A platelet derived growth factor delivery system for boneregeneration

J. J. Delgado • Esther Sanchez • Manuel Baro •

Ricardo Reyes • Carmen Evora • Araceli Delgado

Received: 18 October 2011 / Accepted: 24 April 2012 / Published online: 11 May 2012

� Springer Science+Business Media, LLC 2012

Abstract platelet derived growth factor (PDGF) was

formulated in a calcium phosphate/biodegradable polymer

system for local and controlled delivery to enhance bone

regeneration. Implants with a porosity of 67 %, composed

of hydroxyapatite, PLGA microspheres and Pluronic�,

were obtained by compression. An increase in porosity

with time was expected due to Pluronic� dissolution and

PLGA microsphere degradation. In vivo PDGF release and

tissue distribution were monitored after system implanta-

tion into femurs of rabbits using 125I-PDGF. Most of the

PDGF was released within approximately 5 days and

remained located around the implantation site with negli-

gible systemic exposure. Compared with the reference

groups, an important enhancement of bone regeneration

was found with doses of 600 and 1,200 ng of PDGF,

although no histological differences were observed

between the two doses. In conclusion, the elaborated sys-

tem exhibited good biocompatibility and offered a physi-

ologically relevant PDGF profile that enhances bone

formation compared to the non-treated bone defect.

1 Introduction

The bone regenerative process is characterized by a

remodeling cycle, in which cell populations are recruited

and differentiated for new bone formation as well as

resorption. A concerted system of growth factors (GFs) and

cytokines coordinates and regulates these activities.

Among other GFs, platelet derived growth factor

(PDGF), a potent chemoattractant and mitogen, is consid-

ered to be a key mediator in wound healing and tissue

repair [1]. PDGF-BB is chemotactic and mitogenic for

osteoblast lineage cells and osteoblasts [2]. PDGF stimu-

lates proliferation and differentiation of osteoblasts [3].

Moreover, PDGF, also known for its angiogenic effect,

exerts an indirect angiogenic action by upregulating the

expression of vascular endothelial growth factor (VEGF).

Angiogenesis plays a critical role in skeletal development

and bone fracture repair [4]. Taken together, PDGF

may enhance bone regeneration by attracting osteopro-

genitor cells, inducing their proliferation and stimulating

angiogenesis.

PDGF dimers -AB, -AA, -BB, and -CC are generally

produced by discrete cell populations and act locally to drive

different cellular responses. Ligand binding of PDGF

receptor (PDGFR) promotes dimerization and endocytotic

internalization. Specificity of PDGFR signaling is achieved

through a combination of spatio-temporally regulated

expression and differential engagement of downstream sig-

naling pathways [5]. Apart from the short PDGF half-life

(\2 min) [6], the delivery mode appears to be critical for GF

applications in general [7–9]. Previous studies suggest a

bone regenerative potential of PDGF delivered from differ-

ent materials, such as chitosan (CHT) [10] and its composites

[11, 12], brushite [13, 14], collagen [15], and b-TCP-colla-

gen matrices [2, 16]. These materials or their composites

J. J. Delgado � E. Sanchez � R. Reyes � C. Evora (&) �A. Delgado (&)

Department of Chemical Engineering and Pharmaceutical

Technology, University of La Laguna, Av. Astrofısico Fco.

Sanchez s/n, 38200 La Laguna, Spain

e-mail: cevora@ull.es

A. Delgado

e-mail: adelgado@ull.es

M. Baro

Traumatology Service, Hospiten Rambla, Ltd.,

Santa Cruz de Tenerife, Spain

123

J Mater Sci: Mater Med (2012) 23:1903–1912

DOI 10.1007/s10856-012-4661-z

were capable of extending PDGF release, although the sys-

tems were not designed to mimic the physiological presen-

tation kinetics of PDGF. Apart from our own studies [13, 14],

none of the above cited works provided either a quantitative

analysis of the in vivo release kinetics for PDGF or the

achieved local concentrations in the bone defect, nor were

they supported by data about new bone formation. As our

understanding of the bone forming process evolves, we need

to establish the optimal in vivo GF release kinetics, not only

with respect to local concentrations but also to temporal

presentation, in the damaged tissue.

