Injury, Int. J. Care Injured 43 (2012) 334–342

Effect of triple growth factor controlled delivery by a brushite–PLGA system ona bone defect

Ricardo Reyes a, Beatriz De la Riva a, Araceli Delgado a, Antonio Hernandez b, Esther Sanchez a,Carmen Evora a,*a Department of Chemical Engineering and Pharmaceutical Technology, University of La Laguna, 38200 La Laguna, Spainb Traumatology Service, Hospiten Rambla, Santa Cruz de Tenerife, Spain

A R T I C L E I N F O

Article history:

Accepted 9 October 2011

Keywords:

Vascular endothelial growth factor (VEGF)

Platelet derived growth factor (PDGF)

Transforming growth factor beta 1 (TGF-b1)

Brushite–PLGA delivery system

Bone regeneration

Histology

Immunohistochemistry

Histomorphometry

Mineral apposition rate (MAR)

A B S T R A C T

Bone regeneration is a complex process that involves multiple cell types, growth factors (GFs) and

cytokines. A synergistic contribution of various GFs and a crosstalk between their signalling pathways

was suggested as determinative for the overall osteogenic outcome.

The purpose of this work was to develop a brushite–PLGA system, which controls the release rate of

the integrated growth factors (GFs) to enhance bone formation.

The brushite cement implants were prepared by mixing a phosphate solid phase with an acid liquid

phase. PDGF (250 ng) and TGF-b1 (100 ng) were incorporated into the liquid phase. PLGA microsphere-

encapsulated VEGF (350 ng) was pre-blended with the solid phase. VEGF, PDGF and TGF-b1 release

kinetics and tissue distributions were determined using iodinated (125I) GFs.

In vivo results showed that PDGF and TGF-b1 were delivered more rapidly from these systems

implanted in an intramedullary defect in rabbit femurs than VEGF. The three GFs released from the

brushite–PLGA system remained located around the implantation site (5 cm) with negligible systemic

exposure. Bone peak concentrations of approximately 4 ng/g and 1.5 ng/g of PDGF and TGF-b1,

respectively were achieved on day 3. Thereafter, PDGF and TGF-b1 concentrations stayed above 1 ng/g

during the first week. The scaffolds also provided a VEGF peak concentration of nearly 6 ng/g on day 7 and

a local concentration of approximately 1.5 ng/g during at least 4 weeks. Four weeks post implantation

bone formation was considerably enhanced with the brushite–PLGA system loaded with each of the three

GFs separately as well as with the combination of PDGF and VEGF. The addition of TGF-b1 did not further

improve the outcome.

In conclusion, the herein presented brushite–PLGA system effectively controlled the release

kinetics and localisation of the three GFs within the defect site resulting in markedly enhanced bone

regeneration.

� 2011 Elsevier Ltd. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Injury

jo ur n al ho m epag e: ww w.els evier . c om / lo cat e/ in ju r y

Introduction

A variety of biomolecules like growth factors (GFs) and cytokinesas well as diverse cell types participate in the progression of bonerepair. A synergistic contribution of GFs and a crosstalk betweentheir signalling pathways were suggested to be determinative for theoverall osteogenic outcome.1–3 Many of these factors are expressedduring foetal skeletal development and also induced in response toinjury.4,5 The present study was focused on three GFs with animportant role in osteogenic regeneration, PDGF, TGF-b1 and VEGF.

* Corresponding author at: Av. Astrofısico Francisco Sanchez s/n, Facultad de

Farmacia, 38200, La Laguna, Tenerife, Spain. Tel.: +34 922 318507;

fax: +34 922 318506.

E-mail address: cevora@ull.es (C. Evora).

0020–1383/$ – see front matter � 2011 Elsevier Ltd. All rights reserved.

doi:10.1016/j.injury.2011.10.008

PDGF exhibits a potent stimulatory effect as a chemoattractant andmitogen for mesenchymal and osteogenic cells, along with its abilityto promote angiogenesis.6,7 PDGF also enhances bone regenerationindirectly by stimulating the expression of angiogenic moleculessuch as VEGF,8 an important GF during the initiation of fracturehealing9 and bone regeneration.10 VEGF is predominantly known forits role in vessel formation but also promotes endochondral andintramembranous ossification in bone growth,11,12 being involved inthe recruitment, survival and activity of bone forming cells.13–15

TGF-b1 is present in early stages of bone development as well asrepair and remodelling after trauma, regulating the proliferation,migration and differentiation of bone mesenchymal stem cells.16,17

