Yamamoto et al., 2000). Osteoinductive growth factors such

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140

as bone morphogenetic proteins (BMPs) have been widely studied in bone tissue

engineering (Langer 2009). In particular BMP-2 and BMP-7 were usually carried

(delivered) by bone substitutes, and thus confer osteoinduction on the bone substitutes

(Liu et al., 2010b; Magin and Delling 2001). They have been applied in clinically.

BMP-2 was used to expedite and augment spinal fusion (Shimer et al., 2009), to heal

open tibia fractures (Garrison et al. 2010; Alt et al.,2006), and to augment the alveolar

bone (Casado et al., 2010; Tonetti and Haemmerle et al., 2008); BMP-7 was used to

promote the healing of bony non-unions (Schmidmaier et al., 2009).

1.2. Clinical and economic backgrounds

Nowadays the implantation of bone substitutes for bone repair and augmentation is fairly

routine clinically and the postoperative healing follows a predictable course in most

patients resulting in a good long-term functional outcome (Mordenfeld et al., 2010;

Ozkan et al., 2011). However, the expectations of surgeons and patients are continually

increasing and aspiring to a shortening of the recovery. (Rustemeyer and Bremerich

2007). Large amounts of BMPs are required in some cases for osteoinduction and for a

further improvement in bone formation (Seeherman et al., 2006, Dickerman et al., 2007).

The devices containing BMPs tend to fail in a certain percentage of cases, and thereby

raise concerns about costs and safety (Geesink et al., 1999; Lieberman et al., 2002;

Bridwell et al., 2004). The high price and the rapid degradation of BMPs are its major

shortcomings and limit its use clinically. (Urist 1965; Zhao et al., 2006). Therefore, there

is an impending need to develop alternative methods to overcome these limitations (Zhao

et al., 2008). Attempts have been made to reduce the dose of BMP and so raise the

efficiency, such as the use of biomimetic calcium-phosphate coating (Liu et al., 2010b)

and polymers mixed with calcium-phosphate cements (Ruhe et al., 2005) which give a

sustained release of BMP. Most of these attempts have been effective but remain in a

preclinical stage. There remains the need for improving the loading efficiency of BMP to

reduce the amount of BMP used. All in all, this research is still on the way to developing

a simple, efficient and cost effective method.

1.3. Traditional Chinese Medicine in bone regeneration

Traditional Chinese medicines (TCMs) are considered as good alternatives for bone

regeneration (Shang et al., 1987). It becomes of great interest to combine bone substitutes

with TCMs used for bone regeneration (Zhao et al., 2010). TCMs are divided into single

component and multi-component formulations. One multi-component formulation

contains many kinds of herbs, whereas one single component contains only one herb.

Some of TCMs have been recommended for bone regeneration for hundreds of years

(Putnam et al., 2007). A variety of TCMs for bone regeneration have been widely studied

(see Table 1). They have shown positive effects on the treatment of osteoporosis, and can

stimulate the proliferative activity of osteoblasts, inhibit the formation of osteoclasts,

prevent bone loss, and increase the bone mineral density (Zhu et al., 2012; Qin et al.,

2005; Xu and Lawson 2004; Lee et al., 2005).


141

Table 1. Multi-component formulations and single components for bone health

Product Name

Multi-component

formulations (M) or

Single components (S)

Experimental

subjects Main effects References

Xian ling gu bao M OVX rats;

Postmenopausal

women

BMD↑ Zhu et al., 2012;

Qin et al., 2005

Shu di shan zha M Menopausal

women

BR↓ Xu and Lawson

2004

Shen gu M Osteoporotic

patients

BMD↑; BR↓ Mingyue et al.,

2005

Yang huo gu bao M Osteoporotic

male rats

BMD↑ Liao et al., 2001

Hachimi-jio-gan M OVX rats BR↓ Hidaka et al., 1997

Kami-kihi-to M OVX rats BMD↑ Kanai et al.,

2005

Jian gu M OVX rats BMD↑; TBA↑ Lin et al., 2004

BushenNingxin M Osteoblasts;

OVX mice

PA↑ Wang et al.,

2001

Dang-gui-ji-hwang-yeum M OVX rats TBA↑; OCG↓ Chae et al.,

2004 Hochu-ekki-to M Rats BMD↑ Sakamoto et al.,

2000

Herba Epimedii S Postmenopausal

women;

UMR-106 cells;

Rat osteoblasts;

OVX rats

BR↓; PA↑; mRNA

of OPG↑;

RANKL↓; cbfa1

mRNA↑; OCN↑

Zhang et al.,

2007;

Meng et al.,

2005a;

Liu et al., 2005; Qian et al., 2006

Sambucus williamsii S OVX rats;

UMR106 cells

BMD↑; ALP↑;

OCN↑; OCG↓

Xie et al., 2005b

Cistanche salsa S OVX rats BR↓ Yamaguchi et

al., 1999

Red sage S Osteoclasts OCG↓ Lee et al., 2005

Drynariae rhizoma S Rats and mice;

Human osteoprecursor

cells

Cathepsins K and

L↓

Jeong et al.,

2004; Jeong et al., 2005

Puerariae radix S Castrated mice BMD↑; TBA↑ Wang et al.,

2001

Astragalus

membranaceous

S OVX rats BR↓ Kim et al., 2003

Abelmoschus manihot (L.)

Medik

S OVX rats BR↓ Shirwaikar et

al., 2003;

Puel et al., 2005

Wedelia calendulacea

Less.

S OVX rats BR↓ Annie et al.,

2006

Sophorae fructus S OVX rats BR↓ Joo et al., 2004

Cimicifuga racemosa S OVX rats BR↓ Nisslein and

Freudenstein 2003

Among these TCMs, the Herba Epimedii and its multi-component formulation ‘Xian

Ling Gu Bao’ (XLGB) have icariin as their main ingredient. Recently, it was reported

that icariin is safe, non-toxic, inexpensive and osteoinductive (Wu et al., 2009b; Zhao et

al., 2010), and this makes it a very attractive potential agent for bone tissue engineering.

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142

It was demonstrated that icariin enhanced the osteogenic induction activity of BMP-2 in a

fibroblastic cell line (Zhao et al., 2010) and induced osteogenic differentiation of

preosteoblastic cells (Zhao et al., 2008). After the intramuscular implantation in the backs

of rats for three months, new bone formation was observed in β- tricalcium phosphate

(TCP) ceramic loaded with icariin but not in the β-TCP ceramic alone (Zhang et al.,

2011a). All these studies indicate the highly positive effects of icariin on bone formation.

Especially, the ectopic bone formation strongly proved the potential of osteoinduction of

icariin (Zhang et al., 2011a). Therefore, it is of great interest that icariin may be used as a

substitute for BMP, or as a promoter to enhance the therapeutic effects of BMP and so

reduce the dose of BMP. Potentially there is an application for icariin in bone tissue

engineering.

Hereby, we review the performance of icariin for bone tissue engineering with the

current knowledge relevant to molecular mechanisms and signal pathways. The aim of

this review is to clarify whether icariin has osteoinductive potential. The publications in

the regard of icariin and bone tissue regeneration were selected using following keywords:

icariin AND (bone* OR osteoblasts* OR osteoclasts* OR chondrocytes*). Databases

were searched from the earliest date available until 1 April 2013. The initial literature

search, resulted in 52 articles from PubMed, 104 from ISI, and 2 from Cochrane. After

screening all titles and abstracts, 32 articles from PubMed and 41 from ISI were

considered to be eligible for this study. The exclusion of 32 duplicates resulted in a total

of 41 articles, as shown in Fig. 1. All references in the selected manuscripts were

reviewed in order to ensure that no papers had been missed with the chosen search

strategy.

2. What is icariin?

Icariin (C33H40O15, molecular weight: 676.67) was recorded in the Chinese

pharmacopoeia for the purpose of anti-rheumatics (anti-inflammation), tonics (health

promotion), and aphrodisiacs (Hsieh et al., 2010). It is a prenyl flavonoid glycoside with

a glucosyl group on C-3; a rhamnosyl group on C-7; a methoxyl group on C-4; and a

prenyl group on C-8 position (Fig. 2). This prenyl group on C-8 could be the active group

that takes part in osteoblastic differentiation and explains its greater potency in

osteogenesis and mechanisms of action (Ma et al., 2011). The metabolites profiles in

plasma revealed that glucuronide conjugates of isoflavonoids and flavonoid aglycones

were the major circulating forms of icariin (Qian et al., 2012).

Through the development of modern separation techniques, Icariin has been extracted

successfully as a bone active ingredient from Herba Epimedii (Nian et al., 2009, Hsieh et

al., 2010). A rapid and accurate reversed-phase liquid chromatography-tandem mass

spectrometry method has been developed and validated for the quantitative determination

of the flavonoid glycosides in Herba Epimedii (Islam et al., 2008). Icariin can also be

extracted and purified by an ultrasonic technique (Zhang et al., 2008a, Jia et al., 2011)

and by Dual-Mode HSCCC (Li and Chen 2009).


143

Figure 1. Flow diagram of literature selection process. *E.g. reviews, letters.

Figure 2. Chemical structure of icariin (Ma et al., 2011).

