Available online at www.sciencedirect.com

Biomaterials 29 (2008) 1892e1900www.elsevier.com/locate/biomaterials

The effect of mesenchymal populations and vascular endothelialgrowth factor delivered from biodegradable polymer

scaffolds on bone formation

Janos M. Kanczler a, Patrick J. Ginty b, John J.A. Barry c, Nicholas M.P. Clarke d,Steve M. Howdle e, Kevin M. Shakesheff b, Richard O.C. Oreffo a,*

a Bone and Joint Research Group, Centre for Human Development, Stem Cells and Regeneration, Developmental Origins of Health and Disease,Institute of Developmental Sciences, University of Southampton, Southampton SO16 6YD, UK

b School of Pharmaceutical Sciences, University of Nottingham, University Park Nottingham, NG7 2RD, UKc InMotion Musculoskeletal Institute, South Dudley Street, Memphis, TN 38138, USA

d University Orthopaedics, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, UKe School of Chemistry, University of Nottingham, University Park Nottingham, NG7 2RD, UK

Received 26 October 2007; accepted 22 December 2007

Available online 29 January 2008

Abstract

The capacity to deliver, temporally, bioactive growth factors in combination with appropriate progenitor and stem cells to sites of tissue re-generation promoting angiogenesis and osteogenesis offers therapeutic opportunities in regenerative medicine. We have examined the bone re-generative potential of encapsulated vascular endothelial growth factor (VEGF165) biodegradable poly(DL-lactic acid) (PLA) scaffolds createdusing supercritical CO2 fluid technology to encapsulate and release solvent-sensitive and thermolabile growth factors in combination with humanbone marrow stromal cells (HBMSC) implanted in a mouse femur segmental defect (5 mm) for 4 weeks. HBMSC seeded on VEGF encapsulatedPLA scaffolds showed significant bone regeneration in the femur segmental defect compared to the scaffold alone and scaffold seeded withHBMSC as analysed by indices of increased bone volume (BV mm3), trabecular number (Tb.N/mm) and reduced trabecular separation(Tb.Sp. mm) in the defect region using micro-computed tomography. Histological examination confirmed significant new bone matrix in theHBMSC seeded VEGF encapsulated scaffold group as evidenced by Sirius red/alcian blue and Goldner’s trichrome staining and type I collagenimmunocytochemistry expression in comparison to the other groups. These studies demonstrate the ability to deliver, temporally, a combinationof VEGF released from scaffolds with seeded HBMSC to sites of bone defects, results in enhanced regeneration of a bone defect.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Bone tissue regeneration; Supercritical fluids; PLA scaffolds; VEGF; Segmental femur defect; Human bone marrow stromal cells

1. Introduction

Bone is a highly vascularised tissue with the unique abilityto heal and remodel spontaneously with minimal treatment orscarring [1]. However, segmental bone defects and non-unionsresulting from trauma, resection or pathology represent

* Corresponding author. Tel.: þ44 2380 798502; fax: þ44 2380 5525.

E-mail address: roco@soton.ac.uk (R.O.C. Oreffo).

URL: http://www.mesenchymalstemcells.org

0142-9612/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biomaterials.2007.12.031

significant clinical challenges world-wide, for the orthopedic,reconstructive and maxillofacial surgeons [2,3]. To date, bonegrafting techniques that use such graft materials as autografts,allografts or metallic implants face significant limitations forbone repair. The use of autologous bone grafts while highlysuccessful as a consequence of the transfer of osteoprogenitorcells, increased osteoinductive capacity and mechanical sup-port is limited by lack of sufficient material and/or inabilityto shape the material for the defect site. Alternatively, alloge-neic bone grafts from donors can circumvent some of theselimitations but despite being readily available, have limited

1893J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

osteogenic potential, and present a risk of disease transmission[4] and cell-mediated immune responses. These difficultieshave resulted in the search for alternative bone graftsubstitutes.