The aim of the current study was to evaluate the efficacy

of a porous composite (calcium phosphate/biodegradable

polymer) system, including different doses of PDGF, on

vascularization and regeneration of an intramedullary defect

in rabbit femurs. The system was designed to mimic early

PDGF presentation and provide controlled release into the

local microenvironment of the implantation site, thus

avoiding systemic exposure to the growth factor. Scaffolds

were assessed empty and loaded with two distinct doses of

PDGF. Release kinetics was monitored and vascularization

and bone regeneration quantified in histological specimens.

2 Materials and methods

2.1 Material pre-treatment

First, hydroxyapatite (HA, Merck) was pulverized; the

resulting particle size of 8.10 lm was determined with a

Coulter LS100 Laser Scattering Particle Analyzer and

expressed as volume average mean diameter.

Poly(lactide-co-glycolide) PLGA (Resomer� RG502,

Boehringer Ingelheim) was used in form of microspheres

prepared by an emulsion-solvent evaporation method, as

previously described [17]. Briefly, 2 mL of 200 mg/mL

polymer solution in dichloromethane (DCM) was emulsi-

fied with 50 mL of Pluronic� F68 (4 %) (Sigma) using a

homogenizer (Silverson L4RT) at 6,000 rpm for 15s. DCM

was then evaporated under magnetic stirring at room

temperature. Microspheres were collected by centrifuga-

tion and freeze dried. The microspheres gave a volume

average mean diameter of 35.3 lm (Coulter LS100).

Moreover, materials assigned for implant fabrication

were previously sterilized by c-irradiation, following the

USP recommendations, with a dose of 25 kGy from a 60Co

source (Gamma Sterilization Unit of Aragogamma, Bar-

celona, Spain). Thermometric control confirmed that the

sample temperature during irradiation did not rise appre-

ciably above room temperature. Except for PDGF, liquid

components and laboratory instruments were sterilized by

autoclaving at 121 �C for 30 min. PDGF-BB was recon-

stituted in a solution of 4 mM HCl, containing 0.1 %

bovine serum albumin (BSA) as recommended by the

supplier.

2.2 Elaboration of PDGF-phosphate/polymer implants

PDGF-BB (R&D Systems) and 125I-PDGF (Amersham

Biosciences) were added to a suspension of HA (515 mg/

mL) and PLGA microspheres (57 mg/mL) in a 5 % aqueous

solution of Pluronic� F68 (Sigma), incubated for 9 h at 4 �C

and freeze-dried. The implants (6 mm of diameter 9 2 mm

height) were obtained by compression of 75 mg of the

freeze-dried blend (83 % HA, 9 % PLGA microspheres and

8 % Pluronic�) at 35 MPa, at room temperature for 5 min.

The preparation of the implants, containing 300 or

600 ng of PDGF, was performed in aseptic conditions. All

implants were stored at 4 �C until use.

Uniform distribution of 125I-PDGF/PDGF in each indi-

vidual blend was determined before compression by

radioactivity measurement in a gamma counter (Cobra� II,

Packard) in three aliquots of 10 mg each. After compres-

sion, homogenous PDGF loading of each implant was also

tested by radioactivity measurement.

2.3 Microporous structure of the implants

Implant structure was examined by scanning electron

microscopy (Jeol JSM-6300) at 20 kV. The dried samples

were coated with gold–palladium under argon atmosphere.

Pore diameter distribution and the porosity of the

implants were measured by mercury intrusion porosimetry

(Autopore IV 9500, Micromeritics Instrument Co.).

Determination of pore size distribution was based on the

relationship between the applied pressure and the diameter

of the pores into which mercury intrudes, according to the

Washburn equation [18]. A mercury surface tension of 484

dyn/cm and a contact angle between the mercury and the

pore wall of 1418 were used.

The Implant specific surface area was determined by

nitrogen adsorption (ASAP 2020, Micromeritics Instru-

ment Co.). The BET adsorption theory and the BET

method for data reduction were used to estimate the BET

specific surface area.