Chapter 9

144

3. Icariin-based multi- or single- component formulation

3.1 Xian Ling Gu Bao

Xian Ling Gu Bao (XLGB) is a phytoestrogen-rich multi-component formulation

containing Epimedin B, Epimedin C and icariin (Guan et al., 2011). These three

flavonoids were all from the Herba Epimedii. XLGB is one such herbal medication

officially approved by the Chinese Food and Drug Administration and is orally

administered intermittently in the treatment of osteoporosis (Zhu et al., 2012). The

ingredients of XLGB consist of six non-leguminous herbs with percentages in weight as

follows: Herba Epimedii (70%), Radix Dipsaci (10%), Radix Salviae Miltiorrhizae (5%),

Rhizoma Anemarrahenae (5%), Psoralea Corylifolia L. (5%), and Rehmannia Glutinosa

(5%) (Guan et al. 2011). Recently, both qualitative and quantitative methods were

established for the comprehensive quality control of XLGB. Using high performance

liquid chromatography coupled with diode array detection and electrospray ionization

tandem mass spectrometry, a total of 47 compounds were identified from XLGB (Guan et

al., 2011). XLGB prevented a deterioration of musculoskeletal tissues induced by

ovariectomy (OVX). (Qin et al., 2005). The treatment over one year with the

conventional dose of XLGB demonstrated a safe and a statistically significant increase in

bone mineral density in the lumbar spine after 6 months in postmenopausal women (Zhu

et al., 2012).

3.2 Herba Epimedii

Icariin is the main pharmacological component of Herba Epimedii. Herba Epimedii is a

centuries old traditional medicine herb and its formulation is one of the most frequently

prescribed herbs (Pei and Guo 2007). It is recorded in the Chinese pharmacopoeia as ‘yin

yang huo’ and was used to cure bone diseases such as osteoporosis and bone fracture in

ancient China. Herba Epimedii can be considered as a complementary and alternative

medicine for treatment of postmenopausal osteoporosis (Xie et al., 2005a; Zhang et al.,

2007). It was shown that the Herba Epimedii can promote the proliferation, the

differentiation and the expression of osteoprotegerin (OPG) mRNA of the osteoblasts

cultured in vitro (Liu et al., 2006; Meng et al., 2005a). Core binding factor alpha1 (Cbfa1)

is a member of the runt family of transcription factors, which appears to play a pivotal

role in regulating the differentiation of osteoblastic precursors and the activity of mature

osteoblasts. Herba Epimedii could increase the expression of Cbfa1 mRNA in the bone of

ovariectomized rats depending on the dose. Furthermore, a high dose of Herba Epimedii

of 160 mg/kg administered for 12 weeks in vivo stimulated osteocalcin expression (Qian

et al., 2006).

4. Icariin applications in bone tissue engineering

The applications of icariin in bone tissue engineering are summarized in Table 2. In order

to enhance bone formation for the repair of bone defects, icariin, was loaded into porous

beta-tricalcium phosphate ceramic (ICA/beta-TCP) disks (Zhang et al., 2011a). It was

revealed that loading icariin in Ica/beta-TCP disks hardly affected the attachment and

morphology of rat osteoblast-like (Ros17/28) cells, supporting the proliferation and

differentiation of the cells at a higher level than the porous beta-TCP ceramic (beta-PTCP)

disks. After intramuscular implantation in the back of rats for three months, no obvious

osteogenic evidence was detected in beta-PTCP disks, but new bone formation was


145

observed in ICA/beta-TCP disks. These results indeed prove a potential of osteoinductive

property of icariin.

More and more studies reported the application of icariin combined with calcium

phosphate biomaterials. Calcium phosphate cement (CPC) loaded with Icariin filled in

the mouse calvarial bone defect induced significant new bone formation and increased

bone thickness (Zhao et al., 2010). Obvious blood vessel formation was also observed in

the icariin induced new bone in the calvarial bone defect. Moreover, by the thorough

mixing of icariin and chitosan/hydroxyapatite (ICA-CS/HA) using a freeze-drying

technique, a new bone repair scaffold was generated (Wu et al., 2009b). The results

showed that ICA-CS/HA had favorable cell compatibility and promoted osteogenic

differentiation of human bone marrow stem cells (hBMSCs). The controlled release of

icariin was satisfactory and the release retained after 90 days in vitro. Most interestingly,

ICA-CS/HA scaffolds showed favorable osteoconduction and osteoinduction in vivo.

They could fill bone segment defects and stimulate new born bone tissues formation at

early stage. Recently, another icariin-loaded chitosan/nano-sized hydroxyapatite system

was developed, which also controls the release kinetics of icariin to enhance bone repair

(Fan et al., 2012). The in vitro bioactivity assay revealed that the loaded icariin was

biologically active.

Due to the development of the carrier as mentioned above, icariin administered

locally can be more efficient for the local bone repair than a systemic administration. For

example, the gastrointestine may reduce the therapeutic effect of icariin given orally.

Therefore, the use of icariin for bone tissue engineering should concentrate on

administration locally rather than systemically.

5. Underlying mechanisms of icariin for bone regeneration

5.1 Anti-osteoporosis

Icariin has a definite anti-osteoporotic effect which is similar to estrogen and it is

especially effective for the prevention of bone fractures induced by an estrogen

deficiency (Nian et al., 2009; Liu et al., 2012). The anabolic effects of icariin in bone

possibly result from activating the estrogen receptor in a ligand-independent manner.

Research delineates the mechanism by which icariin prevented bone loss after

ovariectomy. Icariin suppressed the loss of bone mass and increased the strength in distal

femur and the mRNA expression ratio of OPG/RANKL in tibia (Mok et al., 2010). Oral

administration of icariin could promote bone formation during mandibular distraction

osteogenesis and might be a promising method for shortening the course of distraction

osteogenesis (Wei et al., 2011). OVX rats treated orally with icariin could improve the

degree of bone mineralization and bone strength and also prevent the suppression of

serum calcium phosphorus and 17β-oestradiol (Nian et al., 2009). The oral administration

of icariin, limited the metabolism of the medicine due to the gastrointestine. Icariin

propylene glycol-liposome suspension (ICA-PG-liposomes) injected intraperitoneally in

mice changed the pharmacokinetic behavior (Yang et al., 2012). With improved

pharmacokinetics, ICA-PG-liposomes might be developed as promising carriers for

icariin injection. Consequently, the use of icariin locally should be considered for future

clinical applications.

Chapter 9

146

Table 2. Icariin in bone tissue engineering

Experiments Main effects Carrier for loading

icariin References

In vivo Rabbits

BMD↑ Chitosan/hydroxyapatite Wu et al., 2009b

Cartilage formation↑ Cell-hydrogel constructs Li et al., 2012

BMD↑; Volumes of new bone↑;

TBA↑; Trabecular separation↓

- Wei et al., 2011

Rats Bone formation↑ Porous β-TCP ceramic Zhang et al., 2011a

BMD↑; BR↓; Biomechanical strength↑; Serum estrogen,

calcium and phosphorus↑; Root

resorption index↓; Collagen↑;

Osteoclast number and activity↓;

OCN↑; OPG/RANKL↑; Cbfa1↑;

Osterix↑

- Xue et al., 2012a; Xue et al., 2012b;

Wang et al., 2012;

Liu et al., 2012;

Bian et al., 2012;

Nian et al., 2009;

Qin et al., 2008

Mice Bone formation↑;

Bone thickness↑

Calcium phosphate

cement

Zhao et al., 2010

TBA↑; OPG/RANKL↑ - Zheng et al., 2012;

Mok et al., 2010

In vitro BMSCs ALP↑; Mineralized nodules↑;

Proliferation↑

Chitosan/nano-size

hydroxyapatite

Fan et al., 2012

ALP↑; Proliferation↑ Chitosan/hydroxyapatite Wu et al., 2009b

Mineralized nodules↑;

Proliferation↑; Differeatition↑;

Calcium deposition↑; ALP↑; OCN↑; OPN↑; Bone sialoprotein↑;

TGF-β1↑; IGF-I↑; Cbfa1↑;

Collagen I↑

- Fan et al., 2011;

Chen et al., 2007b;

Chen et al., 2005; Bian et al., 2012

Osteoblasts Proliferation↑ PHBV coatings Dai et al., 2011

ALP↑; Runx2↑; BSP↑; OCN↑;

Mineralization↑

Calcium phosphate

cement

Zhao et al., 2010

Proliferation↑;

Mineralization↑;

Osteoblast colonies↑;

Cell viability↑;

Calcified nodules↑;

ALP↑; Cbfa1↑;

BSP↑; BMP-2↑;

OPG↑;

OPG/RANKL↑;

RANKL↑;

Smad4↑;

NO↑; Collagen I↑;

OCN↑; Osterix↑;

OPN↑; ERK1/2↓; IκBα↓; p38↑

- Zhang et al., 2011a;

Zheng et al., 2012;

Mok et al., 2010;

Qin et al., 2008;

Liang et al., 2012;

Cao et al., 2012;

Zhang et al., 2011b; Ma et al., 2011;

Hsieh et al., 2011;

Hsieh et al., 2010;

Zhao et al., 2008;

Zhang et al., 2008b;

Zhang et al., 2008c;

Yin et al., 2007;

Xiao et al., 2005; Meng et al., 2005a;

Meng et al., 2005b;

Huang et al., 2007a;

Yang et al., 2013


147

Osteoclasts Apoptosis and cell cycle arrest ↑;

Osteoclastogenesis↓; Pit areas↓;

Superoxide anion↓;

TRAP↓; MMP-9↓; RANKL↓; OPG↑; IL-6↓; TNF-α↓; COX-2↓;

PGE2↓; HIF-1α↓; p38↓; JNK↓

- Zhang et al., 2012b;

Xue et al., 2012a;

Hsieh et al., 2011;

Huang et al., 2007a; Huang et al., 2007b;

Qin et al., 2008;

Chen et al., 2007a

Chondrocytes Aggrecan↑; Sox9↑; Collagen II↑;

GAGs↑

Cell-hydrogel constructs Li et al., 2012

Viability↑; Extracellular matrix↑; NO↑; MMP-1,3,13↓; COX-2↓;

iNOS↓; Aggrecan↑; Sox9↑;

Collagen II↑; GAGs↑

- Zhang et al., 2012a; Liu et al., 2010a;

5.2 Osteogenesis

The investigation of icariin on rat bone marrow stroma cells revealed an enhancement of

the osteogenic differentiation of these cells. A higher concentration of icariin in the

extract caused more mineralized bone nodules and higher levels of calcium deposition.

