Performance of evacuated calcium phosphate microcarriersloaded with mesenchymal stem cells within a rat calvarium defect

Guang-Zhen Jin • Joong-Hyun Kim •

Jeong-Hui Park • Seong-Jun Choi • Hae-Won Kim •

Ivan Wall

Received: 18 December 2011 / Accepted: 10 April 2012 / Published online: 27 April 2012

� Springer Science+Business Media, LLC 2012

Abstract Tissue engineering of stem cells in concert with

3-dimensional (3D) scaffolds is a promising approach for

regeneration of bone tissues. Bioactive ceramic micro-

spheres are considered effective 3D stem cell carriers for

bone tissue engineering. Here we used evacuated calcium

phosphate (CaP) microspheres as the carrier of mesen-

chymal stem cells (MSCs) derived from rat bone marrow.

The performance of the CaP–MSCs construct in bone

formation within a rat calvarium defect was evaluated.

MSCs were first cultured in combination with the evacu-

ated microcarriers for 7 days in an osteogenic medium,

which was then implanted in the 6 mm-diameter calvarium

defect for 12 weeks. For comparison purposes, a control

defect and cell-free CaP microspheres were also evaluated.

The osteogenic differentiation of MSCs cultivated in the

evacuated CaP microcarriers was confirmed by alkaline

phosphatase staining and real time polymerase chain

reaction. The in vivo results confirmed the highest bone

formation was attained in the CaP microcarriers combined

with MSCs, based on microcomputed tomography and

histological assays. The results suggest that evacuated CaP

microspheres have the potential to be useful as stem cell

carriers for bone tissue engineering.

1 Introduction

For successful healing of nonunion bone defects, tissue

engineering is currently being considered as a promising

alternative to autologous treatments. This mainly involves

the seeding of osteoprogenitor or stem cells within the

3-dimensional (3D) scaffolds, ex vivo culturing and

expansion, and then implantation of the bone-mimicking

constructs into the defective areas. Many 3D-structured

materials, such as porous sponges, fibrous meshes, hydro-

gels and microparticulates have been developed as a carrier

to support and deliver osteoprogenitor or stem cells in the

tissue engineering of bone [1, 2].

Among the scaffolds developed thus far, microspherical

particulates have recently gained great interest for use as a

3D matrix to support cellular expansion and tissue specific

differentiation that is necessary to achieve success after

implantation into damaged tissues [3–9]. In fact, micro-

particulates have commonly been used to augment and fill

bone defects, such as the alveolar ridge and periodontal

pocket [2]. Bioactive ceramics, such as calcium phosphates

and glasses/glass ceramics are the most popularly used

compositions for the treatment of osseous defects. Merits

of using spherical particulates as the carrier of tissue cells

include increased capacity for manipulation to achieve

efficient cell adhesion, migration and proliferation, as well

as increased plasticity of the final required product

dimensions as they can be applied as an injectable sub-

stance to fit the shape of bone defects [10].

G.-Z. Jin � J.-H. Kim � J.-H. Park � S.-J. Choi � H.-W. Kim (&)

Institute of Tissue Regeneration Engineering (ITREN), Dankook

University, Cheonan 330-714, Korea

e-mail: kimhw@dku.edu

G.-Z. Jin � J.-H. Park � H.-W. Kim � I. Wall

Department of Nanobiomedical Science & WCU Research

Center, Dankook University Graduate School, Cheonan 330-714,

Korea

H.-W. Kim

Department of Biomaterials Science, School of Dentistry,

Dankook University, Cheonan 330-714, Korea

I. Wall

Department of Biochemical Engineering, University College

London, Torrington Place, London WC1E 7JE, UK

123

J Mater Sci: Mater Med (2012) 23:1739–1748

DOI 10.1007/s10856-012-4646-y

The chemical composition and surface properties of the

microparticulates are important factors to take into account

during their design; they should properly guide initial cell

adhesion and proliferation, and stimulate specific-tissue

differentiation and matrix synthesis [4, 10, 11]. It has been

shown that the chemical composition of the spherical

particulates, either made from bioactive ceramics or

degradable polymers, can significantly affect their in vivo

bone forming ability [3, 4, 10, 11]. Moreover, the

3-dimensional (3D) shape and morphology of the micro-

particulates are also important to effectively load and

contain tissue cells within the microsphere structure. Some

recent works have reported the development of porous or

evacuated microparticulates containing internal macrop-

ores, in order to contain cells as effectively and as abun-

dantly as possible [6, 7]. This type of spherical particulate

can host a large number of cells, demonstrating potential

usefulness in cell delivery and bone tissue engineering.