2.4 125I-PDGF in vitro release assay

In vitro release assays were carried out by incubating one

implant in 2 mL of Dulbecco’s Modified Eagle’s Medium

(DMEM, Sigma) supplemented with 5 % of fetal bovine

serum (FBS, Gibco) 1 % penicillin–streptomycin (Gibco),

0.5 % 200 mM L-glutamine (PAA Laboratories) and

0.02 % sodium azide at 37 �C, 5 % CO2 and a relative

humidity of 95 %, under 75 rpm of orbital shaking (Orbital

shaking platform POS-300, Grant-bio). In order to avoid a

1904 J Mater Sci: Mater Med (2012) 23:1903–1912

123

potential protein adsorption to the delivery system com-

ponents, the total volume of the medium was withdrawn

and replaced by fresh solution every day. Radioactivity of

the samples was measured using a gamma counter (Cobra

II, Packard). The assay was carried out in triplicate.

In parallel, 125I-PDGF radiolabeling stability in the release

medium was checked by paper chromatography. To this end, a

solution of 125I-PDGF (0.5 lCi/mL) in release medium was

incubated as indicated above. At specific time intervals, an

aliquot (10 lL) was spotted on a paper strip (1 9 15 cm;

3MM Chr., Whatman�) and chromatography carried out with

85 % methanol in water along a distance of 9 cm. Once fin-

ished, the band was cut into six parts (from the starting point to

the front) which were subjected to radioactivity measurement

in the gamma counter. With this chromatographic system, the

free 125I- reaches the front (Rf = 1) and the 125I-PDGF is

retained at the starting point (Rf = 0).

2.5 Animal experiments

All the experiments were carried out in conformity with the

Guidelines on Care and Use of Animals in Experimental

Procedures (Directives 2010/63/EU and the Spanish R.D.

1201/2005). Furthermore, animal experiments were previ-

ously approved by the local committee for animal studies

of the University of La Laguna. All experiments were

carried out in aseptic conditions.

2.5.1 Surgical procedure: bone defect

Surgery to produce the bone defect was performed as pre-

viously described [19]. Briefly, male New Zealand rabbits

(3–4 kg) were anaesthetized intramuscularly with ketamine

(35 mg/kg) and xylazine (5 mg/kg) and their right hind legs

shaved and disinfected. A vertical external parapatellar

incision was made in the knee. Then, a dislocation of the

patellar tendon and quadriceps was performed to allow

access to the femoral condyles. A hole of 1.5–2 cm in depth

to reach the medullar cavity was made in the intercondylar

space with a 6 mm dental burr. The implants were inserted in

the damaged femur, and then the patella and the patellar

tendon were reduced. The surgical wound was closed with

stitches and disinfected. Subcutaneous injections of bupr-

enorphine (50 lg/kg/12 h, Buprex�) were administered for

72 h to reduce post-surgical pain. After recovery from the

operation (20–30 min), the animals were allowed free

movement, food and water uptake.

2.5.2 In vivo release assay and biodistribution

The release assay and determination of PDGF levels were

carried out in a group of 12 rabbits. 125I-PDGF release

kinetics from the implants, inserted in the defect, was

followed up by radioactivity measurements using the

gamma counter. At each sampling time point, three

rabbits were sacrificed, the femur extracted and the

implants removed. The femurs, freed of soft tissues,

were divided into three pieces: distal metaphysis (DM)

corresponding to the implantation site, distal diaphysis

(DD) and the remaining bone (RB). In order to deter-

mine 125I-PDGF biodistribution, muscle around the femur

and blood samples were also collected. Radioactivity of

all samples was measured in the 125I energy range

(15–75 keV) for 30s.

2.5.3 Histological and histomorphometrical analysis

In order to assess potential effects of released PDGF on the

surrounding tissue, four groups of three rabbits each were

prepared for histological examination. Groups were clas-

sified as follows:

– Control group: non-treated bone defects

– Blank group: two implants without PDGF

– Group P600: two implants containing a total dose of

600 ng of PDGF

– Group P1200: two implants containing a total dose of

1,200 ng of PDGF

To label the mineralization front, animals were injected

oxytetracycline-HCl (40 mg/kg, IM) 4, 3, 2, and 1 week

before sacrification.

The rabbits were sacrificed 4 weeks post-implantation

and the implant bearing femurs (three specimens of each

experimental group) prepared for histological evaluation.