The gene expression involved in osteogenesis was also improved, including alkaline

phosphatase, bone matrix protein (osteocalcin, osteopontin, bone sialoprotein) and

cytokines (TGF-β1 and IGF-I) (Chen et al., 2007b). The effect of icariin on the

proliferation of human marrow stroma cells was found to be dependent on the dose and it

could also enhance the osteogenic differentiation of these cells in a suitable range of

concentrations (Fan et al., 2011). Icariin may strengthen the bone by enhancing the

osteogenic differentiation of bone marrow stroma cells, which partially explains the

anti-osteoporotic action of the Epimedium herb.

When icariin was added to osteoblasts, it promoted the proliferation of human

osteoblast and MC3T3-E1 cell lines (Guo et al., 2011; Cao et al., 2012). However, a

certain concentration of icariin showed no effect on the proliferation of rat osteoblasts

(He et al., 2009). Osteoprotegerin (OPG) plays an essential role in beneficial effects of

icariin on bone (Zheng et al., 2012). It was also reported that icariin significantly

promoted the expression of type I collagen and osteopontin (OPN) mRNA in rat

osteoblasts, and the expression was strengthened gradually with increasing concentration

of Icariin (Xiao et al., 2005). Icariin with final concentration of 1 x 10-5

mol/L, which

was the best concentration, significantly enhanced the osteogenic differentiation and

maturation of rat osteoblasts. It improved significantly the secretion of collagen I,

CFU-F(ALP) amounts and mineralized nodules and it also enhanced the mRNA level of

Cbfa1 and Osterix (Zhai et al., 2011; Ming et al., 2011). Furthermore, the Cbfa1, BMP2,

BMP4 and mRNA were significantly up-regulated after icariin treatment (He et al., 2009).

It was suggested that icariin exerts its potent osteogenic effect through the induction of

Cbfa1 expression, the production of BMP-4 and the activation of BMP signaling (Zhao et

al., 2008). The osteogenic effect was inhibited by the introduction of Smad6 or

dominant-negative Cbfa1, as well as Noggin treatment. It was demonstrated that icariin is

a bone anabolic agent that may exert its osteogenic effects through the induction of

BMP-2 and nitric oxide (NO) synthesis, subsequently regulating Cbfa1/Runx2, OPG, and

RANKL gene expressions (Fig. 3) (Hsieh et al., 2010). NO regulates the Cbfa1/Runx2

gene expression, and these effects may contribute to the induction of osteoblasts

proliferation and differentiation. Meanwhile BMP-2/Smad suppresses capsase-3 activities

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148

and thus inhibits apoptosis of osteobalsts and hence improves the survival of osteoblasts.

In a recent study, icariin up-regulated the expression of BMP-2, Smad4, Cbfa1/Runx2,

OPG, RANKL and the OPG/RANKL ratio, indicating that icariin can modulate the

process of bone formation via the BMP-2/Smad4 signal transduction pathway in human

osteoblastic cell line (Liang et al., 2012).

Figure 3. Molecular mechanism of the anabolic effect of icariin on osteoblasts (Hsieh et al., 2010).

5.3 Anti-osteoclastogenesis

Icariin inhibited osteoclastic differentiation in both osteoblast-preosteoclast co-culture

and osteoclast progenitor cell culture, and reduced the motility and bone resorption

activity of isolated osteoclasts (Huang et al., 2007a). It can be concluded that icariin has

the ability to inhibit the formation and bone resorption activity of osteoclasts (Chen et al.,

2007a). This in turn, supports the use icariin as an effective component for strengthening

bone. In a recent study, icariin decreased osteoclast numbers and activity levels, and

increased OPG/RANKL expression ratios, evoking a reparative effect on rapid palatal

expansion induced root resorption in rats (Wang et al., 2012). The detail molecular

mechanisms of icariin on anti-osteoclastogenesis were further examined (Hsieh et al.,

2011). It was demonstrated that a low dose of icariin inhibited LPS-induced

osteoclastogenesis without losing cell viability. Icariin can also inhibit LPS-induced

pro-inflammatory cytokines synthesis and scavenge LPS-induced RANKL up-regulation

and OPG down-regulation. Icariin decreased LPS-mediated prostaglandin E2 (PGE2)

production by inhibiting the cyclooxygenase-2 (COX-2) synthesis of osteoblasts and

osteoclasts. In osteoclasts, icariin suppressed LPS-mediated activation of the IκB, Jun

N-terminal kinase (JNK), extracellular regulated protein kinases (ERK1/2), p38, and


149

Hypoxia-inducible factor 1α (HIF-1α) pathways. While in osteoblasts, only IκB and

ERK1/2 pathways were involved. It can be concluded that Icariin inhibited LPS-induced

osteoclastogenesis by suppressing the activation of the p38 and JNK pathway (Fig. 4).

Figure 4. Molecular mechanism of icariin on the LPS-induced osteoclastogensis (Hsieh et al.,

2011).

5.4 Chondrogenesis

Icariin is a safe anabolic agent for chondrogenesis (Liu et al., 2010a). When rabbit

chondrocytes isolated from articular cartilage were cultured in vitro with different

concentrations of icariin, the higher concentration of icariin produced more extracellular

matrix synthesis and expression of chondrogenesis genes of chondrocytes (Zhang et al.,

2012a). The effect of icariin on the synthesis of glycosaminoglycans (GAGs) and

collagen of chondrocytes, and its potent chondrogenic effect, might be due to its ability to

up-regulate the expression of aggrecan, collagen II and Sox9 genes and to down-regulate

the expression of the collagen I gene of chondrocytes (Li et al., 2012). It also improves

the efficiency of restoring of supercritical-sized osteochondral defects in adult rabbit

model, and enhances the integration of newly formed cartilage with subchondral bone (Li

et al., 2012). These preliminary studies imply that icariin might be an effective accelerant

for chondrogenesis and a substitute for the use of some growth factors. The biomaterials

loaded with icariin might have a potential in bone and cartilage tissue engineering.

It is known that there are two mechanisms for bone formation. They are

intramembranous, which is direct bone formation, and endochondral ossification which is

indirect bone formation on a cartilage intermediate. (Einhorn TA, 1998). The potential

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150

different mechanisms between chondrogenic and osteogenic differentiation are associated

with two transcription factors, Sox9 and Cbfa1. The transcription factor Sox9 acts during

early chondrogenic differentiation (Bi et al., 1999), while Cbfa1 is essential for osteoblast

differentiation (Qian et al., 2006). Icariin can up-regulate the expression of Sox9 and

Cbfa1 in controlling osteogenesis and chondrogenesis. However, more mechanisms need

to be investigated in detail.

5.5 Angiogenesis

Vascularization is considered to be a crucial step in bone formation (Wernike et al., 2010).

Icariin stimulated in vitro endothelial cell proliferation, migration, and tubulogenesis, as

well as increasing in vivo angiogenesis (Chung et al., 2008). It was shown that Icariin has

the protective effect on injured vascular endothelial cells, which may be related to its

anti-apoptosis effect (Ji et al., 2005, Wang and Huang 2005). Icariin increases the

endothelial nitric oxide synthase (eNOS) expression through activating the EGF-EGFR

pathway in porcine aorta endothelial cells, by which the endothelial cell function could

be regulated (Liu et al., 2011). Moreover, icariin activated the angiogenic signal

modulators, ERK, phosphatidylinositol 3-kinase (PI3K), Akt, and eNOS, and increased

NO production, without affecting the expression of vascular endothelial growth factor.

This indicates that icariin may stimulate angiogenesis directly (Xu and Huang 2007,

Chung et al., 2008). Therefore, it should be noted that Icariin stimulated angiogenesis by

activating the MEK/ERK- and PI3K/Akt/eNOS-dependent signal pathways and it may

also have a potential as a drug in angiogenic therapy (Koizumi et al., 2010, Chung et al.

2008).

5.6 Anti-inflammatory

Anti-inflammation plays an important role in bone healing. For example, the treatment of

bone defects in peri-implantitis in dentistry particularly needs anti-inflammation (Park

2011). Icariin has displayed its anti-inflammatory potential (Wu et al., 2011). The partial

mechanism could be the multiple link intervention on pro-inflammatory cytokines

(TNF-α, IL-6), inflammatory mediators (NO) and adhesion molecules (CD11b) (Wu et

al., 2009a). Research on the anti-inflammatory effects of icariin on LPS-induced acute

inflammatory and its molecular mechanism, suggests that activation of the PI3K/Akt

pathway and the inhibition of NF-kappaB are involved in the protective effects of icariin

on lipopolysaccharide (LPS)-induced acute inflammatory responses (Xu et al., 2010).

Icariin may exert its protective effects through the inhibition of nitric oxide and matrix

metalloproteinase (MMP) synthesis, and it may then reduce the destruction of the

extracellular matrix (Liu et al., 2010a). Recently, researchers have found an

anti-inflammatory property of a novel derivative of icariin, 3, 5,

7-Trihydroxy-4'-methoxy-8-(3-hydroxy-3-methylbutyl)-flavone (ICT) (Wu et al., 2012).