Therefore, TGF-b1 affects osteoblast differentiation, matrix forma-tion and mineralisation.18

Although GFs are clearly implicated in harnessing and controllingcellular functions in tissue regeneration, the appropriate rate and

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342 335

combination to be presented at the desired site is crucial. Theapplication of delivery systems is a good strategy to control andlocalise the factors at the damage site during a defined period.Moreover, these systems avoid or reduce systemic distribution of theGFs, which could lead to undesirable side effects, insufficient localconcentrations and/or fast degradation. Concretely, a GF deliverysystem for bone tissue engineering should be fabricated withosteoinductive and osteoconductive biomaterials. Besides, it shouldbe resorbed and gradually replaced by newly formed bone. Calcium-phosphate compounds (CPCs), such as b-tricalcium phosphate (b-TCP) and brushite exhibit a higher resorption rate in vivo thanhydroxyapatite (HA) materials, allowing simultaneous materialresorption and bone formation.19,20 According to the literature, twobrushite systems were recently described; one for releasing thecytokine RANKL (Receptor activator of nuclear factor kappa bligand), a key stimulator within the bone remodelling process21 andanother one, fabricated by our group to release PDGF and VEGF.22

The latter provided a one week PDGF release, followed by a sustainedrelease of VEGF during more than four weeks and led to synergicbone formation.

Evidently, the release kinetics of multiple GFs from a systemshould be controlled temporally and spatially to obtain the desiredeffects. For this reason, the aim of the present study was toexamine the biological efficacy of a brushite–PLGA delivery systemcapable of releasing a combination of PDGF, TGF-b1 and VEGF in anorchestrated sequence in a bone defect created in rabbit femurs.

Materials and methods

Preparation of brushite implants

Brushite implants were made as previously described.22,23 First,1.428 g of b-tricalcium phosphate (b-TCP, Fluka) and 0.8 g ofmonocalcium phosphate (Sigma–Aldrich) were mixed with0.012 g of the chemical retardant, sodium pyrophosphate (Sig-ma–Aldrich). Cement was prepared by mixing 200 mg of the solidphase with 100 ml of 0.5 M citric acid solution using a mixer(Headolph Reax) for 30 s. To prepare the implants, one-face-openpolyethylene syringes (5 mm of internal diameter) were filled withthe cement paste and were left to harden at room temperatureduring 1 h. The implants were kept at 4 8C during 12 h before use.The setting temperature was approximately 20 8C.

PDGF (250 ng, Chemicon) and TGF-b1 (100 ng, Chemicon) wereincorporated in the liquid phase, whilst VEGF (350 ng, Chemicon),pre-encapsulated in 20 mg of PLGA (Resomer1 RG504, Boehringer-Ingelheim) microspheres, was included in the solid phase. 0.5–2 mCi of 125I-PDGF, 125I-TGF-b1 and 125I-VEGF (Perkin-Elmer) wereadditionally incorporated as tracers together with the pure GF. Allformulations contained PLGA microspheres with or without VEGF,depending on the experimental group.

PLGA microsphere fabrication

PLGA microspheres with a fixed VEGF/polymer loading of50 ng/mg were prepared using a double emulsion (w/o/w) process.Briefly, the first emulsion was made by vortexing (3 min) a VEGFsolution in 0.07% poly(vinyl)alcohol (PVA) with a PLGA methylenechloride solution. Then, the first emulsion was poured into 100 mlof a 0.1% PVA solution under constant stirring (2 h). Then,microspheres were collected by filtration, lyophilised and keptat 4 8C until use.

Mercury porosimetry

Pore diameter distribution and the porosity of the fabricatedsystems were measured using a mercury intrusion porosimeter

(Autopore IV 9500, Micromeritics Instrument Co.). Porosity wascalculated according to the Washburn equation.24

Scaffold loading

First, encapsulation efficiency of VEGF microspheres wasdetermined by measuring the radioactivity levels of three aliquotsusing a gamma counter (Cobra1 II, Packard). To determine totalloading GF, radioactivity of each implant containing one individualfactor was counted. These data were assumed to be valid forcalculating the dose of PDGF, TGF-b1 and VEGF included in thetriple systems. The spatial distribution of each GF in the brushite–PLGA systems was assessed by cutting them into three sections(upper–medium–low) and measuring the radioactivity of eachsection.23

In vitro release experiments

The in vitro release assays were carried out in triplicates. Briefly,implants or samples of PGLA microspheres were incubatedindividually in DMEM medium, supplemented with 2% FBS (Gibco)and 0.02% sodium azide, at 37 8C in a 5% CO2 atmosphere andrelative humidity of 95%, under orbital shaking at 75 rpm. Therelease of each GF was studied separately and the amount ofreleased GF calculated by measuring the radioactivity of eachsample. Radiolabelling stability of 125I-GF in the release mediumwas checked by thin layer chromatography (TLC), as previouslydescribed.21 The TLC was carried out in plastic silica gel (60 F254,Merck) (0.9 � 8 cm) stripes with 85% methanol in water. Theradioactivity of the 3 parts (starting point, medium and front) ofthe band was measured. With this chromatographic system, thefree 125I� reaches the front and the 125I-GF is retained at thestarting point.