The femurs were fixed in 10 % formalin solution (pH 7.4),

dehydrated in a graded series of ethanol, and embedded in

methyl methacrylate. Following polymerization, 10 lm

thick longitudinal sections were prepared throughout the

implant, using a sawing microtome (LEICA SM 2500).

Sections were stained with Goldner’s Trichrome to identify

new bone formation or left unstained for detection of

fluorochrome labels or immunolabeled with an anti-Von

Willebrand factor polyclonal antiserum, a blood vessel

marker, to identify neovascularization. Specimens were

inspected with a light microscope (LEICA DM 4000B)

coupled to a digital camera. All sections per specimen were

evaluated for histomorphometrical analysis, using com-

puter based image analysis software (Leica Q-win V3 Pro-

image analysis system, Barcelona, Spain). Bone neofor-

mation was quantified in a predetermined region of interest

(ROI), defined as the tissue within the defect site and the

transition zone to the host bone. The ROI was set using a

square of 7 9 7 mm covering the defect site (see Fig. 1).

New bone formation was distinguished from the implant

through structure and color differences. Quantification was

carried out in four adjacent sections in the middle of the

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implant, selecting a fixed threshold for positive stain (green

for Goldner’s Trichrome), and values expressed in mm2.

The distance between tetracycline labels was also

measured inside the ROI, in ultraviolet light for calculation

of the mineral apposition rate (MAR). MAR was expressed

in lm/day.

Quantitative evaluation of neovascularization was per-

formed by determining blood vessel density and vessel

surface area within the ROI. To this end, sections were

immunolabeled with an anti-Von Willebrand factor poly-

clonal antiserum (DAKO, Barcelona, Spain). Briefly, sec-

tions were deplastified in xylene–chloroform (1:1) and

rehydrated in a gradient of ethanol and distilled water and

then transferred to TBS buffer (pH 7.4, 0.1 M), which was

also used for all further incubation and rinse steps. Sections

underwent antigen retrieval in Tris–EDTA buffer (pH 9.0,

10:1 mM) at 65 �C for 20 min and were then blocked in

bovine fetal serum at 2 % in a TBS-Triton X-100 solution.

The indirect immunohistochemical procedure was carried

out by incubating the sections overnight at 4 8C with the

above mentioned antiserum (1/50). After a rinse step,

sections were incubated sequentially with biotin-SP con-

jugated F (ab’) fragment Donkey anti-Rabbit (Millipore,

Barcelona, Spain) (1/1,000) for 60 min and a streptavidin–

peroxidase complex (Millipore, Barcelona, Spain)

(1/1,000) for another 60 min. Peroxidase activity was

revealed in Tris–HCl buffer (pH 7.6, 0.05 M), containing

0.04 % of 4-chloro-1-naphtol (Sigma, Poole, UK) and

0.01 % of hydrogen peroxide. Labeling specificity was

assessed by replacing the specific antiserum by normal

serum. Vascular density and vessel surface area were

determined simultaneously in four adjacent sections in the

middle of the implant, selecting a fixed threshold for

positive stain (blue for immunohistochemical label). The

specific immunolabeling allowed counting by image

analysis software and determination of the blood vessel

areas. Blood vessel density was expressed in absolute

values and vessel surface area in mm2.

Statistical analysis was performed with SPSS software

using one-way analysis of variance (ANOVA) with a

Tukey multiple comparison post-test. Significance was set

at P \ 0.05. Data are presented as mean ± SD.

3 Results

3.1 Implant characteristics

The variability of 125I-PDGF distribution in the freeze

dried blend of 83 % HA, 9 % PLGA microspheres and 8 %

Pluronic� F-68, expressed as mean variation coefficient,

was 6.2 % before compression. PDGF radioactivity, mea-

sured in individual implants after compression, confirmed a

similar variability in GF content, indicating that the fabri-

cation process allowed homogeneous 125I-PDGF/PDGF

loading.

The implants presented 67 % of porosity with a uni-

modal pore size distribution, a median pore diameter in

volume of 50.9 nm (Fig. 2), and a BET specific surface

area reaching 32.09 ± 0.24 m2/g.

3.2 In vitro and in vivo release assays

Release kinetics for PDGF was analyzed using 125I-PDGF.

Approximately 13 % of free 125I- was detected at the end

of the in vitro release assay, demonstrating good radiola-

beling stability.