Recent advances in the field of regenerative medicine haveled to the possibility of the successful repair and restoration offunction in damaged or diseased tissues [5,6], especially in thefield of orthopedics. A variety of porous scaffold polymers,naturally derived or synthetic matrices have shown potentialfor bone tissue engineering applications offering new [7e9]approaches to cell and growth factor delivery. Scaffolds canact as a delivery vehicle for cells and a three-dimensionalstructure for tissue regeneration, providing specific cues toregulate osteogenesis [10e12]. One of the key issues in de-signing clinically translatable regenerative tissue is the gener-ation of a functional microvascular network within theengineered constructs to provide oxygen and nutrients to facil-itate growth, differentiation, and tissue functionality [1,13]. Aninadequate microvascular network will result in the hypoxiccell death of engineered tissues [14] leading to total implantfailure [15].

Angiogenesis is a fundamental process for osseous forma-tion and repair, and an early vascular response is essentialfor the normal progression of fracture healing. Vascular endo-thelial derived growth factor (VEGF), a potent growth factorinvolved in angiogenesis has been extensively investigatedand shown to be involved in osteogenesis and bone repair[16]. During endochondral bone formation VEGF modulatesangiogenesis, chondrocyte apoptosis, cartilage remodeling, os-teoblast migration and endochondral growth plate ossification[17e19]. Conversely, inhibition of angiogenesis has beenshown to prevent fracture healing [20] while inhibition ofVEGF decreases angiogenesis, callus mineralisation andbone healing [21].

A number of tissue engineered bone regenerative studieshave been undertaken at non-load-bearing sites [22], or in de-fects stabilized externally or by bone plates [23]. In this cur-rent study we have used an innovative mouse critical-sizedfemur defect model (load-bearing) to investigate the potentialbone regeneration capacity of a combination therapy of humanbone marrow stromal cells (HBMSC) seeded onto VEGF en-capsulated porous biodegradable poly(DL-lactic acid) (PLA)scaffolds. The scaffolds were prepared using supercriticalCO2 methods that in a single step process can prepare scaf-folds with controlled and interconnected porosity [24] andthat ensures that the bioactive VEGF is encapsulated withoutloss of activity [25,26].

2. Methods and materials

2.1. Materials

Recombinant human vascular endothelial growth factor-165 (VEGF) was

purchased from Tebu-bio, Peterborough, UK. Vibrant� carboxy fluorescein di-

acetate, succinimidyl ester (CFDA SE) cell tracer kit and Ethidium Homo-

dimer-1 were purchased from Molecular Probes, Leiden, The Netherlands.

Fetal calf serum was purchased from Invitrogen Ltd, Paisley, UK. All other

materials were obtained form SigmaeAldrich, Poole, UK, unless stated.

2.2. VEGF incorporation into poly(DL-lactic acid) (PLA)scaffolds using supercritical CO2 mixing technology

Recombinant human VEGF (10 mg) was resuspended in 120 ml of dH2O.

Twenty microliters of the reconstituted VEGF was pipetted onto 0.13 g of Pol-

y(DL-lactic acid) (MW 65 000) and mixed in Teflon moulds (10 mm deep and

10 mm in diameter). The VEGF/PLA mix was then lyophilised overnight in

a freeze drier. The PLA was then foamed using scCO2 to create VEGF encap-

sulated scaffolds as previously described [24]. For this, the polymer was plas-

ticised at 35 �C under a pressure of 17.32 MPa. Upon release of the pressure,

the pores are formed and fixed in the polymer structure thus creating a porous

monolith composite with the encapsulate VEGF (Fig. 1A, B). Scaffolds were

prepared for scanning electron microscopy (SEM) by cutting the scaffolds

transversely and gold coating using a Balzers Union SCD030 sputter coater.

Scaffolds were scanned at 20 kV with a Philips 505 scanning electron micro-

scope to view the inner pore structure of the scaffold. An average pore size of

250 mm and an average porosity of 70% was determined by micro-CT.