The biological activity of the released VEGF was determinedusing human umbilical vein endothelial cells (HUVECs) aspreviously described.22 HUVECs were cultured in complete culturemedium, containing Medium 199 with Hanks’ BSS, 100 mg/l L-glutamine, 25 mM HEPES and 1.4 g/l NaHCO3 (BioWhittaker),supplemented with 20% of the standardised foetal bovine serumGold (PAA), 50 UI/ml penicillin and 50 mg/ml streptomycin.5 � 103 cells per well were seeded in 150 ml of medium in 96-well plates. Then, 150 ml of adequately diluted media directlyobtained from PLGA microspheres after a release period of 1 and 7days or of defined VEGF standard solutions (0–4 ng/ml) wereadded in triplicates. The VEGF concentrations in the collectedmedia were calculated according to the in vitro release kinetics. Onday five, cells were quantified using the XTT tetrazolium assay(Roche Molecular Biochemicals). Bioactivity of the released VEGFfrom the implants was determined by comparing the induced cellproliferation to the one with its equivalent VEGF standard solution.Untreated control cells served to verify the growth stimulatoryeffect of VEGF on HUVECs.

Scaffold water uptake and mass loss

Implants were incubated under the same conditions as in therelease assays. Water uptake and mass loss were determinedgravimetrically. At specific time points, three samples werewithdrawn, weighted, freeze-dried, the dried weight recordedand the percentages of water uptake and mass loss calculated.23

Animal experiments

All the experiments were carried out in conformity with the E.C.Guideline (86/609/CEE) on care and use of animals in experimentalprocedures. Furthermore, the animal experiments were previously

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342336

approved by the local committee for animal studies of theUniversity of La Laguna. All experiments were performed inaseptic conditions.

Surgical procedure: bone defect

The surgery to produce the intramedullary femur defect inrabbits was performed as previously described.25 Briefly, maleNew Zealand rabbits (3–4 kg) were anaesthetised with ketamine(35 mg/kg) and xylazine (5 mg/kg). A vertical external parapatellarincision was made in the knee of their right hind legs. Then, adislocation of the patellar tendon and quadriceps was performed toallow access to the femoral condyles. A hole in the intercondylarspace of 1.5–2 cm depth was made with a 6 mm dental burr andthe implant was inserted in the medullar cavity. The surgicalwound was closed with stitches and disinfected. Buprenorphine(Buprex1; 0.05 mg/kg, SC) was used pre-operatively (between 10and 30 min before surgical procedure) for preventive analgesia andpost-operatively every 8–12 h during 48 h. Ketoprofen (Ketofen1

1%; 4 mg/kg, SC) was injected at the end of surgical procedure toreduce post-surgical pain during the first night.

In vivo release and bio-distribution of PDGF, TGF-b1 and VEGF

These experiments were carried out with three groups ofrabbits (3–4 kg). One group of 15 animals received implantscontaining dispersed 125I-PDGF/PDGF and 20 mg of blank PLGAmicrospheres. The second group of 18 rabbits received implantscontaining dispersed 125I-TGF-b1/TGF-b1 and 20 mg of blankPLGA microspheres. The other group of 24 rabbits was implantedsolely with 125I-VEGF/VEGF microspheres incorporated in thebrushite implants.

Three rabbits per sampling time point were sacrificed, thefemurs extracted and the implants removed. The release kinetics ofeach GF was followed up by radioactivity measurements of theextracted implants and determined experimental durations (3weeks for PDGF, 4 weeks for TGF-b and 8 weeks for VEGF). Todetermine GF bio-distribution, the femurs were divided into 2pieces: implant area (the implant including piece of bone of 5 cm)and the remaining bone. Additionally, the radioactivity of musclearound the femur and blood samples was also assessed.

Histological and histomorphometrical analysis

To investigate the effect of the released PDGF, TGF-b1 and VEGFon the regeneration of the rabbit femurs, 8 groups of 6 animalseach were examined (Table 1).