The in vitro and in vivo release profiles were practically

superimposed (Fig. 3a) and a linear correlation was

observed (y = 0.96 9 -2.62, R2 = 0.9896). The implants

led to a rapid burst release of approximately 40 % of the

loaded PDGF within the first 24 h, followed by a release

rate of approximately 6 % per day during the next 4 days,

reaching about 65 % of release at day 5. Thereafter, a

slower release was observed, and approximately 80 % of

the loaded PDGF was liberated by day 10. Afterwards,

practically no further PDGF release was detected (data not

shown).

3.3 Biodistribution

Tissue distribution of the PDGF, released from 2 implants

(600 ng of PDGF), was analyzed in femurs, surrounding

muscle and blood (Fig. 3b). The PDGF concentration was

higher at the implantation site and declined with distance.

Maximum PDGF levels (around 7 ng/g bone tissue)

were observed at the defect site (distal metaphysis) 24 h

post-implantation and did not decline below 6 ng/g during

Fig. 1 Horizontal section of a rabbit femur with implants in the

region of the epiphysis and metaphysis. The two small rectangles in

dark grey represent the two implants and the big rectangle of

7 9 7 mm in soft grey indicates the ROI, wherein new bone

formation, mineral apposition rate, vessel surface area, and blood

vessel density were measured at 4 weeks post-implantation

1906 J Mater Sci: Mater Med (2012) 23:1903–1912

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the first 5 days. From then on, GF levels diminished con-

tinuously, reaching a bone concentration of 2 ng/g at day

10 of the experiment. The concentrations found in the rest

of the femur were lower. PDGF levels in distal diaphysis

stayed around 1 ng/g during the 10 days of assay duration

(Fig. 3b). On the other hand, PDGF concentrations in the

remaining femur, muscle and blood were very low and did

not differ significantly from the background of the gamma

counter.

3.4 Histological and histomorphometrical evaluation

Histological evaluation was performed in the region of

interest (ROI), 4 weeks post-implantation. All treated

groups demonstrated good compatibility of the material

with the host tissue; no signs of inflammation were

observed.

No significant differences in new bone formation

(Fig. 4) and mineral apposition rate (Fig. 5) between ani-

mals with non-treated bone defects (control group) and

animals with two implants without PDGF (blank group)

were observed. However, significant differences in blood

vessel density and vessel surface area (Fig. 6) were

observed between the control and the blank group. Con-

nective tissue filled the defect site in the control group and

surrounded the implants in the blank group (Fig. 4). Sev-

eral small areas of new bone formation were detected at the

defect sites of the control group and around the implants of

the blank group (Fig. 4).

Fig. 2 SEM microphotograph of phosphate/polymer implant surface

(a). Pore size distribution of implants determined by mercury

intrusion porosimetry (b)Fig. 3 Cumulative 125I-PDGF release profiles obtained from

implants incubated in DMEM medium (in vitro) or after implantation

in a rabbit femur defect (in vivo) (a). PDGF concentrations in

different tissues of rabbits implanted with systems containing 600 ng

of PDGF (b). DM distal metaphysis (implantation site), DD distal

diaphysis, RB remaining bone

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Significant differences in all evaluated parameters were

found between the control and the blank group with respect

to the groups with two implants containing 600 ng of

PDGF (P600) and also the ones containing 1,200 ng of

PDGF (P1200). More extensive areas of new bone for-

mation were found in the PDGF treated groups (P600 and

P1200) compared to groups control and blank (Fig. 4).

Mineralized bone trabeculae of variable size around the

implants and in between the fragments were observed in

both PDGF treated groups (P600 and P1200) (Fig. 4).

Likewise, bone ingrowth into the implants was also

detected (Fig. 4). Osteoblastic activity was high in both

groups (P600 and P1200), being reflected by the presence

of areas of non-mineralized bone matrix (osteoid) (Fig. 4).

Osteoclasts on bone surface trabeculae, indicated a

resorption and remodeling process.

Histomorphometrical evaluation of the ROI revealed

significant differences in new bone formation between the

control and the blank group, on the one hand, with areas of

newly formed bone of 0.36 ± 0.11 and 0.26 ± 0.11 mm2,

and the groups P600 and P1200, with 1.89 ± 0.38 and

1.77 ± 0.55 mm2, on the other hand (Fig. 4).