In a recent study, we demonstrated that the evacuated

form of calcium phosphate (CaP) microspheres was

effective in enhancing proliferation and osteogenic induc-

tion of mesenchymal stem cells (MSCs) [12]. Moreover,

new bone formation was observed within the evacuated

parts of the microspheres [12]. The main benefit of using

CaP microspheres for bone tissue engineering is that,

unlike conventional methods using pre-fabricated scaf-

folds, the approach is more dynamic in terms of graft

specifications that can be produced for the transplantable

product. Thus, size and shape do not have to be pre-

defined, as the microspheres can be arranged in the

appropriate conformation after cell seeding and

differentiation.

To this end, we hypothesized that the beneficial effects

of evacuated microspheres on new bone formation could be

achieved by applying them in combination with MSCs.

Specifically, we used the evacuated microspheres with a

composition of hydroxyapatite (HA)/b-tricalcium phos-

phate (b-TCP) biphasic, as this composition has been

widely used as bone substitutes for medical applications

[13, 14]. Moreover, the MSCs were induced into osteo-

blasts under in vitro osteogenic conditions, to enhance

osteogenesis and bone formation in vivo. While in the

previous work we investigated the efficacy of the evacu-

ated microspheres in providing scaffold conditions for cells

in vitro and the tissue compatibility in vivo [12], here we

approached tissue-engineering of the evacuated micro-

spheres with MSCs by pre-culturing the cells under oste-

ogenic conditions and then implantation in a rat calvarium

defect model. The feasibility of using the evacuated CaP

microspheres loaded with pre-differentiated MSCs was

investigated for 12 weeks of implantation by means of

microcomputed tomography and histological assessment.

2 Materials and methods

2.1 Overview

The flow chart of this study was shown in Fig. 1. MSCs

derived bone marrow were harvested from the femora and

tibiae of adult rats. 2.0 9 105 MSCs per 10 mg of micro-

spheres were incubated overnight in control medium

(a-minimum essential medium, 1 % penicillin/streptomy-

cin, and 10 % fetal bovine serum). The cell-microsphere

constructs were then treated with either control media or

osteogenic medium (control medium ? 50 lg/ml ascorbic

acid ? 10 mM b-glycerophosphate ? 100 nM dexameth-

asone) for 7 days in vitro. Thereafter, the constructs were

transplanted into calvarial defects of rats. The samples

were collected after sacrifice, at 12 weeks post-implanta-

tion. Meanwhile, microcomputed tomography (micro-CT)

and histological analysis were performed for evaluation of

new bone formation.

2.2 Preparation of evacuated CaP microspheres

Preparation of microspheres and evaluation has been

described in a previous study [12]. Briefly, the initial

powder of biphasic calcium phosphate (BCP) made of HA

Fig. 1 Schematic drawing of the tissue engineering application of

evacuated CaP microspheres in the rat calvarial defect model. Starting

in the upper left-hand corner and proceeding along the arrowdirection: rat MSCs were isolated from bone marrow of the femur and