It was reported that Icariin and ICT exert anti-inflammatory and anti-tumor effects, and

modulate myeloid derived suppressive cell (MDSC) functions (Zhou et al., 2011). The

anti-inflammatory effects of ICT were mediated, at least partially, via inhibition of the

CD14/TLR4 signaling pathway. ICT reduced NO and PGE2 levels by inhibiting

inducible NO synthase and cyclooxygenase-2 protein expression (Wu et al., 2011). This

icariin derivative inhibits tumor necrosis factor-alpha (TNF-α) production, inducible

nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression, and


151

protein expression in LPS stimulated macrophages. Furthermore, ICT suppresses the

activation of mitogen-activated protein kinase and inhibits translocation of nuclear factor

(NF)-kappaB p65 to the nucleus through decreasing the phosphorylation of IkappaBalpha

(Chen et al., 2010). As a result of all these properties, icariin and its derivative can be

considered as a potential drug for inflammatory diseases.

6. Toxicity of icariin

There was no cytotoxicity toward hBMSCs when the concentration of icariin was smaller

than 10−6

M, whereas icariin can limit the cell viability when the concentration was larger

than 10-5

M (Fan et al., 2011). The cytotoxicity test of icariin on MC3T3-E1 cells (a

pre-osteoblastic cell line) revealed that the cell viabilities varied from 88% to 98% on

both days 1 and 3 after treating with different concentrations of icariin (range from

10-10

M to 10-5

M) for 72 hours (Zhao et al., 2008). Icariin at a concentration of 5×10-5

M

strongly inhibited the proliferation of osteoblast-like (Ros17/28) cells (Zhang et al.,

2011a). However, many studies demonstrated that icariin with concentration of 10-5

M

had positive effect on the proliferation of UMR106 cell and human osteoblast (Meng et

al., 2005a; Huang et al., 2007a; Yin et al., 2005). Therefore, the optimal concentration of

icariin with low cytotoxicity toward osteoblasts was equal to or less than 10-5

M (6.8

μg/ml) (Zhang et al., 2011a). Additionally, more than 90% of murine macrophages

(ANA-1) can survive at concentrations up to 80 μg/ml icariin (Li et al., 2011). In general,

icariin is safe and non-toxic at low doses (Wu et al., 2009b; Zhao et al., 2010). At doses

up to 120 mg/kg in rats given orally administration, icariin has low toxicity, but without

overt toxic effects (Luo et al., 2007)

7. Concluding remarks and perspectives

It is well known that BMPs induce a sequential cascade of events leading to

chondrogenesis, osteogenesis, angiogenesis and the controlled synthesis of extracellular

matrix (Kang et al., 2004). BMP-2 and BMP-7 are the most extensively evaluated BMPs

with a very high price (Wang et al., 2011). Although BMPs have an outstanding

performance in bone formation, they also could result in some cases in negative effects

(Kao et al., 2012). Research has developed a variety of methods in bone tissue

engineering to reduce the use of BMPs and to improve the osteoinductive effects of

BMPs by a slow delivery (Liu et al., 2010b; Ruhe et al., 2005). However, one of the

simplest ways could be to search for an effective and low cost substitute for the

expensive BMPs. The perspectives discussed herein demonstrate the importance of

exploiting an inexpensive osteoinductive drug.

More and more researches show that icariin has an osteoinductive potential, due to its

properties of inducing osteogenesis, chondrogenesis and angiogenesis. The multiple

function of icariin, especially the induction of osteogenesis, is remarkable. The loading of

icariin in calcium phosphate biomaterials provides a good alternative to for delivering

icariin locally for bone repair, since the calcium phosphate materials have been used as

osteoconductive scaffolds. It has been known that the local use of icariin demonstrated

positive effects in bone formation at an early stage (Wu et al., 2009b). Several studies

have tried to clarify the molecular mechanisms underlying the osteogenic effects. In

summary, icariin may exert its osteogenic effects through the induction of BMP-2 and

NO synthesis and the BMP-2/Smad4 signal transduction pathway, by up-regulating the

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expression of BMP-2, BMP-4, Smad4, Cbfa1/Runx2, OPG, RANKL and the

OPG/RANKL ratio (Hsieh et al., 2010; Liang et al., 2012). Icariin can inhibit

LPS-induced osteoclastogenesis by suppressing the activation of the p38 and JNK

pathway (Hsieh et al., 2011), which in turn contributes to strengthening the bone. The

positive effects of icariin on a potent chondrogenic effect might be the up-regulation of

the expression of aggrecan, collagen II and Sox9 genes and down-regulation the

expression of the collagen I gene of chondrocytes (Zhang et al., 2012a). However, the

more detailed osteoinductive mechanisms and the clinical applications of icariin need to

be investigated further.

Compared with BMPs, icariin is cheaper and has low adverse effects (Zhao et al.,

2010; Wu et al., 2009b). The extremely low cost and the high abundance of icariin and its

excellent function for bone regeneration make it very appealing for clinical applications.

Therefore, it could be candidate for an assistant of BMPs or as a substitution.

Nevertheless, the effects of local use of icariin still need to be continually investigated

and there is also a need for an appropriate carrier for the most effective delivery.

According to the current studies and knowledge, it can be concluded that icariin can be a

potential osteoinductive agent. We would like to prove that icariin indeed has a potential

for bone tissue engineering. Several projects are running in our lab, both in vitro and in

vivo. One of our studies was to use icariin that was incorporated into a biomimetic

calcium phosphate bone substitute for the repair of critical-sized bone defects in the rat

calvaria. On the whole, the developing techniques give us the confidence to believe that

icariin might have a very bright future in bone tissue engineering.

Acknowledgments

We would like to thank Prof. Dr. Tony Hearn for his scientific input and English editing

as a native speaker for this publication.

Conflict of Interest

The authors declare that there are no conflicts of interest.


153

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127. Zhao BH, Katagiri T, Toyoda H, et al. 2006. Heparin potentiates the in vivo ectopic bone

formation induced by bone morphogenetic protein-2. Journal of Biological Chemistry 281:

23246-23253.

128. Zhao J, Ohba S, Komiyama Y, Shinkai M, Chung UI, Nagamune T. 2010. Icariin: a potential

osteoinductive compound for bone tissue engineering. Tissue engineering. Part A 16:

233-243.

129. Zhao J, Ohba S, Shinkai M, Chung UI, Nagamune T. 2008. Icariin induces osteogenic

differentiation in vitro in a BMP- and Runx2-dependent manner. Biochemical and

Biophysical Research Communications 369: 444-448.

130. Zheng D, Peng S, Yang SH, et al. 2012. The beneficial effect of Icariin on bone is diminished

in osteoprotegerin-deficient mice. Bone 51: 85-92.

131. Zhou J, Wu J, Chen X, et al. 2011. Icariin and its derivative, ICT, exert anti-inflammatory,

anti-tumor effects, and modulate myeloid derived suppressive cells (MDSCs) functions.

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132. Zhu HM, Qin L, Garnero P, et al. 2012. The first multicenter and randomized clinical trial of

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Osteoporosis and the National Osteoporosis Foundation of the USA 23: 1317-1327.

161

Chapter 10

General discussion

Chapter 10

162

GENERAL DISCUSSION

The preparation procedure of biomimetic calcium phosphate material

In this thesis (Chapters 2-4), a biomimetic calcium phosphate material (BioCaP) was

developed as a biodegradable bone substitute which is based on the protocol of

biomimetic calcium phosphate coating. This is a breakthrough in modifying the

biomimetic coating approach. BioCaP is prepared in a biomimetic environment which

can retain the bioactivity of growth factors [1, 2]. This biomimetic environment [(200

mM HCl, 20 mM CaCl2·2H2O, 680 mM NaCl, and 10 mM Na2HPO4) buffered by TRIS

(250 mM) to a pH of 7.4] is the key point in the preparation of BioCaP. It was found that

BioCaP has two phases of precipitations with different crystalline morphologies during

the preparation in Chapter 3. It was shown that BioCaP is a compound including different

crystalline structures of calcium phosphate. The biomimetic preparation methods resulted

in the bone-like mechanical strength (enough hardness) of BioCaP, whereas other

calcium phosphate biomaterials need sintering at high temperature to achieve enough

hardness [3]. More importantly, the use of BioCaP can simply and slowly deliver

proteins/drug without requiring other materials such as polymers and chitosan.

During the preparation, the sterility of biomedical materials is very important for

clinical trials. Accordingly, we took strict measures to guarantee the sterility of our

biomaterials. The whole processing of this material, including vacuum filtering, was

carried in a biological safety cabinet. For sterile vacuum filtering, we used the sterile

“Bottle Top Filter”. Therefore, the whole process was performed under sterile condition.

In our cell experiments we never encountered the presence of bacteria in the culture

media. Consequently, our in vitro as well as in vivo results in Chapter 2-4 indicated the

sterility of BioCaP materials.

The characteristics of BioCaP

In BioCaP, protein and calcium phosphate were precipitated together to form BioCaP

granules in which a depot of protein was incorporated in the center of the granules as an

internal depot. Next, protein and calcium phosphate were co-precipitated onto the surface

of these granules, thus creating a surface coated depot. This dual system provides an ideal

vehicle for the delivery of different protein/drugs in two phases, an initial slow delivery

phase (surface coated depot) and a delayed phase (internal depot). This thesis evaluated

the BioCaP with incorporated bone morphogenetic protein-2 (BMP-2). The two delivery

modes of BMP-2 were studied separately in Chapters 2-4. The simultaneous delivery of

two or multiple growth factors in vivo is needed to be investigated further in the future.

Since bone regeneration is a coordinated cascade of events regulated by several growth

factors, the local sequential delivery of vascular endothelial growth factor (VEGF) and

BMP-2 could enhance bone formation compared with BMP-2 alone [4]. Therefore, it will

be interesting to study the dual release of BMP-2 and VEGF from BioCaP.