To label the mineralisation front, the animals were injectedoxytetracycline–HCl (40 mg/kg, IM) and calcein blue (15 mg/kg,SC) twelve and four days before sacrification, respectively. Therabbits were sacrificed 4 weeks after implantation. The scaffoldbearing femurs (six specimens of each experimental group) wereprepared for histological evaluation. The femurs were fixed in 10%

Table 1Experimental groups for histological analysis (N = 48 animals).

Group Implant Dose (ng)

C None None

B Non-loaded None

P PDGF 250

V VEGF 320

T TGF-b1 100

VT VEGF + TGF-b1 320 + 100

VP VEGF + PDGF 320 + 250

VPT VEGF + PDGF + TGF-b1 320 + 250 + 10

formalin solution (pH 7.4), dehydrated in a graded series ofethanol and embedded in methyl methacrylate. Followingpolymerisation, 10 mm thick longitudinal sections were preparedthroughout the scaffold, using a sawing microtome (LEICA SM2500). New bone formation was identified by von Kossa andGoldner’s trichrome staining. Unstained sections were used todetect fluorochrome labelling and vascularisation was deter-mined by immunollabeling with anti-von Willebrand factorantiserum. The specimens were inspected by light microscopy(LEICA DM 4000B) and documented by means of a coupled adigital camera, LEICA DFC300 FX.

For histomorphometrical analysis, all sections per specimenwere evaluated using computer based image analysis software(Leica Q-win V3 Pro-image Analysis System, Barcelona, Spain). Thequantitative evaluation of newly formed bone was done bydetermining a region of interest (ROI), defined as the tissue withinthe defect site and the transition zone to the host bone. The ROIwas set using a rectangle of 5 � 5.5 mm positioned in the upperhalf of the defect, the region corresponding to the epiphysis (seeFig. 1) where new bone formation and osteointegration are betterevaluated. Within this ROI, new bone formation was distinguishedfrom the scaffold through structure and colour differences. Newbone tissue was quantified from six neighboured sections from thecentral zone of the scaffold by selecting a fixed threshold forpositive stain (black for von Kossa and green for Goldner’strichrome). Bone formation was expressed in mm2 based on thequantitative evaluation of the ROI.

The distance between oxytetracycline and calcein blue labelswas measured in ultraviolet light for calculation of mineralappositional rate (MAR). MAR was expressed in mm/day based onthe quantitative evaluation of the ROI.

The quantitative evaluation of neovascularisation was done bydetermining blood vessel density and vessel surface area withinthe ROI. For this purpose, sections were immunolabelled with ananti-von Willebrand factor polyclonal antiserum (DAKO, Barce-lona, Spain). Briefly, section were deplastified and rehydrated inTBS buffer (pH 7.4, 0.1 M), which was also used for all furtherincubation and wash steps. The sections underwent antigenretrieval in Tris–EDTA buffer (pH 9.0, 10:1 mM) at 65 8C for 20 minand were then blocked in bovine foetal serum at 2% in TBS–TritonX-100 solution. The indirect immunohistochemical procedure wascarried out by incubating the sections overnight at 4 8C with theanti-von Willebrand factor antiserum (1/50). After rising, sectionswere incubated sequentially with biotin–SP conjugated F (ab0)fragment donkey anti-rabbit (Millipore, Barcelona, Spain) (1/1000)for 60 min and streptavidin–peroxidase complex (Millipore,Barcelona, Spain) (1/1000) for another 60 min. Peroxidase activitywas 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% hydrogenperoxide. Immunolabelling specificity was controlled by replacingthe specific antiserum by normal serum. Blood vessel density wasexpressed in absolute value and vessel surface area in mm2 basedon the quantitative evaluation of the ROI.

Commentaries

Empty defect (control group)

Blank group

PDGF dispersed

VEGF-microspheres

TGF-b1 dispersed

VEGF-microspheres + TGF-b1 dispersed

VEGF-microspheres + PDGF dispersed

0 VEGF-microspheres + PDGF dispersed + TGF-b1 dispersed

Fig. 1. Horizontal section of a rabbit femur implanted with a brushite scaffold (Sc) in the regions of the epiphysis and metaphysis. The large rectangle indicates the scaffolds

location and the small rectangle defines the region of interest (ROI) in which the repair response (new bone formation, mineral apposition rate and neovascularisation) were

quantified. Scale bar: 0.5 mm.

Fig. 2. Evolution of the porosity and pore size distribution in brushite–PLGA system

during in vitro release. Data were obtained before (t = 0) and after 4 weeks of the

assay.