tibia. The cells were seeded onto CaP microspheres at a quantity of

2.0 9 105 cells. The MSC-microsphere constructs were cultured in

the presence of osteogenic medium for 7 days. For the ease of

handling, the MSC-microsphere constructs were embedded in diluted

collagen gel. Thereafter, the constructs were implanted into the rat

calvarial defect and specimens were harvested after 12 weeks

1740 J Mater Sci: Mater Med (2012) 23:1739–1748

123

and b(TCP) at 50:50 was prepared from a solution-based

reaction of precursors of calcium nitrate and ammonium

hydrogen phosphate [15]. Evacuated microspheres were

prepared using the BCP powder by the oil-in-water emul-

sification technique [12, 16]. The powder (at 20 wt%) was

added in the oil phase that consisted of chloroform and

poly(vinyl butyral) (PVB, 5 wt%). The powder mixture

solution was then dropped into a water bath containing

poly(vinyl alcohol) (PVA) at a concentration of 2 wt%,

while stirring at 350 rpm, for 10 min. The volume ratio of

the water bath to the oil solution was set at 50. The

microspheres that were floating on the water bath were then

gathered and dropped into a new bath containing 0.5 %

PVA to stabilize them. The stabilized microspheres were

then washed and dried in an oven at 50 �C overnight. The

dried microspheres were placed in an alumina crucible and

heat treated in a furnace by subjecting the samples to the

following conditions: heating to 800 �C at a rate of 1�/min,

where they were held for 3 h; heating to 1,250 �C at a rate

of 10�/min, where they were held for 2 h; and then the

furnace was cooled to room temperature.

2.3 Culture of MSCs on the microspheres

The MSCs were harvested from the excised proximal and

distal epiphyses of the femora and tibiae of adult rats (8-

week-old) and cultured according to the procedures in a

previous study, with a slight modification [17]. The MSCs

were used for the following experiments when they were at

the 3–4 passages. The size of the microspheres for cell

culture was selected around 1,000 lm using sieves. Ten mg

of microspheres were placed in each well of a 96-well

plate, sterilized with 70 % ethanol, and then dried under a

laminar flow. The microspheres contained in each well

were shown to be stacked in 2–3 layers, covering the well

surface almost completely. A 50 ll aliquot of cell sus-

pension with 2.0 9 105 cells in control medium (a-mini-

mum essential medium, 1 % penicillin/streptomycin, and

10 % fetal bovine serum) was then seeded into each well

and incubated overnight. Next day, the cell-microsphere

constructs were treated with either control medium or

osteogenic medium (control medium plus 50 lg/ml

ascorbic acid, 10 mM b-glycerophosphate, and 100 nM

dexamethasone) statically for 7 days in vitro, and the

medium was refreshed once during the culture period.

2.4 Scanning electron microscopy (SEM)

The constructs cultured for 7 days were fixed in 2.5 %

glutaraldehyde for 10 min, at room temperature. The fixed

samples were then dehydrated with increasing concentra-

tions of ethanol (75, 95 and 100 %) for 5 min each, and

treated with hexamethyldisilazane for 10 min. Finally,

SEM was conducted at an accelerating voltage of 15 kV,

after coating the sample surface with gold.

2.5 Alkaline phosphatase (ALP) staining

The level of ALP expression was observed at day 7 by

staining the cell-scaffold constructs with a chemical regent

(Cat.# MK300, Takara). After washing with phosphate

buffered saline, a fixation solution was added to each

sample, followed by addition of the ALP reaction substrate.

The samples stained in purple-blue color were observed by

optical microscopy.

2.6 Semiquantitative reverse transcription-polymerase

chain reaction (RT-PCR)

At the osteogenic period of 7 days, ALP mRNA expres-

sion, an early osteogenic marker, was detected by RT-PCR.

b-actin was used as internal control. Total cellular RNA

was extracted using TRIzol reagent, according to the

manufacturer’s protocol (Invitrogen). An aliquot of 2 lg of

total RNA was used in cDNA synthesis using an Accu-

PowerTM

RT Premix kit, according to the manufacturer’s

instructions (Cat.# K-2042, Bioneer Co., Korea). The pri-

mer sequences of the genes were as follows: for ALP,

sense: 50-AGGCAGGATTGACCACGG-30 and antisense:

30-TGTAGTTCTGCTCATGGA-50 (440 bp); for b-actin,

sense: 50-ACGTTGACATCCGTAAAGAC-30 and anti-

sense: 30-TAATCTCCTTCTGCATCCTG-50 (96 bp). The

PCR reactions were conducted using an AccuPower PCR

PreMix (Cat.# K-2016, Bioneer Co., Korea) under the

following conditions: initial denaturation at 95 �C for

1 min followed by 35 cycles at 95 �C for 30 s, with

annealing temperatures of 56.0 �C (ALP) and 50.0 �C

(b-actin) for 30 s, and a final extension at 72 �C for 1 min.