In order to evaluate the mineral apposition rate, fluoro-

chrome labels were administered. Four labels of tetracy-

cline can be clearly observed, demonstrating progressive

new bone formation during the 4 weeks. By the end of the

forth week, mineralization rates differed significantly

between groups control and blank, on the one hand, with

mineral apposition rates of 2.16 ± 0.37 and 2.15 ±

0.40 lm/day, respectively and groups P600 and P1200,

with mineral apposition rates of 2.74 ± 0.42 and

2.79 ± 0.39 lm/day, on the other hand (Fig. 5). Extensive

fluorochrome labels were found throughout the specimens

of the groups treated with PDGF (P600 and P1200),

whereas the implants without PDGF (blank group) and the

non-treated bone defects (control group) only stimulated

bone repair at the defect site and its border zones (data not

shown).

Neovascularization was identified in the defects of the

control group and adjacent to the implants of the blank

group. Big blood vessels had formed in the groups treated

with PDGF (P600 and P1200) adjacent to and in between

the fragments of the implants. Blood vessels were identi-

fied in Goldner’s Trichrome sections (Fig. 6 upper row) as

well as in sections immunolabeled with anti-Von Wille-

brand factor polyclonal antiserum (Fig. 6 bottom row),

Fig. 4 Histological specimens of rabbit femurs and new bone

formation within the ROI at 4 weeks post-implantation in the control

group (non-treated bone defects), the blank group (implants without

PDGF), the group P600 (implants containing 600 ng of PDGF), and

the group P1200 (implants containing 1,200 ng of PDGF). Images of

horizontal sections at lower magnification (Left column; scale bar160 lm), showing the bone defect area in specimens from each group.

Magnification (Right column; scale bar 50 lm) of the bounded areas

demonstrates fibrous tissue and intramembranous ossification foci

(arrows) in the control and the blank group, and morphology of newly

formed bone trabeculae (arrows) in the groups P600 and P1200. BMabone marrow, FbT fibrous tissue, MB mineral bone, Imp: implant, Ostosteoid, Ot osteocites (arrowheads), v blood vessels

c

1908 J Mater Sci: Mater Med (2012) 23:1903–1912

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which were both applied for counting and measuring.

Blood vessel density and vessel surface area differed sig-

nificantly in the control and the blank group with respect to

groups P600 and P1200 (Fig. 6). Significant differences in

both parameters were also found between the control and

the blank group (Fig. 6).

Groups P600 and P1200 did not differ significantly from

each other in any of the evaluated parameters.

4 Discussion

The objective of this study was to optimize a PDGF for-

mulation, taking into account three aspects. First, system

characteristics should enhance or at least not hinder defect

healing. Second, the system should be capable of mim-

icking physiological presence of PDGF in damaged tissue.

And third, the applied doses should be adequate to produce

Fig. 5 Mineral apposition rates,

determined within the ROI in

histological specimens from

rabbit femurs at 4 weeks post-

implantation in the control

group (non-treated bone

defects), the blank group

(implants without PDGF), the

group P600 (implants

containing 600 ng of PDGF),

and the group P1200 (implants

containing 1,200 ng of PDGF).

Fluorochrome (tetracycline)

labeling of the mineralization

front; doses were given weekly

throughout the experimental

period of 4 weeks. Inter-label

thickness was determined by

image analysis to calculate the

mineral apposition rate.

Scale bar 50 lm

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123

angiogenic and osteogenic effects, leading to a good bone

repair response.

Implants, employed for bone regeneration, should ide-

ally serve as delivery systems as well as scaffolds for bone

ingrowth. Consequently, a porous matrix is needed to

promote tissue formation. Despite the fact that no porogen

was used for implant fabrication in this study, the inclusion

of microspheres, made of PLGA and Pluronic� F68, may

have increased the overall porosity with time and thus

favored bone formation [20]. Pluronic dissolves and the

polymer degrades, forming short, soluble chains. Conse-

quently, a progressive increase in porosity was expected.

Moreover, inclusion of a surfactant in the composition of

implants decreases PDGF adsorption. It should be noted

Fig. 6 Neovascularization

determined within the ROI in

histological specimens at

4 weeks post-implantation in

the control group (non-treated

bone defects), the blank group

(implants without PDGF), the

group P600 (implants

containing 600 ng of PDGF),

and the group P1200 (implants

containing 1,200 ng of PDGF).