2.3. Isolation and culture of adult human bone marrow stromalcells (HBMSC)

Adult human bone marrow samples were obtained from hematological

normal patients undergoing routine elective hip replacement surgery for oste-

oarthritis. Only tissue that would have been discarded was used with the ap-

proval of the Southampton and South West Hampshire Local Research

Ethics Committee. Primary cultures of bone marrow cells were established

as previously described [27]. Cultures were maintained in basal medium

(a-MEM containing 10% FCS) at 37 �C in humidified air with 5% CO2.

2.4. In vivo studies: segmental femur defect model

Prior to implantation, PLA scaffolds were sterilized in an antibiotic/anti-

mycotic solution for 24 h and washed with 1� PBS. HBMSC (2 � 105)

were labelled with Vybrant (CFDA SE) and ethidium homodimer-1 according

to the manufacturer’s instructions prior to seeding onto the scaffolds. A 21-G

needle was inserted into the centre of the scaffold and placed at the bottom of

a 30 ml universal tube. Dropwise, half of the HBMSC suspended in culture

medium (1 ml) was added to the scaffold and agitated for 3 h. The scaffolds

were then rotated and again dropwise the rest of the HBMSC in TCM were

added to the other side of the scaffold with further agitation. The seeded scaf-

folds were then cultured in basal conditions for 24 h and examined for cell vi-

ability and cell necrosis (Fig. 1C).

For all in vivo studies male MF-1 nu/nu immunodeficient mice were pur-

chased from Harlan, Loughborough, UK and acclimatized for a minimum of 1

week prior to experimentation. Animals had ad libitum access to standard

mouse chow and water at all times, and all procedures were performed with

prior received ethical approval and carried out in accordance with the regula-

tions as laid down in the Animals (Scientific Procedures) Act 1986, UK

(Project License number: 70/6348). The animals were anaesthetised intraper-

itoneally with fentanylefluanisone (Hypnorm; JansseneCilag Ltd) and mida-

zolam (Hypnovel; Roche Ltd) in sterile water at a ratio of 1:1 and a dose of

10 ml/kg. The skin of the femur was cleansed by swabbing with Hydrex (Chlo-

rhexidine Gluconate 0.5% w/v in denatured ethanol B 70%). The transverse

skin incision made over the posterior aspect of the thigh and the femur was

exposed.

A 21-G needle was threaded through the greater trochanter and inserted

into the intramedullary canal, distally into the diaphysis of the femur, to

lock into the supracondylar metaphysis. Using a Sagittal saw, a 5 mm defect

was created in the femur (Fig. 1D). The stability of construct in both angula-

tory and rotational planes was tested manually and if found to be insufficient

the needle was reinserted. The 21-G needle was carefully withdrawn from the

distal end of the femur into the defect region being careful not to draw-out

fully from the femur. The needle was then threaded through the hole in the

centre of the scaffold (with or without seeded cells) which was carefully being

held using forceps. The needle was then threaded through the distal end of the

femur, fixed at both ends, and stability was ensured by palpation. Under asep-

tic technique the wounds were closed using interrupted Polydioxanone

Fig. 1. Critical-sized segmental femur defect model. SEM images at low magnification (A) and high magnification (B) of a representative PLA scaffold with en-

capsulated VEGF. Scale bar¼ 1 mm and 200 mm, respectively. HBMSC seeded onto VEGF encapsulated PLA scaffolds in vitro stained with Vybrant (CFDA SE)

(green) and ethidium homodimer-1 (red) prior to explanting into the femur segmental defect (C), and examined by fluorescent microscopy showing viable cells

(green) and some associated necrosis (red) on the seeded scaffold. Scale bar¼ 100 mm. Faxitron X-ray image of the MF-1 nu/nu mouse critical-sized femur seg-

mental defect model (z 5 mm) (D). Scale bar¼ 5 mm.