All samples were run in triplicates in each experiment. The

PCR products were separated electrophoretically in a

1.5 % agarose gel and then stained with ethidium bromide.

2.7 Quantitative real-time PCR

Quantitative analysis of the ALP gene expressed at day 7

was conducted by quantitative real-time PCR. The first

strand cDNA was synthesized from the total RNA (1 lg)

using the AccuPowerTM

RT Premix kit (Cat.# K-2042, Bi-

oneer Co., Korea). Real-time PCR was performed using

SYBR GreenER qPCR SuperMix reagents (Invitrogen) in

20 ll reactions. The primer sequences of the genes were, as

follows: for ALP, sense: 50-AGCTGCCCGCATCCTTAA-

30 and antisense: 30-TGTAC GTCTTGGAGAGAGCCA-

50; for b-actin, sense: 50-ACGTTGACATCCGTAAAGA

C-30 and antisense: 30-TAATCTCCTTCTGCATCCTG-50.The reaction was performed under the following

J Mater Sci: Mater Med (2012) 23:1739–1748 1741

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conditions: 95 �C for 10 min followed by 40 cycles of

95 �C for 10 s, 55 �C for 15 s, and 72 �C for 20 s. The

relative transcript quantities were calculated using the

DDCt method with b-actin as the endogenous reference

gene amplified from the samples [18].

2.8 Surgical procedure

The experimental procedures for the in vivo animal study

were approved by the Animal Care and Use Committee of

Dankook University. Nine adult male Sprague–Dawley rats

(weighing 250–300 g) were used. They were divided ran-

domly into Group I (control), Group II (CaP microspheres),

and Group III (CaP microspheres with cells). To facilitate

the handling and implantation of the CaP microspheres,

both CaP microspheres and cell-seeded CaP microspheres

were treated with collagen solution diluted at 1 mg/ml

(type I collagen from rat tail, BD Biosciences). After

placing the microspheres within a cylindrical plastic mold

(diameter of 6 mm), the collagen mixture solution with

109 Dulbecco’s modified eagle medium (DMEM) and 1 N

NaOH buffered at pH 7.4 was added, and was then incu-

bated at 37 �C for 30 min, to allow gelation. The size of

the gelled sample was confirmed to be 6 mm in diameter

and *2 mm in thickness. As a control, the gelled collagen

without any microspheres was used. Therefore, three dif-

ferent sample groups (control, CaP microspheres, and CaP

microspheres with cells) were prepared for the implanta-

tion in a rat calvarium defect.

The animals were anesthetized by an intraperitoneal

injection with a mixture of ketamine (7 mg/100 g; Ket-

ara�, Yuhan Corp., Seoul, Korea) and xylazine (1 mg/

100 g; Rompun�, Bayer Korea, Ltd., Seoul, Korea). A skin

incision of 15 mm was made and the periosteum was ele-

vated for the trephine operation. Two critical-sized, full-

thickness bone defects (6 mm diameter) were prepared in

each rat, using a saline-cooled trephine drill. Care was

taken not to damage the underlying sagittal sinus and dura

matter. Each defect was randomly filled with either of the

three different groups (control, CaP microspheres, and CaP

microspheres with cells). The control defect was kept with

collagen gel without the use of microspheres. The incisions

were then sutured. The samples were collected after sac-

rifice at 12 weeks post-implantation for microcomputed

tomography (micro-CT) evaluation and histological

analysis.

2.9 Micro-computed tomography and histological

analysis

Twelve weeks after surgery, the radiographic evaluations

of new bone formation within the defect region were car-

ried out using a micro-CT system (Skyscan 1072, Skyscan,

Aartselaar, Belgium) [19]. The harvested samples were

fixed in 10 % neutral formalin solution for 24 h at room

temperature and scanned in a direction parallel to the

coronal and horizontal aspect of the calvarial bone sur-

rounding the defect area. A cylindrical region of interest

was precisely positioned over the center of each defect,

encompassing all new bone within the defect site. Three

samples for each group were evaluated (n = 3).