General discussion

163

The work in this thesis brought us the following highlights of BioCaP:

Enough hardness / mechanical stability / easy of handling

Good biodegradability

A slow release system for the delivery of single or multiple proteins/drugs

Osteoconductivity

Osteoinductive potential

Micro-porosity

These characteristics make BioCaP an attractive or idea bone substitute. However, the

limitation is that it has no macro-porosity which can enable cell ingrowth into the

composite. The use of granular form of BioCaP may overcome this disadvantage, since

the space between the granules could be equal to the macro-porosity.

Osteoinductivity of BioCaP

The osteoinductivity of BioCaP was mainly conferred by using osteogenic growth factors

such as bone morphogenetic protein-2 (BMP-2). The findings in Chapters 2-4 imply that

the in vivo BMP-2 release from BioCaP may be in a slow release pattern, since the

adsorption way always has a burst release which results in poor osteoinduction [5, 6].

However, the in vivo BMP-2 release kinetic needs to be investigated further. In previous

studies, growth factors were co-precipitated into the latticework of crystalline calcium

phosphate coating and then were shown to be released locally in a slow manner [2, 5, 7].

The slow release of BMP-2 can enhance the osteoinductivity of BioCaP [1]. In Chapter 4,

we implanted BioCaP with the two delivery modes of BMP-2 into the bone defects in

sheep. The findings demonstrated that the therapeutic effect of this material was excellent,

which was significantly better than the deproteinized bovine bone (DBB, Bio-Oss®) and

was comparable with the autologous bone. All the finding indicated that osteoinducive

BioCaP by carrying BMP-2 can be a good alternative to autologous bone (gold standard).

Osteoinductive promoter

Biomimetic BMP2-coprecipitated calcium phosphate (BMP2-cop.BioCaP) particles were

developed and evaluated in Chapters 4 and 5. It was shown that this material serves as a

biodegradable and efficient “osteoinducer” for DBB which need effective osteoinduction

[8, 9]. The directly mixing BMP2-cop.BioCaP with other bone substitutes is one of the

simplest operations for clinicians. However, this calcium phosphate material may not be

used as an independent bone substitute, since its mechanical property still needs to be

improved and the preparation time also needs to be shortened.

Multinucleated giant cells

The cells involved in the degradation of calcium phosphate materials are mainly

multinucleated giant cells (osteoclasts or foreign body giant cells), macrophages, and

monocytes [7, 10]. It is important to understand how and to what extent these cells might

influence the protein release from a drug-delivery system [11, 12]. It has been

demonstrated that the monocytes/macrophages has no significant effects on the protein

release from calcium phosphate ceramics, but osteoclasts do [11]. When the resorbing

cells digest BioCaP, the protein release from BioCaP could be elevated. The findings in

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164

Chapter 2 indicate osteoclasts are able to increase the protein release from BioCaP.

However, when BioCaP delivers BMP-2, this growth factor could also influence the

proliferation and differentiation of these cells. This needs further investigated.

When biomaterials were implanted into body, multinucleated giant cells (MGCs)

formed on biomaterials could be osteoclasts or foreign body giant cells (FBGCs).

However, FBGCs is the end stage of the inflammatory responses following the

implantation of biomaterial. FBGCs can also release inflammatory cytokines which

stimulate circulating stem cells to become osteoprogenitors [10, 13]. Osteoclast has

become the common term to denote any cell that has a unique function to break down

mineralized matrices [14, 15]. However, it is difficult to precisely identify FBGCs and

osteoclasts, since both of them are tartrate resistant acid phosphatase (TRAP) positive.

This is very interesting to investigate further.

Icariin: an osteoinductive traditional Chinese medicine

Traditional Chinese Medicines (TCMs) have been recommended for bone regeneration

and repair for thousands of years. Icariin, a typical flavonol glycoside, is considered to be

the main active ingredient of the Herba Epimedii from which icariin has been

successfully extracted. Because of its osteoinductive potential and the low price, it can be

a very attractive candidate as a substitute of BMP-2 (Chapter 8). We thought about that

icariin can be slowly delivered by BioCaP, since icariin has been incorporated into the

interior of BioCaP. In our on-going study, we are going to investigate the therapeutic

effects of icariin-incorporated BioCaP with or without BMP-2 in the treatment of

critical-sized bone defects in rats.

Micro-CT and histological analysis

Histological analyses are derived from stereological analysis of a few 2D sections,

usually assuming that the underlying structure is plate-like [16]. The inclusion of dental

implants may further lessen the histomorphometric information of bone because only one

central section of each implant can be used for analysis [17]. In contrast, micro-computed

tomography (micro-CT) can directly measure bone micro-architectures independent of

stereological models [18]. The high positive correlation (Pearson coefficient=0.992,

p<0.001) between these two parameters was found (Chapter 9). This validated the

reliability of micro-CT. This finding is consistent with previous studies in animal [19]

and human specimens [20]. However, when new bone was deposited on bone substitute,

single threshold setting cannot distinguish bone and materials. The micro-CT method in

Chapter 3 successfully separated BioCaP and new bone by using an “onion-peeling”

algorithm (Scanco Medical AG) and specific threshold settings. The results were

confirmed by the histological analysis in Chapter 2. However, the correlation between the

two parameters needs to be evaluated further.

General discussion

165

CONCLUSION

The main scope of the work conducted in this thesis was to develop osteoinductive

bone substitutes.

1. It was shown that BioCaP with an internal or surface coated depot of protein has the

capacity to maintain a slow and sustained protein release. Both modes of delivering

of BMP-2 with the use of BioCaP make these granules efficient osteoinductive

compounds. Benefiting from these two delivery modes, BioCaP with BMP-2 can be

a promising alternative to the autografts. The findings also showed that BioCaP has

good biocompatibility and degradability.

2. The findings indicate BMP2-cop.BioCaP can serve as a highly efficient

osteoinducer for inducing bone formation with DBB and for suppressing the

foreign-body reaction in a critical-sized bone defect. BMP2-cop.BioCaP also

showed good biocompatibility and degradability. This material has a very promising

clinical potential to enhance significantly the therapeutic effects of bone substitutes

for filling bone defects.

3. Our findings show the excellent biocompatibility and osteoconductivity of DBB.

Incorporating BMP-2 into the calcium phosphate coating of DBB induced strong

bone formation around DBB in critical-sized bone defects. This functionalization

approach renders DBB efficiently osteoinductive and could greatly enhance the

clinical potential of DBB to be an alternative to bone autografts in the repair of

large or critical-sized bone defects.

4. Low-dose BMP2/7 heterodimer facilitated more rapid bone regeneration in better

quality in peri-implant bone defects than BMP2 and BMP7 homodimers. Micro-CT

results were confirmed by histological analysis.

5. The osteoinductive potential and the low price of icariin make it a very attractive

candidate as a substitute of osteoinductive protein − BMPs, or as a promoter for

enhancing the therapeutic effects of BMPs. However, the effectiveness of the local

delivery of icariin needs to be investigated further.

FUTURE SCOPE

We are on the way to developing an ideal osteoinductive bone substitute. Osteoinductive

BioCaP with incorporated BMP-2 is expected to be evaluated in clinical trials in the

future. BioCaP granule might also be considered as a promising tool for the orderly

delivery of multiple therapeutic agents, such as antibiotics, osteogenic agents, and

anti-cancer drugs for different clinical applications.

Chapter 10

166

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8. Schwartz Z, Weesner T, van Dijk S, Cochran DL, Mellonig JT, Lohmann CH, et al. Ability of

deproteinized cancellous bovine bone to induce new bone formation. J Periodontol

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Immunol 2008;20:86-100.



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13. Le Nihouannen D, Saffarzadeh A, Gauthier O, Moreau F, Pilet P, Spaethe R, et al. Bone

tissue formation in sheep muscles induced by a biphasic calcium phosphate ceramic and

fibrin glue composite. J Mater Sci-Mater M 2008;19:667-75.

14. Basle MF, Chappard D, Grizon F, Filmon R, Delecrin J, Daculsi G, et al. Osteoclastic

resorption of Ca-P biomaterials implanted in rabbit bone. Calcif Tissue Int 1993;53:348-56.



16. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche H, Meunier PJ, et al. Bone

histomorphometry: standardization of nomenclature, symbols, and units. Report of the

ASBMR Histomorphometry Nomenclature Committee. 1987;2:595-610.

17. Sennerby L, Dasmah A, Larsson B, Iverhed M. Bone tissue responses to surface-modified

zirconia implants: A histomorphometric and removal torque study in the rabbit. 2005;7 Suppl

1:S13-20.

18. Bouxsein ML, Boyd SK, Christiansen BA, Guldberg RE, Jepsen KJ, Muller R. Guidelines for

assessment of bone microstructure in rodents using micro-computed tomography.

2010;25:1468-86.

19. Waarsing JH, Day JS, Weinans H. An improved segmentation method for in vivo microCT

imaging. 2004;19:1640-50.

20. Chappard D, Retailleau-Gaborit N, Legrand E, Basle MF, Audran M. Comparison insight

bone measurements by histomorphometry and microCT. 2005;20:1177-84.

167

Chapter 11

General summary

Algemene samenvatting

Chapter 11

168

GENERAL SUMMARY

In the treatment of bone defects an adequate volume of bone tissue is of paramount

importance to achieve bone regeneration. When the bone defects are too large to allow

self-healing or post-traumatic complications occur such as delayed union, non-union or

malunion, bone grafting is indicated in order regenerate the defect. Autografts (gold

standard), allografts, xenografts, and synthetic materials are available to assist in the

repair of bone defects. Synthetic calcium phosphate (CaP) biomaterials are widely used

in the regeneration of bone defects because of their chemical similarity to native bone

tissue. To achieve bone regeneration in large or critical sized bone defects, (i) osteogenic

cells (e.g. progenitor cells or osteoblasts); (ii) osteoinductive signals (growth factors); (iii)

a biocompatible, biodegradable and osteoconductive matrix (scaffold); and (iv) adequate

blood and nutrient supply are required. Bone grafts are often associated with the terms

biocompatibility, biodegradability, osteoconductivity and osteoinductivity.