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342 337

Statistical analysis

Statistical analysis was performed with SPSS 18.0 softwareusing one-way analysis of variance (ANOVA) with a Tukey multiplecomparison post-test. Significance was set at p < 0.05. The resultsare shown as mean � SD.

Results

System characteristics

VEGF microsphere-encapsulation efficiency was 61.7 � 4.9%.The size of the brushite–PLGA system was 4.8 � 10.9 mm with anaverage weight of 224.9 � 2.5 mg. Radioactivity levels in the threesections of the implants ranged between 30% and 35% each, indicatinga homogeneous distribution of the GFs throughout the systems. Theporosity of brushite implants and brushite–PLGA systems wereapproximately 50%.

In vitro release

Release kinetics for the GFs encapsulated within the brushite–PLGA implants were subsequently analysed using 125I-VEGF or125I-TGF-b1 or 125I-PDGF separately. TGF-b1 and PDGF exhibitedsimilar release profiles. During the first day, the release rate ofPDGF was faster (47%) compared to 35% of TGF-b1; then,approximately 80% of both GFs were released by the end of thenext 2 weeks.

The release kinetics of VEGF from the PLGA microspheres wasstudied first. The release was approximately 35% during the first24 h and approximately 60% of VEGF were released within the firstweek. The inclusion of the microspheres into brushite implantsreduced the VEGF release rate. Primarily, the burst release wasreduced to less than 10%. Then, 45% of VEGF were deliveredthroughout the following two weeks of the experiment. During thelast weeks the release rate lowered down to less than 1% per day.

About 95% of the expected VEGF bioactivity was preservedthroughout the experiments and about 10–15% of free 125I� was

detected. Hence, during the in vitro release assays good radi-olabelling stability as well as VEGF bioactivity were observed.

Porosity, mass loss and water uptake

After 4 weeks of incubation in release medium, the porosity ofthe brushite–PLGA system resembled that of the intact system,which was about 50%. However, a slight shift of pore sizedistribution to smaller sizes was detected (Fig. 2). These resultsfit well with the moderate water uptake capacity of approximately10% and mass loss of about 30%.

In vivo release

VEGF, TGF-b1 and PDGF release kinetics from the implants weremonitored separately. The remaining 125I-PDGF or 125I-TGF-b1 in

Fig. 4. 125I-VEGF, 125I-TGFb1 and 125I-PDGF concentrations achieved in the different

areas of rabbit femurs after brushite–PLGA implantation (n = 3).

Fig. 3. In vivo release profiles of 125I-PDGF, 125I-TGFb1 and 125I-VEGF incorporated

in brushite–PLGA system. Inset: in vitro–in vivo PDGF, TGFb1, VEGF correlation

obtained from the brushite–PLGA system (n = 3).

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342338

the systems was measured throughout 3 weeks. PDGF and TGF-b1were delivered rapidly from these scaffolds; the release profileswere practically superimposed. Similarly as in vitro, approximately40% of the GFs were liberated within the first 24 h (Fig. 3). Thereafter,the release rates of both GFs were reduced to 5.5% per day during thenext 6 days, followed by a slower rate. A total release of 90% of the GFwas achieved after three weeks.

Furthermore, the release of VEGF, pre-encapsulated in PLGAmicrospheres from the brushite implants, was monitored. Acontrolled release of 7% per day took place during the first week.Then, the release rate dropped and stayed at 1.2% per day duringthe following three weeks. A release of almost 80% of VEGF wasachieved within the experimental period (Fig. 3).

A good in vitro–in vivo correlation was obtained for all three GFs(Fig. 3). The values of the slopes are situated within the 95% of theconfidence interval of 1.

GF bio-distribution

The distribution of the three GFs was analysed in femur and insurrounding muscle and blood. The highest GF concentrationswere located at the defect site. Then, the levels declined withdistance from that location; concentrations in the rest of the femurwere lower. GF concentrations obtained from different tissuesamples indicated a negligible systemic exposure (Fig. 4).

PDGF and TGF-b1 bio-distribution profiles were similar, asexpected according to their similar release kinetics. PDGF and TGF-b1 concentrations increased until reaching a bone peak level ofapproximately 4.5 and 1.5 ng/g, respectively on day 3. After thefirst week, the levels of both GFs stayed at about 1 ng/g until to theend of the experiment. A constant GF level of less than 0.5 ng/g wasobserved in the remaining bone throughout the whole experi-mental period (Fig. 4).