The constructs were collected at 12 weeks after trans-

plantation and fixed in 10 % neutral formalin, and then

decalcified by dipping into Planko–Rychlo solution,

including formic and hydrochloric acids, for about 72 h.

Decalcified samples were embedded in paraffin block, and

serially sectioned with microtome (LEICATM

) at 5-lm

thickness. Finally, slides with tissue sections were stained

with hematoxylin & eosin (H&E). The slides were exam-

ined under an optical microscope (OlympusTM

). Histomor-

phometric analyses were performed to evaluate the

quantity of newly formed bone in the samples using image

analysis software (DP2-BSW, Olympus Co.), and the mean

values were then calculated.

2.10 Statistics

The results are expressed as the mean ± standard devia-

tion. Differences between the experimental groups were

assessed by one-way analysis of variance (ANOVA) using

the SPSS software package version 13.0 (SPSS Inc., Chi-

cago, IL). P values of either \ 0.05, \ 0.01, or \ 0.001

were considered as statistically significant.

3 Results and discussion

3.1 Culture of MSCs on the evacuated CaP

microspheres

Calcium phosphate-based bioactive ceramics including HA

and TCP have been shown to be osteoconductive and

highly biocompatible. Therefore, they can potentially be

employed as carriers for cells and growth factors to treat

and augment defective bone tissues. Our previous study

demonstrated that the evacuated form of HA–TCP micro-

spheres was highly effective in facilitating growth of cells

in vitro, both on the outer surface and throughout the inner

surface of the microspheres [12]. Moreover, de novo bone

formation in vivo was effectively induced within the

evacuated portion of the microspheres, proving tissue

compatibility.

Here, we utilized evacuated bioactive ceramic micro-

spheres as a novel carrier of MSCs for bone tissue engi-

neering. Prior to in vivo implantation of the bioceramic

carriers, we loaded a 50 ll aliquot of 2.0 9 105 cells onto

1742 J Mater Sci: Mater Med (2012) 23:1739–1748

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10 mg of microspheres and cultured them under osteogenic

conditions for 7 days. Subsequent to this, the construct was

formulated with diluted collagen gel to endow the mobile

microspheres a level of malleability for implantation. In

particular, the in vitro osteogenic culture stage for the

MSCs was aimed at deriving populations of cells with

enhanced capacity to induce new bone formation in vivo.

Studies have highlighted that the pretreatment of stem

cells, including MSCs, under osteogenic conditions prior to

the in vivo implantation is of importance to direct appro-

priate osteogenic lineage specification and enhance the

bone-forming capacity of these cells and their progeny in

vivo [20–22].

The morphology of the MSCs adhered to and prolifer-

ated on the evacuated microspheres was examined with

SEM, as shown in Fig. 2. The MSCs were found to grow

actively on both the outer and inner surfaces of the evac-

uated CaP microspheres. Importantly, the cells migrated

and populated well on the inner surface of the microspheres

and formed physical cell–cell contacts within the micro-

spheres. Upon proliferation, the cells appeared to stack in

layers and bridged the gaps of adjacent microspheres.

After culture for 7 days under osteogenic conditions, the

expression of ALP, an early osteogenic marker, was

assessed. The results showed that ALP staining in the

differentiated MSCs-microsphere construct was much

more intense than that in the control cultured in the normal

medium (Fig. 3a). Meanwhile, ALP was also assessed at

the gene expression level. Using semi-quantitative RT-

PCR, the band for the ALP 440 bp product was observed to

have greater intensity in the differentiated group than in the

control (Fig. 3b). These semi-quantitative results were then

confirmed using quantitative real-time PCR with statisti-

cally significant differences evident (Fig. 3c).