This thesis discusses the research on a biomimetic calcium phosphate bone substitute

(BioCaP). For the first time we have developed a dual protein release system using this

material. In this system protein and calcium phosphate were precipitated together to form

BioCaP granules in which, in the center of the granules a depot of protein was

incorporated, a so called internal depot. Next, protein and calcium phosphate were

co-precipitated onto the surface of these granules, thus creating a surface coated depot.

This dual system provides an ideal model for delivery of different proteins/drugs in two

phases, an initial slow delivery phase (surface coated depot) and a delayed phase (internal

depot). By adopting this system, a single drug can be administered in a more consistent

manner or two different drugs can be administered simultaneously. Therefore, BioCaP

granules might be considered as a promising tool for the systematic delivery of multiple

therapeutic agents, such as antibiotics, osteogenic agents, and anti-cancer drugs for

different clinical applications. Moreover, we developed particles of biomimetic

BMP-2-coprecipitated calcium phosphate (BMP2-cop.BioCaP) as an osteoinductive

promoter for commercial bone substitutes such as deproteinized bovine bone (DBB). We

also functionalized DBB with a coating-incorporated depot of BMP-2 for the repair of

critical-sized bone defects. All the products developed and discussed in this thesis were

based on the principle of biomimetic calcium phosphate coating.

The general aim of this thesis includes 5 aspects:

1. To develop a biomimetic calcium phosphate (BioCaP) bone substitute as a dual

delivery model with two protein-delivery modes: one mode by which protein was

incorporated in the interior of BioCaP; and one by which protein was coated on the

outside of BioCaP. We hypothesize that using this model the release of the protein

can be sequential and slow, and that the two delivery modes of BMP-2 could

efficiently accelerate bone formation.

2. To develop particles of biomimetic BMP-2-coprecipitated calcium phosphate

(BMP2-cop.BioCaP). We hypothesize that these particles could serve as an

independent and biodegradable osteoinducer.

3. To evaluate the therapeutic effect of the deproteinized bovine bone functionalized

with coating-incorporated BMP-2 in the repair of critical-sized bone defect in

sheep.

General summary

169

4. To delineate the dynamic micro-architectures of bone induced by low-dose bone

morphogenetic protein (BMP)-2/7 heterodimer in peri-implant bone defects

compared to BMP2 and BMP7 homodimer.

5. To determine the present evidence of the osteoinductive potential of a Chinese

traditional medicine, icariin.

Cells responsible for bone resorption such as osteoclasts may accelerate the

degradation of bone substitutes and so increase the protein release. In Chapter 2, mouse

osteoclasts were used to test the cell-mediated protein release from BioCaP which was

produced by refining a well-established biomimetic protocol. It was shown that BioCaP

with the proteins retained in the internal and surface coatings resulted in a sustained

osteoclast-mediated release, while the adsorbed protein was rapidly released. This release

of the adsorbed protein was not affected by osteoclasts seeded on BioCaP. Next, granules

of BioCaP with an internal or a surface coated depot of BMP-2 were implanted

subcutaneously in rats. Histological analysis showed that the volume densities of bone,

bone marrow, and blood vessels were significantly higher in samples where BMP-2 was

incorporated internally or in the coating compared with granules with adsorbed growth

factor. In the latter samples fibrous capsular tissue proved to be significantly higher.

Osteoclast-like cells and the resorption lacunae were observed on BioCaP granules in

vivo. Different modes of incorporation of BMP-2 on and in BioCaP granules had a

beneficial effect on the formation of ectopic bone. This dual drug release system makes

the BioCaP granule a promising tool for delivering multiple therapeutic agents, such as

osteogenic agents, antibiotics, and anti-cancer drugs for different clinical applications.

In Chapter 3, the physicochemical properties of BioCaP were investigated. Two

phases of precipitation of BioCaP were observed by scanning electron microscopy.

BioCaP exhibited bone-like mechanical strength and the characteristics of

calcium-deficient apatite. The granules with internally- or coating-incorporated protein

exhibited a slow release in vitro (35 days). Human osteoclasts seeded on the granules

were shown to resorb the BioCaP. This finding is consistent with the results in the

previous study using mouse osteoclasts in Chapter 2. Micro-CT analysis using an

“onion-peeling” algorithm can distinguish between BioCaP and newly formed bone. In a

rat ectopic model Micro-CT results showed that significantly more bone formation was

present in the samples containing BioCaP with internally- or coating- incorporated

BMP-2 than those with adsorbed BMP-2. BioCaP with BMP-2 showed slower

degradation than that without BMP-2. These results confirmed the histological results in

Chapter 2.

In Chapter 4 we investigated the therapeutic effect of BioCaP with two delivery

modes of BMP-2 in the repair of a large cylindrical bone defects (Ø8×13mm) in sheep.

Both delivery modes of BMP-2 accelerated the bone formation within a period of 4

weeks. The internally-incorporated protein mode enhanced bone formation after 8 weeks,

showing to be more efficient than DBB. BioCaP with BMP-2 showed equal efficacy as

autologous bone in the bone defect repair at 8 weeks post-implantation. BioCaP with

BMP-2 showed significant degradation at both time points. Benefiting from these two

delivery modes, BioCaP might be a promising alternative to autografts.

Novel particles of biomimetic BMP2-coprecipitated calcium phosphate

(BMP2-cop.BioCaP) were developed to serve as an independent and biodegradable

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170

osteoinducer with the idea to induce bone formation more efficiently for

bone-defect-filling materials such as DBB. To prepare BMP2-cop.BioCaP, we alternately

layer-by-layer assembled amorphous and crystalline CaP triply to enable a “bamboolike”

growth of the particles. BMP2 was incorporated into the outermost layer of BioCaP. We

monitored the degradation, osteoinductivity, and foreign-body reaction of both

BMP2-cop.BioCaP and its combination with DBB in an ectopic site in rats. After 5

weeks, the BMP2-cop.BioCaP significantly induced new bone formation not only when

applied as a solitary product but also when mixed with DBB. Its osteoinductive

efficiency was 10-fold higher than the BioCaP with adsorbed BMP2. More than 90% of

BMP2-cop.BioCaP degraded. Moreover, BMP2-cop.BioCaP also significantly reduced

the host foreign-body reaction to DBB in comparison with the adsorbed BMP2. These

findings indicate a promising clinical potential for BMP2-cop.BioCaP in the repair of

large (critical)-sized bone defects (Chapter 5).

To enhance the therapeutic effect of DBB, we mixed DBB with BMP2-cop.BioCaP in

critical-sized bone defects in sheep. Histological results confirmed the excellent

biocompatibility and osteoconductivity of BMP2-cop.BioCaP and DBB. DBB mixed

with BMP2-cop.BioCaP displayed significantly more bone formation when compared to

DBB, and the induced bone formation was comparable with autologous bone at 8 weeks

post-implantation. At this time point, about 95% BMP2-cop.BioCaP was degraded. It was

shown that the BMP2-cop.BioCaP, as an osteoinductive promoter, has excellent

biocompatibility, biodegradability, osteoconductivity, and a strong capacity to induce

bone formation. It might be possible to substitute an autograft by DBB mixed with

BMP2-cop.BioCaP in the repair of critical-sized bone defects (Chapter 6).

In order to render DBB osteoinductive, BMP-2 has previously been incorporated into

a three dimensional reservoir (a biomimetic calcium phosphate coating) on DBB, which

effectively promoted the osteogenic response by the slow delivery of BMP-2 as described

in Chapter 7. The aim of this chapter was to investigate the therapeutic effectiveness of

such coatings on the DBB granules in repairing large cylindrical bone defects in sheep.

Histological results confirmed the excellent biocompatibility and osteoconductivity of the

coated DBB. Incorporating BMP-2 into the calcium phosphate coating of DBB induced

strong bone formation around DBB when repairing bone defects. 8 Weeks after

implantation, the volume of newly-formed bone around DBB that bore a

coating-incorporated depot of BMP-2 was comparable to that of autologous bone.

Multinucleated giant cells were observed in the resorption process around DBB, whereas

histomorphometric analysis revealed no significant degradation of the DBB.

More rapid repair of peri-implant bone defects has been pursued for years.

Heterodimeric BMPs exhibited several- or even multiple- effect than the respective

homodimers in inducing in vitro osteoblastogenesis. We administrated collagen sponges

with adsorbed low-dose (30 ng/mm3) BMP2/7 to treat the peri-implant defects in the

calvaria of minipig. After 6 weeks post-operation, BMP2/7 showed a significantly higher

relative bone volume and significantly lower structure mode index than BMP-2 and

BMP-7 respectively. The findings indicate that low-dose BMP2/7 heterodimer facilitated

more rapid bone regeneration in better quality in peri-implant bone defects than BMP-2

and BMP-7 homodimers (Chapter 8).

BMP-2 and BMP-7 are the most extensively evaluated BMPs and they are very costly.

Although BMPs have an outstanding performance in bone formation, in some cases when

General summary

171

used in a high concentration could also cause some negative effects. Research has

developed a variety of methods in bone tissue engineering to reduce the use of BMPs and

to improve the osteoinductive effects of BMPs by a slow delivery. Icariin, a typical

flavonol glycoside, is considered to be the main active ingredient of the Herba Epimedii

(a Traditional Chinese Medicine) from which icariin has been extracted. Most

interestingly, it has been reported that icariin can be delivered locally by biomaterials and

that it has an osteoinductive potential for bone tissue engineering. Therefore, we

reviewed the performance of icariin in bone tissue engineering and blended this

information with the current knowledge relevant to molecular mechanisms and signal

pathways. The osteoinductive potential of icariin could be attributed to its multiple

functions in the musculoskeletal system which is involved in the regulation of multiple

signaling pathways in anti-osteoporosis, osteogenesis, anti-osteoclastogenesis,

chondrogenesis, angiogenesis, and anti-inflammation. The osteoinductive potential and

the low price of icariin make it a very attractive candidate as a substitute of

osteoinductive proteins − BMPs, or as a promoter for enhancing the therapeutic effects of

BMPs (Chapter 9).