The bone peak concentration of VEGF was reached on day 7,again in the area where the brushite–PLGA system had been set.During the first 12–13 days, VEGF levels stayed high, above 2 ng/gtissue, with a maximum of 6 ng/g. Afterwards, VEGF concentra-tions close to 1 ng/g were maintained in the implantation zone aswell as in the remaining area throughout the rest of the experiment(Fig. 4).

Histological and histomorphometrical evaluation

Histological evaluation was performed in the ROI. Significantdifferences amongst groups B, P, V, T, VT, VP and VPT were found.No significant differences in new bone formation (Fig. 6g) andmineral apposition rates (Fig. 7g) between groups C and B wereobserved. Four weeks after implantation, limited bone formationadjacent to the defect site and bone ingrowth into peripheral zonesof the scaffold were detected in groups C and B. Zones of fibrosiswith fibroblasts located between the collagen fibres were observedin group C around the defect site (Fig. 5a) and in group B around thescaffold (Fig. 5b). Some regions displayed osteoblastic differentia-tion with newly formed bone invading the peripheral zones of thescaffold (Fig. 5b). Thus, there was good compatibility between thescaffold and the host tissue.

The repair response of groups P, T, V and VT was higher than ingroups C and B with very few signs of fibrosis. New bone formationwas detected adjacent to and beginning to invade the scaffold(Fig. 5c–f), with extensive zones of mineralised bone matrix in the GFgroups than in C and B. No differences amongst the GF groups weredetected (Fig. 6g). Groups VP and VPT were characterised by a higherdegree of new bone formation adjacent to and invading the scaffoldthan the other groups (Fig. 5g and h). Large zones of non-mineralisedbone matrix (osteoid), result of intense osteoblastic activity hadformed in all experimental groups, more markedly in VP and VPT(Fig. 6a). A high degree of osteointegration of the material wasobserved in all experimental groups. Tight interaction between thescaffold and the newly formed mineral bone (Fig. 6b) together withbrushite fragments integrated in the new bone tissue (Fig. 6d)emerged. Phagocytosis of scaffold fragments occurred (Fig. 6e). Lowbone resorption in the regions of newly formed bone was observedin all experimental groups (Fig. 6f). Histomorphometrical evaluationof the ROI revealed significantly larger areas of new bone formationin all treated groups compared to C and B. Additionally, bone neo-formation was significantly enhanced in groups VP and VPTcompared to groups P, T, V and VT (Fig. 6g).The evaluation of themineral apposition rate showed clear double labels of tetracyclineand calcein (Fig. 7a–f), demonstrating new bone formation at 4weeks post-surgery. The mineralisation rate slightly differedbetween groups C and B with respect to groups P, T, V, VT, VPand VPT. However, these differences were not statistically signifi-cant (Fig. 7g). Extensive fluorochrome labels were found throughoutthe specimen of the treated groups, whereas the scaffold alone and

Fig. 5. Histological evaluation. Histological specimens of rabbit femurs with empty defects (C) (a), implanted with brushite scaffolds (B) (b), PDGF loaded brushite scaffolds (P)

(c), TGFb1 loaded brushite scaffolds (T) (d), VEGF loaded brushite scaffolds (V) (e), TGFb1/VEGF loaded brushite scaffolds (VT) (f), PDGF/VEGF loaded brushite scaffolds (VP)

(g) and PDGF/TGFb1/VEGF loaded brushite scaffolds (VPT) (h). (a–g) Goldner’s trichrome staining, (h) von Kossa staining. The photomicrographs of horizontal sections

demonstrate bone neoformation (arrows) around the empty defect in group C (a) and around and invading the scaffolds in the other six groups (b–h). Fibrosis with fibroblasts

(arrowheads) amongst the collagen fibres can be observed in the area around the empty defect (a) and around the scaffold (b) in group B. There is only little fibrosis in group P

(c) and no fibrosis at all in the rest of the groups. Scale bars: (a–h) 60 mm; BMa, bone marrow; ED, empty defect; Fb, fibrosis; MdB, mineralised bone; O, osteoid; Ob,

osteoblast; Sc, scaffold.

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342 339

the empty defect only stimulated bone repair in the periphery of thedefect (data not shown).

Neovascularisation was evaluated in all experimental groups.Many blood vessels of variable size were seen amongst mineralisedbone trabeculae in groups P, V, VT, VP and VPT (Fig. 8b and d)compared to C and B (Fig. 8a and c) and group T. After 4 weeks,blood vessel density and vessel surface area differed significantly

between groups C, B and T compared to groups P, V, VT, VP and VPT(Fig. 8e and f).