Culturing the MSCs on evacuated CaP microspheres in

osteogenic growth medium is an important step to gain

comprehensive in vivo osteogenic potential, because not all

cells within an ex vivo expanded MSC population will

have the capacity to differentiate along the osteogenic

lineage and so in vitro culture enables osteogenic differ-

entiation of cells prior to transplantation. Studies reporting

the effects of pre-culture period of MSCs prior to in vivo

implantation have thus been well documented [23–26]. Rat

MSCs derived from bone marrow were pre-cultured in

osteogenic medium for 4, 10 and 16 days and then loaded

into a titanium fiber mesh scaffold, which was then

implanted into a rat calvarium defect. After 4 weeks, the

group containing MSCs pre-cultured for 4 days showed the

Fig. 2 SEM morphology of the MSCs grown on the evacuated CaP

microspheres for 7 days. Cells were seeded within a single well of a

96-well plate, at a density of 2.0 9 105 cells per 10 mg of micro-

sphere sample. Representative images of cells growing on the outer

(a, b) and inner (c, d) surface of the microspheres demonstrate

favorable signs of cell attachment and proliferation. The cells

underwent adherence, migration, population and contact with each

other, formed a thick cell layer and bridged the gaps of adjacent

microspheres

J Mater Sci: Mater Med (2012) 23:1739–1748 1743

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highest bone formation compared with other groups, sug-

gesting that the culture period is a critical factor to enhance

in vivo bone regeneration capacity [23]. Kruyt et al. [24]

also cultured MSCs in porous HA scaffolds for 6 days to

allow cell differentiation and matrix formation, and con-

sequently to obtain an effective tissue-engineered construct

before implantation in a goat intramuscular pocket. The in

vivo osteogenic potential of human adipose-derived stem

cells was also confirmed to be improved when cells were

pre-cultured in the polymer matrix for 7 days in osteogenic

medium (compared with controls without pre-culture or

pre-culture for 1 or 14 days) [20]. Yang et al. [25] used

dental pulp-derived cells and observed that the greatest

amount of hard tissue formation after transplantation into

subcutaneous tissue of mice occurred when an appropriate

osteogenic pre-culture time of 4 days was used (vs. 8 days

or controls lacking pre-culture), highlighting the impor-

tance of in vitro pre-culture of MSCs under osteogenic

conditions. While the relatively short pre-culture times

(mostly less than a week) for MSCs in vitro in an osteo-

genic medium have been found effective in enhancing in

vivo capability of bone regeneration, more extended pre-

culture time periods (over 8–16 days) have generally

resulted in a limited amount of in vivo bone formation [26,

27]. It therefore seems that ensuring the MSCs are induced

to have a degree of functional specialization before deliv-

ering them in vivo would improve their efficacy during

bone regeneration.

3.2 In vivo bone regeneration in rat calvarium defects

In the in vivo study, the experimental design was divided

into the control group (using collagen gel alone), the CaP

microsphere group, and the cell-combined CaP micro-

sphere group, as shown in Fig. 4. Twelve weeks after

implantation, rats were sacrificed and tissue recovery and

bone regeneration at the defect site were evaluated using

micro-CT imaging. X-ray photographs were obtained

(Fig. 5a, b) and then 2D and 3D reconstructed micro-CT

images (Fig. 5c, d) were produced for the representative

samples. Although evacuated microspheres and newly

formed bone that accompanied them were not easily

Fig. 3 Confirmation of the

early osteogenic marker ALP in

MSC-microsphere constructs

after 7 days of in vitro

differentiation; a ALP staining

image suggesting the

differentiation of MSCs in

osteogenic medium (right well),compared to MSC-microspheres

in normal medium (control, leftwell). b Semiquantitative RT-

PCR and c quantitative real-

time PCR analysis of ALP

expression in cultures of MSC-

microsphere constructs in

osteogenic or normal growth

medium.

Values = mean ± SD, n = 3,

** P \ 0.01 by one-way

ANOVA

Fig. 4 Images of the surgical procedure show where the specimen

was placed within the 6 mm defect in a rat cranium. The study was

conducted in three experimental groups: control group, CaP micro-

sphere group, and cell-combined CaP microsphere group

1744 J Mater Sci: Mater Med (2012) 23:1739–1748

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discerned in the X-ray images (a, b), there was difference

in the radio-density of the newly-formed bone within the

microspheres based on the cross-section panels (a0, b0);much more intense when the MSC-derived cells were

incorporated (Fig. 5a0, b0, arrows), compared with CaP

microspheres alone (Fig. 5b0, arrowheads). Micro-CT

images also showed better connection of newly-formed

bone along the microspheres when MSCs were combined

(Fig. 5c, d). These results demonstrated that the CaP

microspheres were effective in inducing new bone forma-

tion, and the cell-combination with the CaP microspheres

was more effective.