172

ALGEMENE SAMENVATTING

Bij de behandeling van botdefecten is een voldoende hoeveelheid botweefsel van belang

om botregeneratie te bereiken. In het geval van botdefecten van een dusdanige grootte dat

spontaan herstel niet optreedt, of in geval van post-traumatische complicaties als delayed

union, malunion en nonunion. is transplantatie van bot geïndiceerd om het defect te

regenereren. Autograft (de gouden standaard), allografts, xenografts en synthetische

materialen zijn hiervoor beschikbaar. Dankzij de chemische overeenkomst met menselijk

bot, worden synthetische calciumfosfaat (CaP) biomaterialen wijdverspreid gebruikt bij

de regeneratie van botdefecten. Om botregeneratie te bewerkstelligen in grote of

critical-size botdefecten zijn (i) osteogenetische cellen (progenitor cellen of

osteoblasten); (ii) osteoinductieve signalen (groeifactoren); (iii) een biocompatibele,

biologisch afbreekbare en osteoconductieve matrix (geraamte); en (iv) een voldoende

toevoer van blood en voedingsstoffen vereist. Bottransplantaten zijn veelal geassocieerd

met de termen biocompatibiliteit, biologische afbreekbaarheid, osteoconductiviteit en

osteoinductiviteit.

Dit proefschrift behandelt het onderzoek naar een biomimetisch calciumfosfaat

botsubstituut. Voor de eerste keer is dit materiaal gebruikt om een

dual-protein-release-systeem te ontwikkelen. In dit systeem, een zogenoemd intern depot

bestaande uit proteïnen, is gebruikt als basis om de proteïne en calciumfosfaat, samen

BioCaP granulaat vormend, op te laten neerslaan, wat tot incorporatie van het depot

leidt. Vervolgens is dit proces herhaalt maar met het hiervoor gevormde granulaat als

basis, leidend tot een depot met oppervlakte coating. Dit tweeledige systeem geeft een

ideaal model om proteïnen/stoffen in twee fases toe te dienen, een langzame initiële fase

(oppervlakte coating) en een vertraagde fase (intern depot). Door dit systeem te

gebruiken kan een enkele stof geleidelijker worden toegediend, of twee verschillende

stoffen tegelijkertijd. Hierom kan BioCaP granulaat gezien worden als een veelbelovend

systeem voor de systematische toediening van diverse therapeutische middelen zoals

antibiotica, osteogenetische middelen, en kanker medicatie voor diverse klinische

doeleinden. Daarbij hebben we biomimetische BMP-2-coprecipitated calciumfosfaat

(BMP2-cop.BioCaP) ontwikkeld als een osteoinductieve promotor voor commerciële

botsubstituten zoals deproteinized bovine bone (DBB). Tevens hebben we DBB

gefunctionaliseerd met een coating-incorporated depot van BMP-2 voor de regeneratie

van critical-sized botdefecten. Alle ontwikkelde en in dit proefschrift besproken

producten zijn gebaseerd op het principe van biomimetische calciumfosfaat coating.

Het doel van dit proefschrift bestaat uit 5 aspecten:

1. De ontwikkeling van een biomimetisch calciumfosfaat (BioCaP) botsubstituut

als een dual delivery model met twee proteïne afgifte methoden: een methode waarbij het

proteïne is geïncorporeerd in de kern van de BioCaP; en een methode waarbij het

proteïne als een coating aan de buitenzijde van de BioCaP was aangebracht. De

hypothese luidt dat het gebruik van dit model de afgifte van het proteïne geleidelijk en

langzaam is, en dat de twee methoden van BMP-2 toediening botvorming efficiënt kan

versnellen.

2. De ontwikkeling van biomimetische BMP-2-coprecipitated calciumfosfaat

(BMP2-cop.BioCaP) granules. De hypothese luidt dat deze granules kunnen dienen als


173

een op zichzelf staande en biologisch afbreekbare osteoinducer.

3. De evaluatie van het therapeutische effect van het deproteinized bovine bone

gefunctionaliseerd met coating-incorporated BMP-2 bij het herstel van critical-size

botdefecten in schapen.

4. Het in kaart brengen van de dynamische micro architectuur van bot

geïnduceerd door lage doses bone morphogenetic protein (BMP)-2/7 heterodimeer in peri

implantaire botdefecten in vergelijking met BMP2 en BMP7 homodimeer.

5. Het vaststellen van het reeds bestaande bewijs van het osteoinductieve

potentieel van een Chinees traditioneel medicijn, icariin.

De cellen die verantwoordelijk zijn voor bot resorptie, zoals osteoclasten, kunnen de

degeneratie van bot substituten versnellen en zo de proteïne afgifte vergroten. In

hoofdstuk twee zijn osteoclasten afkomstig van muizen gebruikt om de cell-mediated

protein release van BioCaP te testen, welke is geproduceerd door het verfijnen van een

goed gegrond biomimetisch protocol. Het is aangetoond dat BioCaP, met de proteïnen

vastgehouden als kern en als oppervlakte coating, resulteert in een stabiele

osteoclast-gemedieerde afgifte, terwijl de geadsobeerde proteïnen snel worden

afgegeven. Het loslaten van de geadsorbeerde proteïnen is niet beïnvloed door

osteoclasten op de BioCaP. Vervolgens zijn granules van BioCaP met een intern depot of

coating van BMP-2 subcutaan aangebracht in ratten. Histologische analyse toont aan dat

de volume dichtheid van bot, merg en bloedvaten significant hoger is in de samples

waarin BMP-2 intern of in de coating is geïncorporeerd in vergelijking met granules met

geadsorbeerde groei factoren. In verdere samples werd een fibreus kapsel significant

vaker aangetroffen. Osteoclast-achtige cellen en resorptielagunes zijn in vivo

aangetroffen op BioCaP granules. Verschillende methode van incorporatie van BMP-2 op

en in BioCaP granules hebben een positief effect op ectopische botformatie. Dit dual drug

delivery system maakt de BioCaP granule een veelbelovend middel om verschillende

therapeutische middelen, zoals osteogenetische middelen, antibiotica en kankermedicatie

voor diverse klinische doeleinden toe te dienen.

In hoofdstuk drie worden de fysiochemische eigenschappen van BioCap onderzocht.

Twee fasen van het neerslaan van BioCaP zijn geobserveerd met behulp van een scanning

electronen microscoop. BioCaP vertoont mechanische sterkte vergelijkbaar met bot en de

karakteristieken van calciumdeficiënt apatiet. De granules met interne of

coating-incorporated proteïnen laten een langzame afgifte zien in vitro (35 dagen). De

menselijke osteoclasten gezeten op de granules resorberen de BioCaP. De bevinding

komt overeen met de resultaten van een eerdere studie uit hoofdstuk twee waarbij

osteoclasten afkomstig van muizen zijn gebruikt. Micro-CT analyse waarbij gebruik is

gemaakt van een “union-peeling” algoritme kan onderscheid maken tussen BioCaP en

nieuw gevormd bot. Micro-CT resultaten uit een ectopisch rat model laten zien dat er

significant meer botformatie is in samples die BioCaP met intern of coating-incorporated

BMP-2 bevatten, in vergelijking met samples met geadsorbeerd BMP-2. BioCaP met

BMP-2 vertoont een tragere degeneratie dan BioCaP zonder BMP-2. Deze resultaten

bevestigen de histologische resultaten uit hoofdstuk 2.

In hoofdstuk vier onderzoeken we het therapeutisch effect van BioCaP met twee

afgifte methoden van BMP-2 bij de reparatie van grote cilindrische botdefecten

(Ø8×13mm) in schapen. Beide methoden versnellen de botformatie over een periode van

Chapter 11

174

vier weken. De intern geïncorporeerde proteïne methode versnelt botformatie over een

periode van acht weken, waarmee is aangetoond dat dit efficiënter is dan DBB. Acht

weken na implantatie vertoont BioCaP met BMP-2 een gelijke efficiëntie als autoloog bot

in de reparatie van het botdefect. BioCaP met BMP-2 vertoont significante degeneratie

op beide tijdstippen. Gezien het voordeel dat BioCaP heeft bij beide methoden, zou dit

een veelbelovend alternatief kunnen zijn voor autografts.

Nieuwe deeltjes van biomimetisch BMP2-coprecipitated calciumfosfaat

(BMP2-cop.BioCaP) zijn ontwikkeld om te dienen als een zelfstandig en biologisch

afbreekbare osteoinducer met het idee de efficiëntie van botformatie door botsubstituten

als DBB te vergroten. Om BMP2-cop.BioCaP te creëren hebben we om en om cq laag

voor laag amorf en gekristalliseerd calciumfosfaat in drievoud aangebracht om zo een

“bamboe-achtige” groeimethode te realiseren. BMP-2 is geïncorporeerd in de buitenste

laag van de BioCaP. De degeneratie, osteoinductiviteit en vreemd-lichaam-reactie is

gevolgd van zowel BMP2-cop.BioCaP als BMP2-cop.BioCaP gecombineerd met DBB in

ectopische locaties in ratten. Na vijf weken vertoont de BMP2-cop.BioCaP een

significante inductie van nieuwe botformatie, niet alleen als solitair product, maar ook

gemixt met DBB. De osteoinductieve efficiëntie is een tienvoud hoger dan BioCaP met

adsorbed BMP-2. Meer dan 90% van de BMP2-cop.BioCaP is gedegenereerd.