Discussion

In this study, brushite was used as a carrier for the delivery ofPDGF, VEGF and TGF-b1. The elaborated release system intended

Fig. 6. Histological and histomorphometrical evaluation. Histological specimens of

rabbit femurs from group VP. a–c,e,f) Goldner’s Trichrome staining, (d) von Kossa

staining. Photomicrographs of horizontal section showing the morphology of newly

formed bone trabeculae and large zones of non-mineralised bone matrix (osteoid)

(O) (a). Details at high magnification pointing out the tight interaction between the

scaffold and the newly formed mineral bone (arrows) (b) and the presence of

mesenchymal cells (arrowheads) and blood vessels (V) within the scaffold structure

(c). A high degree of osteointegration was found in all experimental groups,

apparent for example by the presence of scaffold fragments integrated into the bone

structure as it shows here in group VP (d), phagocytosis of scaffold fragments

(arrows) (e) and specific resorption processes (f). Scale bar: (a) 40 mm; (b and c)

20 mm; (d) 30 mm; (e and f) 10 mm. BMa, bone marrow; MdB, mineralised bone; O,

osteoid; Ob, osteoblast; Oc, osteoclast; Sc, scaffold. V: blood vessel. (g)

Quantification of the new bone formation within the ROI of all experimental

groups. Data represent means � SD, **p < 0.001 (n = 6).

Fig. 7. Mineral apposition rates determined in histological specimens of rabbit

femurs of all experimental groups. (a–h) Fluorochrome labelling of the

mineralisation front (green = tetracycline, blue = calcein). Doses were given 12

and 4 days prior to euthanasia and interlabel thickness was determined by image

analysis to calculate the mineral apposition rate. Scale bar: 50 mm. (i)

Quantification of mineral apposition rates within the ROI of all experimental

groups. Data presented as means � SD. No significant differences were detected

(p < 0.05) (n = 6). (For interpretation of the references to colour in this figure legend,

the reader is referred to the web version of the article.)

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342340

to mimic the sequential interactions amongst three GFs involved innew bone formation. The efficacy of the system would stronglydepend on the GFs release kinetics. The present results provideevidence that distinct release rates for GFs can be achieved usingdifferent technological strategies. Moreover, the good in vivo–in

vitro correlations demonstrate that the in vitro release kineticsallowed for predicting the release profile in the bone defect. The in

vitro assay using cell culture conditions proved to be a good tool fortesting possible effects of changes in the formulation on the releaseprofile of the GFs.

The in vivo release profile of PDGF and TGF-b1 fitted well withthe expected early liberation. Clearly, the porous matrix structurerequired for the promotion of tissue formation provided fastrelease of PDGF and TGF-b1. On the contrary, as a consequence ofthe system-design, the achieved VEGF release rate from the

brushite–PLGA constructs was slower. The incorporation of thePLGA microspheres within the brushite cement efficiently reducedVEGF burst release and prolonged the delivery of the GF. The VEGFrelease profile from the brushite–PLGA system resembled the onepreviously obtained with VEGF pre-encapsulated in alginatemicrospheres included in brushite–chitosan composites.22 How-ever, the elaboration process of this new brushite–PLGA systemwas much simpler than the one used to prepare the brushite–chitosan constructs.

The GF levels obtained from the bone tissue samples reflectedwell their in vivo release profiles. Evidently, the parameters dose,release profile and pharmacokinetics of each GF define theconcentration detected in bone as well as the shape of the tissuedistribution curve. The release rates of PDGF and TGF-b1 duringthe first days were fast enough to achieve early maximumconcentrations at the implantation site, as expected. Afterwards,the elimination rates for both GFs were higher than the releaserates. Their local concentrations decreased due to their short half-lives of about 2 min.26,27 The differences in the area under the twobone level curves were mainly due to the distinct doses included inthe system. The 2.5 higher dose of PDGF compared to TGF-b1 wasclearly reflected in the values of the areas under the bone levelcurves. By contrast, higher bone concentrations of VEGF weremaintained due the higher dose and its longer half-life togetherwith its slower and continuous release from the system. Moreover,the GF levels obtained from different tissue samples verified that

Fig. 8. Neovascularisation determined in histological specimens of rabbit femurs of

all experimental groups. Representative images demonstrating the presence of

blood vessels in Goldner’s Trichrome sections of groups B (a) and VPT (b) and in

anti-von Willebrand immunostained sections of the same groups, B (c) and VPT (d).

Scale bar: 100 mm. BMa, bone marrow, MdB, mineralised bone, V, blood vessel.