The tissue formed within the calvarium defect zone was

further evaluated histologically by H&E staining. At

12 weeks, a thin layer of fibrous connective tissue was

formed in the collagen gel control group. Only a limited

minimal area was seen with formation of new bone, which

was similar to the vacant defect (free of any materials)

observed at this time of prolonged healing period in this

model. Results illustrated that the collagen gel did not play

any significant role in inducing new bone formation in the

critical-sized calvarium defect model (Fig. 6a). This is

mainly due to that the collagen diluted gel degrades rapidly

(within days to few weeks), thus not playing any scaf-

folding roles in the cell recruitment and bone formation.

However, in the CaP-microsphere group, the areas of new

bone became substantially increased and were mostly

found near the dura mater surface over the defect area

(Fig. 6b), suggesting that the CaP microspheres played an

effective scaffolding role in new bone formation in vivo.

The highest level of bone formation was seen in the cell-

combined CaP group at this time point (Fig. 6c, d). This

new bone formation was not only formed near the dura

mater side, but also at the periosteal side. Meanwhile, new

bone formation was found not only on the external areas,

but also within the evacuated parts of the CaP micro-

spheres. After the 12 week implantation period, histology

revealed that in both CaP and cell-combined CaP groups,

most of the microparticles still remained without any sig-

nificant signs of degradation. As it appears that the in vivo

period used herein was not sufficient to induce substantial

materials degradation, the use of other compositions, such

Fig. 5 Micro-CT images of the harvested samples at 12 weeks post-

operation. Sample groups are control, CaP miscrophere, and cell-

combined CaP microsphere; a, b X-ray photographs of the defect site

after 12 weeks, a0, b0 frontal section visualizations of the defects in

(a, b), (c, d) 2D reconstructed images from (a, b), respectively, and

(c0, d0) 3D reconstructed images from c, d, respectively. Arrowsindicate higher gray levels. Arrowheads indicate lower gray levels.

Scale bars: 1.5 mm

J Mater Sci: Mater Med (2012) 23:1739–1748 1745

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as pure b-TCP, will favor degradation, which might be

useful to tune the regeneration profile in need. The histo-

logical images were quantitatively assessed for areas of

new bone formation, as shown in Fig. 7. A significant

improvement in the amount of new bone formation was

observed with the use of CaP microspheres and, further-

more, the combination of cells significantly enhanced bone

regeneration capacity of the microspheres.

In a previous study [12], we reported the in vivo bone

inducing ability of evacuated CaP microspheres within a

6 mm diameter rabbit calvarium defect, with positive

results showing almost complete regeneration of bone

tissue after 6 weeks. In comparison, here the bone regen-

eration ability of the evacuated CaP microspheres (without

cells) within the 6 mm diameter rat calvarial defect was not

as effective as in the rabbit model; furthermore, the cell-

combined CaP group also failed to promote complete res-

toration of the lamellar bone structure, although the tissue

regeneration effects were clearly seen. This is considered

mainly due to the difference in the defect models. In the

rabbit model, a calvarial defect of 6 mm is not actually the

critical-size in rabbit and thus is considered to support

better in vivo conditions to allow neo-bone tissue forma-

tion, whereas in the current rat model, a calvarial defect of

Fig. 6 Representative

photographs of tissue section

taken at 12 weeks, after staining

different sample groups with

haemotoxylin and eosin;

a control, b CaP and c CaP/

MSC. d is higher-magnification

images from box in (c). Asteriskrepresents newly formed bone

and ‘CaP’ is evacuated

microsphere, upwardsarrow = cranial defect edges.