1894 J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

monofilament absorbable suture (Ethicon Edinburgh, UK). Postoperatively, the

mice were placed in an incubator where they were closely monitored until

fully recovered from the anaesthetic. All mice recovered fully from surgery

without any complications. The initial position of the defect was documented

radiologically and the presence and density of new bone formation in the fem-

oral defect was monitored radiographically in the mice thereafter. Four groups

were examined (i) control group without PLA scaffold (n¼ 4); (ii) control

PLA scaffold group (PLA) (n¼ 4); (iii) PLA seeded with HBMSC (PLAeHBMSC) group (n¼ 4); and (iv) VEGF encapsulated PLA seeded with

HBMSC (VEGF/PLAeHBMSC) (n¼ 4) using a Faxitron� Specimen Radiog-

raphy System (MX-20) (Qados Ltd, Sandhurst, UK) and histologically at 4

weeks postoperatively. Femurs were dissected and placed in periodatee

lysineeparaformaldehyde (PLP) fixative and analysed using micro-computed

tomography.

2.5. Micro-computed tomography

Quantitative three-dimensional analysis of the femur defect samples were

analysed using an X-TEK BenchTop 160Xi CT scanning system for micro-

computed tomography (X-TEK Systems Ltd, Tring, Hertfordshire, UK) equip-

ped with a Hamamatsu C7943 X-ray flat panel sensor (Hamamatsu Photonics,

Welwyn Garden City, Hertfordshire, UK). Defect samples were held securely

in a 5-ml Bijoux tube and centered in the middle of the mCT X-TEK machine.

Samples were focused, calibrated and adjusted to prevent X-ray saturation of

the sample. Following calibration, samples were scanned using settings

100 kV, 70 mA using a molybdenum target with an exposure time of

2134 m/s and 1� digital gain with the number of frames at 16 and the number

of angular positions at 1813 and scans were performed with minimised ring

artifacts. Raw data were collected and reconstructed using Next Generation

Imaging (NGI) software package version 1.4.59 (X-TEK Systems Ltd) with

a 16 mm voxel resolution. All the voxels which formed the structure were au-

tomatically assigned Hounsfield units.

The reconstructed images were visualised and analysed using Volume

Graphics (VG) Studio Max 1.2.1 software package (Volume Graphics,

GmbH, Heidelberg, Germany) for Bone Volume (BV), Bone Surface/Bone

Volume (BS/BV), Bone Volume/Total Volume (BV/TV), Trabecular Number

(Tb.N) Trabecular Thickness (Tb.Th), Trabecular Spacing (Tb.Sp.). Three-di-

mensional images were created and saved as TIFF and JPEG interchangeable

files. After the reconstruction a constant region of interest (ROI) was centered

over the defect region and sections of bone segmentation selected for structural

analysis. Thresholds were selected manually to exclude soft tissue and scaffold

allowing for bone and total volume to be quantified.

2.6. Histology

Following mCT analysis, femurs were washed and decalcified as detailed

by Miao and Scutt [28] and progression of decalcification was checked by mi-

cro-X-ray analysis (Faxitron). Following decalcification, samples were dehy-

drated in a graded series of alcohols and embedded in low-melting point

paraffin using an automated Shandon Citadel 2000. Sections (6 mm) were

cut and stained for the nuclear counterstain Weigert’s haematoxylin, followed

by staining with 0.5% Alcian blue 8GX for proteoglycan-rich cartilage matrix

and 1% Sirius red F3B for collagenous matrix or stained for Goldner’s

trichrome to detect bone and osteoid according to standard protocols.

Alkaline phosphatase activity quantification was performed as previously

described [28]. Briefly, deparaffinised and hydrated sections were preincu-

bated overnight in 1% magnesium chloride in 100 mM Trisemaleate buffer

(pH 9.2) and then incubated for 2 h at room temperature in freshly prepared

1895J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

ALP solution (100 mM Trisemaleate buffer, pH 9.2þ 0.2 mg/ml naphthol AS-

MX phosphate and 0.4 mg/ml Fast Red TR). Sections were washed and

mounted using DPX.