Quantification of blood vessel density (e) and vessel surface area (f) of all

experimental groups. Data represent means � SD, *p < 0.05, **p < 0.001 (n = 6).

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342 341

they remained located around the implantation site with negligi-ble systemic exposure.

The effects of each individual GF and their combinationsencapsulated in the brushite–PLGA system on bone regenerationwere evaluated histologically. In agreement with other authorsusing various animal models, including rabbits28,29 a post-implantation period of 4 weeks with experimental brushitesystems in rabbit bone defects is long enough to observeangiogenesis and new bone formation.22 In the current experi-ments, the histological changes throughout the 4 weeks post-implantation also allowed for evaluation of induced bone

regeneration and angiogenesis. The results indicate that blankbrushite–PLGA scaffolds (group B) are not capable of significantlyenhancing bone formation compared to the empty defects (groupC). Nonetheless, several authors report that brushite is welltolerated, inducing only mild foreign-body reaction, which doesnot impair its replacement by newly formed bone within a fewweeks thus, permitting simultaneous bone formation and materialresorption.19,20,29,30

Contrasting with the blank implants, considerably enhancedbone formation was observed with the individual GFs. Thecombination of TGF-b1 and VEGF did not significantly augmentthe development of new bone tissue compared to the same dose ofTGF-b1 or VEGF alone. However, the dual application of PDGF/VEGF increased the formation of new bone compared to theindividual factors. In contrast, no further bone formation wasachieved by adding TGF-b1 as a third factor. Consequently, VEGFseems to be the essential factor to successfully regenerate bone.

The current results suggest that VEGF and its combination withPDGF are implicated in new bone formation and result in additiveeffects due to their significant impact on blood vessel density andvessel surface area. The herein presented data confirm ourprevious study with the two GFs in a brushite–chitosan system22

and corroborate the direct effects of VEGF and PDGF on bloodvessel maturation31 and indirect enhancement of bone regenera-tion. As expected, TGF-b1 and its combinations did not affect neo-vascularisation.

In spite of a positive effect of TGF-b1 on bone formation, theexpected synergist action of the dual and triple combinations wasnot observed. Analogous findings with TGF-b1 and VEGFcombinations had been reported in an Achilles tendon regenera-tion study.32 This lack of outcome could be due to various reasonssuch as a low dose, non-adequate release kinetics or both. A broadvariety of dosage of TGF-b1 was found in the literature, rangingfrom 20 ng to 120 mg doses,33–36 depending on the experimentalconditions such as animal species, type of defect, etc. The 100 ngdose, a conservative dose, was chosen according to previous workscarried out in rabbits where a bone-inducing effect had beenreported.34–36 It was also taken into account that repeatedsystemic administration of high doses of TGF-b1 to rats hadproduced lesions in multiple target organs including liver andkidney.37 Hence, we had expected a synergistic effect from theprolonged presence of TGF-b1 at the target site. However,antagonistic effects of TGF-b1 on VEGF activity reported by otherauthors32,38 were not detected in the present study, probably dueto the different release kinetics of TGF-b1 and VEGF. Therefore,further work must be done to definitely determine the optimalcombination and/or release kinetics to improve dual actions ofthese GFs.

Finally, although the results obtained with the injectedfluorochrome were not statistically significant different betweenthe study groups, an increase in the mineral apposition rate wasobserved in the groups treated with the different GFs alone or incombination. Again, the addition of TGF-b1 did not increase theeffect of the VEGF/PDGF combination.

Conclusions

Our results indicate that a delivery system which efficientlycontrols GF release during the required time can maintain effectiveGF concentrations in situ. This way, the application of GFs for boneregeneration can improve tissue response. It needs to be highlightedthat the effect depends on the material system, combination of GFs,their in vivo GF release kinetics, achieved local concentrations andtime frame in the damaged tissue. Herein, we suggest a brushiteformulation containing a combination of three GFs released in asequential rate around the implantation site with negligible

R. Reyes et al. / Injury, Int. J. Care Injured 43 (2012) 334–342342

systemic exposure. Our data further suggest that VEGF is a crucial GFin such systems to achieve successful tissue regeneration.

Conflict of interest statement

The authors declare that they have no conflict of interest relatedto this article.

Acknowledgements

This work was supported by the Ministry of Science andTechnology (MAT2008-02632/MAT to C.E.). R.R. was financed bythe Proyecto Motiva de la ACIISI. The authors would like to thankProf. Francisco Collia for technical support with histologicalspecimen processing. We thank Martina K. Pec for assistance withmanuscript preparation.

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