Scale bar in (a, b), and c is

400 lm, and that in d is 200 lm

1746 J Mater Sci: Mater Med (2012) 23:1739–1748

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6 mm is considered to be a critical-sized defect, due to the

increased defect size relative to the size of the animal,

therefore creating more difficult conditions for bone

regeneration. Another reason might be due to the use of

different-sized microspheres. Previously, we used micro-

spheres in the range of 200–500 lm diameter, whereas

here we selected more regular and larger size microspheres

of *1,000 lm. Although we selected this size (within the

limit of sizes relevant to the calvarium defect thickness) in

an attempt to seed cells more easily and uniformly, the in

vivo function of the tissue-engineered microspheres

appeared not as optimal as expected. The longer distance

(and hence more space) between the microspheres as well

as the larger inner space associated with the enlarged

sphere size is considered less effective in allowing in vivo

bone formation. One possible strategy for improved func-

tion could be to populate the inside space and outer surface

of the microspheres with a greater number of cells,

increasing initial cellularity and therefore attaining greater

functional osteogenesis by the engineered cells. It is

therefore considered that the in vivo osteoconductive

potential of the evacuated microspheres is probably highly

dependent on their size and will likely be improved if

optimal microsphere sizes are used. The effects of micro-

carrier size on bone formation is thus considered of interest

study to follow, as there have been few works on this type

of microspherical carriers, particularly on the macro-por-

ous or evacuated types.

Although most studies have used 3D porous scaffolds

made of bioactive ceramics as carriers for hard tissue

engineering, here we reported for the first time on the

utility of microspherical carriers, with the inner part totally

evacuated, in combination with MSCs. Rather than defin-

ing at the outset what the desired bone graft shape should

be, microsphere-based tissue engineering offers a large

degree of freedom to modify and manipulate the desired

shape of the construct later on, after single functional units

of the graft have been produced. Moreover, the evacuated

form of the microspherical scaffolds should be a better

choice in delivering stem cells, compared to solid micro-

spheres, as the evacuated part provides additional space for

cellular uptake. When cultured in favorable conditions, the

space can become populated with cells and extracellular

components, leading ultimately to osteogenesis; if not so

fully during ex vivo culture, it can be achieved after in vivo

implantation. The evacuated portion of the CaP micro-

spheres developed herein amounts to 60–70 % of the whole

sphere volume; therefore, the relatively lower volume

space of the material’s part is also considered to be more

favorable in terms of regenerative capacity, as the newly-

formed biologic tissues will have a smaller quantity of

artificial material to replace. These aspects indicate a

promising future for the use of evacuated microspheres in

bone tissue engineering. However, compared to 3D porous

scaffolds, the initial uniform cell seeding and ex vivo cell

cultivation techniques are not a simple issue. Due to dis-

continuity and motility of the microspheres, the static

seeding and subsequent culturing of cells are not optimal,

particularly when a large volume of tissue-engineered

microspheres is needed to fill large-sized defects. There-

fore, further development such as exploration of growth in

dynamic culture systems is required and this is currently

underway.

4 Conclusions

Newly-developed evacuated CaP microspheres were

applied for bone tissue engineering in combination with the

MSCs. After in vitro culture for a week under osteogenic

conditions, the constructs were implanted in 6-mm-diam-

eter rat calvarium defect for 12 weeks. When the evacuated

CaP microspheres were combined with the differentiated

MSCs, new bone formation in vivo was significantly

improved. Based on these results, the evacuated micro-

spheres are considered to have great potential to be used

as stem/osteoprogenitor cell carrier for bone tissue

engineering.

Acknowledgments This work was supported by Priority Research

Centers Program (No. 2009-0093829) and WCU program (R31-

10069) through the National Research Foundation of Korea (NRF)

funded by the Ministry of Education, Science and Technology.

Authors thank Dr. Kim T. H. for his kind help in preparation of tissue

samples.

Fig. 7 Quantitative analysis of the newly formed bone in the defects

after 12 weeks performed with image analysis software (DP2-BSW,

Olympus Co.). Experimental values were expressed as mean ± SD.

**P \ 0.01; ***P \ 0.001 by one-way ANOVA

J Mater Sci: Mater Med (2012) 23:1739–1748 1747

123

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