BMP2-cop.BioCaP zorgt tevens voor een reductie in de vreemd-lichaam reactie tegen

DBB in vergelijking met het geadsorbeerde BMP-2. Deze bevindingen indiceren een

veelbelovend klinisch potentieel voor BMP2-cop.BioCaP bij de reparatie van grote

(critical-sized) botdefecten (Hoofdstuk vijf).

Om het therapeutisch effect van DBB te vergroten hebben we DBB gemixt met

BMP2-cop.BioCaP in critical-sized botdefecten in schapen. Histologische resultaten

hebben de perfecte biocompatibiliteit en osteoconductiviteit van BMP2-cop.BioCaP en

DBB bevestigd. DBB gemixt met BMP2-cop.BioCaP laat een significant hogere

botformatie zien in vergelijking met DBB, en de geïnduceerde botformatie is

vergelijkbaar met autoloog bot acht weken na aanbrengen. Op dat moment is ongeveer

95% BMP2-cop.BioCaP gedegenereerd. Het is aangetoond dat BMP2-cop.BioCaP als

een osteoinductieve promotor een perfecte biocompatibiliteit, biologische

afbreekbaarheid en osteoconductiviteit heeft, evenals een grote capaciteit tot het

induceren van botformatie. Het is bij de reparatie van critical-sized botdefecten wellicht

mogelijk om een autograft te vervangen door een mix van BMP2-cop.BioCaP en DBB.

(Hoofdstuk zes).

Om DBB osteoinductief te maken is BMP-2 al eerder geïncorporeerd in een

driedimensionaal reservoir (een biomimetische calciumfosfaat coating) op DBB, wat de

osteogenetische respons van de langzame afgifte van BMP-2, zoals beschreven in

hoofdstuk zeven, effectief bevordert. Het doel van dit hoofdstuk is het onderzoeken van

de therapeutische effectiviteit van dit soort coating op de DBB granules bij de reparatie

van grote cilindrische bot defecten in schapen. Histologische resultaten bevestigen de

goede biocompatibiliteit en osteoconductiviteit van de gecoate DBB. Het incorporeren

van BMP-2 in de calciumfosfaatcoating van DBB induceert een duidelijke botformatie

rond DBB bij de reparatie van botdefecten. Acht weken na implantatie is het volume van

nieuw gevormd bot rond DBB dat een coating-incorporated depot van BMP-2 droeg,

vergelijkbaar met dat van autoloog bot. Multinucleaire reuscellen zijn aangetroffen in het

resorptieproces rond DBB, terwijl histomorphometrische analyse geen significante


175

afbraak van de DBB aantoont.

Een sneller herstel van peri implantaire botdefecten wordt al jaren nagestreefd.

Heterodimeric BMP’s bevatten verschillende – of zelfs meer – effecten dan de

respectievelijke homodimeren met betrekking tot induceren van in vitro

osteoblastogenese. We hebben sponsen aangebracht met geadsorbeerde BMP2/7 in lage

doses om de peri implantaire defecten in caviaschedels te behandelen. Zes weken post

operatief laat de BMP2/7 een significant hoger relatief bot volume zien en een significant

lagere structure mode index in vergelijking met BMP-2 en BMP-7 respectievelijk. Deze

bevindingen indiceren dat een lage dosis BMP2/7 heterodimeer een snellere bot

regeneratie van betere kwaliteit in peri implantaire botdefecten faciliteert dan BMP-2 en

BMP-7 homodimeren (Hoofdstuk acht).

BMP-2 en BMP-7 zijn de meest uitgebreid geëvalueerde BMP’s en zijn erg kostbaar.

Hoewel BMP’s een buitengewone staat van dienst hebben qua botformatie, kunnen ze in

sommige situaties als ze in hoge concentraties gebruikt worden negatieve effecten

hebben. Onderzoek heeft geleid tot de ontwikkeling van tal aan methoden in bone tissue

engineering om het gebruik van BMP’s te beperken en de osteoinductieve effecten van

BMP’s te vergroten door middel van een vertraagde toediening. Icariin, een typisch

flavonol glycoside wordt gezien als het voornaamste actieve ingrediënt van de Herba

Epimedii (een traditioneel Chinees medicijn), waar deze icariin uit gewonnen wordt. Het

is interessant dat er wordt gemeld dat icariin lokaal kan worden afgegeven door

biomaterialen en dat het een osteoinductief potentieel heeft voor bone tissue engineering.

Daarom hebben we de werking van icariin in de bone tissue engineering beoordeeld en

hebben we deze informatie samengevoegd met de huidige kennis met betrekking tot

moleculaire mechanismen en signaal routes. Het osteoinductieve potentieel van icariin

kan worden toegedicht aan zijn vele functies in het musculoskeletale systeem dat is

betrokken bij de regulatie van verschillende signalering routes in anti-osteoporose,

osteogenese, anti-osteoclastogenese, chondrogenese, angiogenese en anti-inflammatie.

Het osteoinductieve potentieel en de lage prijs van icariin maken het een erg

aanlokkelijke kandidaat als vervanging voor osteoinductieve proteïnen – BMP’s, of als

een promotor om de therapeutische effecten van BMP’s te versterken (Hoofdstuk negen).

Chapter 11

176

Acknowledgments

177

ACKNOWLEDGEMENTS

First and foremost, I would like to express my sincerest gratitude to my supervisors, prof.

dr. Daniel Wismeijer and dr. Yuelian (Maria) Liu, who kindly provided me with the

opportunity to do this research in ACTA.

Dear Daniel, it is my great honor to have you as my supervisor. Thank you for fully and

strongly supporting me to complete my research and thesis. Your concern, guidance and

encouragement always gave me great power to keep working. I am deeply inspired by

your intellectual elegance and amiable personality. Despite your role as the head of the

department of oral implantology and all other obligations, you always had time to assist

me, making everything go silky smooth.

Dear Maria, thank you for leading me through my whole Ph.D. pursuit. Words have not

been able to express the gratitude that I feel for your tireless help and encouragement.

You are a wonderful guider, providing me so many ideas. You always stood beside me,

encourage and guide me, whenever I met with difficulties. I greatly appreciate your

efforts to make my doctorial journey productive.

Dear prof. Zhiyuan Gu, thank you for introducing me to ACTA. It is so lucky that I have

you as my supervisor. I have had great respect for your noble personality, since I was a

master student.

Dear prof. Vincent Everts, thank you for being a knowledgeable advisor. You are an

amazing advisor. I always asked questions, and you always gave me the right answers. I

really learned a lot from you. Thank you for your insightful questions and comments on

those manuscripts, and thus make me know how to write a good article.

Dear Ton Broncker, thank you for teaching me about histological studies. You always

provided very nice ideas to me.

Dear Gang, thank you for your company. We had lived together for almost two years.

Everything seems like just happened yesterday. I enjoyed discussing research issues with

you. I could see your attitude on research is very serious.

Dear Afsheen, thank you for your suggestions on the manuscripts. I learned many writing

skills from you.

Dear Sven, I would like to thank you for the translation. Without your help, I cannot

complete the thesis.

In regards to the cell biology studies, I especially would like to thank Jolanda, Cor,

Dirk-Jan, Teun, Ton, Ineke, Behrouz, and Marion. I also would like to thank Leo and Jan

Harm for your kind help. All of you provided me techniques and supports to complete

my research.

Acknowledgments

178

Jenny, Janak, Alejandra, Marjolein, Ceylin, Nawal, Greetje, Sepanta, I am lucky to have

your help and to share Ph.D. life with you.

Chenfeng, Qilong, Lei, Xingnan, Xiao, Dongyun, thank you for the passionate friendship.

We had a very good time.

Special appreciate should be given to all my Chinese and Dutch friends in ACTA or once

in ACTA.

Lastly, I would like to thank my parents, my parents-in-low, and my sweet wife for your

endless love and support.

至此，再次衷心感谢我的导师刘月莲老师，多谢您在这四年来的悉心指导。

最后，我希望对我的父母，岳父岳母，以及我最爱的妻子表达深深的感谢。

你们是我心中的动力。没有你们的支持，我无法完成这四年的学业。

特别感谢我可爱的妻子陆倩，感谢你的理解，支持和奉献。

Curriculum vitae

179

CURRICULUM VITAE

Name: Tie Liu

Date of birth: 17th February, 1983,

Place of birth: Yuyao, Zhejiang province, China

Contact

Mobile phone: (86)13588135231 (China)

Email: [email protected]

[email protected]

Scientific education

2009.09-2013.09 PhD-student. Department of Oral Implantology and

Prosthetic Dentistry, Academic Centre for Dentistry

Amsterdam (ACTA), Research Institute MOVE,

University of Amsterdam and VU University Amsterdam,

The Netherlands.

2007.09 Exchange student. School of Dentistry, Medical College

of Georgia, USA

2007.08-2009.07 Master degree. Oral and Maxillofacial Surgery / Oral

Implantology, Hospital/School of Stomatology, Zhejiang

University.

2002.09-2007.07 Bachelor degree. General Dentistry, Hospital/School of

Stomatology, Zhejiang University.

Clinical education

2007.05-2009.07 Hospital/School of Stomatology, Zhejiang University.

Internship.

2007.02-2007.04 Department of Oral and Maxillofacial Surgery. The First

Affiliated Hospital of College of Medicine, Zhejiang

University (First Hospital of Zhejiang Province).

Internship.

2006.06-2007.01 Hospital/School of Stomatology, Zhejiang University.

Internship.

mailto:[email protected]

mailto:[email protected]

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