Horizontal sections demonstrate

the presence of blood vessels in

connective tissue in the control

and the blank group

(arrowheads), and in between

mineral bone trabeculae in the

group P600. Goldner’s

Trichrome staining (upper line)

and anti-Von Willebrand factor

immunolabeling (bottom line).

Scale bar 50 lm. BMa bone

marrow, FbT fibrous tissue,

MB mineral bone, Imp implant,

v blood vessels

1910 J Mater Sci: Mater Med (2012) 23:1903–1912

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that in vivo GF adsorption might be low or non-existent, as

many of the physiologically present proteins and ions may

act as surfactants and/or compete for binding sites or

change the ionic interaction, thus inhibiting or reducing

such adsorption [21]. However, in the current study, the in

vitro and in vivo release profiles were similar, probably due

to the components of the implant and composition of the

release medium. All these technological strategies also lead

to a lack of GF retention within the matrix. Clearly, the

porous matrix structure, required to promote tissue for-

mation, provided a fast release of PDGF. However, it must

be highlighted that PDGF acts early in the wound-healing

cascade [22], and a too late temporal presentation could

even hamper the regeneration process. In the present work,

in addition to achieving a release profile of PDGF that

fitted well with the temporal expression pattern of PDGF

during fracture healing [23], the effective levels and their

period of maintenance were monitored.

After administration of two implants, containing a total

dose of 600 ng of GF, PDGF levels in different tissue

samples revealed that the GF remained located around the

implantation site with a negligible systemic exposure. The

PDGF release kinetics was found to be perfectly reflected

on the tissue level (Fig. 3a, b). The bone peak PDGF

concentration was reached on the first day with a release of

40 %, and similar levels were maintained in the bone

defect throughout the first five days. Afterwards, PDGF

levels in the target defect diminished rapidly as the reduced

release could no longer overcome the fast clearance from

the bone.

Histological analyses revealed similar effects with doses

of 600 ng compared to 1,200 ng of PDGF, demonstrating

that doses higher than 600 ng do not produce an increase in

the repair response. This result can be explained by

described regulatory mechanisms of action of PDGF [24,

25]. Too high doses of PDGF cannot induce a further

increased response; they possibly will even inhibit acti-

vating signaling through regulatory mechanisms. However,

both doses tested in this study, induced a higher degree of

bone regeneration and vascularization in terms of new bone

formation, mineral apposition rate, vessel surface area, and

blood vessel density than the blank implants. These results

suggest that the PDGF dose–response in vivo may have

reached a plateau. Importantly, comparison of PDGF

treated groups with normal animal bone revealed that

approximately 50 % of the defect was filled with newly

formed bone after 4 weeks. Accordingly, a very big

amount of bone was formed during this short period. The

present results demonstrate the benefit of optimizing the

PDGF dose and the in vivo release kinetics to control local

concentrations and persistence of PDGF in the damaged

tissue. Therefore, our results match with Jin et al. [26], who

provided evidence of the importance to control dose and

release kinetics of PDGF to induce tissue formation and

neo-angiogenesis in a non-bone rat model.

5 Conclusions

The present study demonstrates that dose and controlled

release rate form the key elements in promoting tissue

repair in an in vivo application of PDGF. The formulation,

suggested in this work, was designed to offer a physio-

logically relevant PDGF profile for bone repair. As a result,

a high level of regeneration was achieved within only four

weeks. In conclusion, PDGF showed evidence of a sig-

nificant potential for hard tissue engineering application,

assigning the GF a possibly higher efficacy within short

periods of time than other osteogenic factors. Nonetheless,

further studies are still required to determine whether

PDGF alone could be enough for bone repair, or if com-

binations with other bioactive substances are still required.

Acknowledgments This work was supported by the Ministry of

Science and Technology (MAT2008-02632/MAT to CE) and Hos-

piten Holding (Convenio ULL-CI02320701). R. Reyes was financed

by the Motiva Project of ACIISI and thanks FUNCIS for continuous

support. We thank Martina K. Pec for assistance with manuscript

preparation.

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