2.7. Immunocytochemistry

Bone samples were stained for type I collagen and Von Willebrand factor

as previously detailed [26]. In brief, after quenching endogenous peroxidase

activity with 3% H2O2 and blocking with 1% bovine serum albumin (BSA)

in PBS, sections were incubated with either LF67 type I collagen antibody

(1:300) or Von Willebrand factor (1:100) (DAKO A/S, Denmark) followed

by anti-rabbit IgG biotin-conjugated secondary antibody (DAKO A/S, Den-

mark; 1:200 with 1% BSA in PBS). Visualization of the immune complex in-

volved the avidinebiotin method linked to peroxidase and 3-amino-9-

ethylcarbazole (AEC), resulting in a reddish brown reaction product. Sections

were counterstained for light green and alcian blue and mounted in glycerol

jelly. No staining was observed in any control sections in which the primary

antibody was omitted.

2.8. Statistical analysis

The statistical analyses were performed using GraphPad Prism software.

Differences among groups were determined by one-way ANOVA with a post-

Dunnett’s test according to experimental design and were considered to be sig-

nificantly different if P< 0.05.

3. Results

3.1. Temporal imaging of segmental bone defect repair

Fig. 2. Faxitron digital in vivo two-dimensional X-rays of mouse MF-1 nu/nu

segmental femur defects at 0 and 28 days postoperation. The control segmental

defect without a PLA scaffold (A), PLA scaffold treated femur defect (B),

PLA seeded with HBMSC treated femur defect (C), and VEGF encapsulated

PLA scaffold seeded with HBMSC treated segmental femur defect (D). Scale

bar¼ 2 mm.

After 28 days postimplantation, two-dimensional in vivodigital X-rays (Faxitron) were used to initially evaluate the de-gree and extent of bone healing in the femoral defects withoutPLA scaffolds (Fig. 2A); with added PLA scaffolds (Fig. 2B);PLA scaffolds with HBMSC (Fig. 2C) and VEGF/PLA scaf-foldsþHBMSC (Fig. 2D). Detailed images generated bymCT at day 28 showed some bone regeneration within the de-fect region in the PLA and PLAeHBMSC femur defectgroups (Fig. 3A, B). However, the VEGF/PLAeHBMSCgroup showed significant osteogenic growth and repair ofthe femoral femur defect gap (Fig. 3C) compared to thePLA and PLAeHBMSC femur defects. Femur defects thathad no scaffolds implanted into the defect region showed neg-ligible bone repair (data not shown).

3.2. Micro-computed tomography analysis of boneformation in segmental bone defects

mCT analysis at day 28 indicated the extent of new boneformation in the VEGF/PLAeHBMSC scaffolds group, withextensive new bone growth in comparison to PLA scaffoldsor PLAeHBMSC (Fig. 4). Indicators of new bone formation,using histomorphometric indices in cell/scaffold constructswere quantitated using three-dimensional reconstructed im-ages from mCT. A significant increase in bone volume (BV)was observed in VEGF/PLAeHBMSC (12.620 mm3� s.d1.919; ***P< 0.001; *P< 0.05) compared to PLA defectgroup (7.429 mm3� s.d. 1.655) and PLAeHBMSC defectgroup (9.070 mm3� s.d. 1.148), respectively (Fig. 4A). Simi-larly BV/TV showed significant increases in the VEGF/PLAe

HBMSC group (0.279� s.d. 0.043; *P< 0.05) compared toPLA defect group (0.205� s.d. 0.029) and PLAeHBMSC de-fect group (0.202� s.d. 0.026) (Fig. 4C). Furthermore, exam-ination of trabecular number (Tb.N) showed a significantincrease (***P< 0.001) in Tb.N in the defect ROI of theVEGF/PLAeHBMSC scaffold group (2.316� s.d. 0.203)compared to PLA scaffolds alone (1.620� s.d. 0.151) orPLAeHBMSC scaffold group (1.615� s.d. 0.317) (Fig. 4E). In support of the observed increase in bone volume and tra-becular number, a significant decrease in trabecular spacing(Tb.Sp.) in the femoral defect region of the VEGF/PLAeHBMSC scaffolds (0.313� 0.036; *P< 0.05) comparedto the implanted PLAeHBMSC (0.484� s.d. 0.070) andPLA only scaffold groups (0.528� s.d. 0.122) was found

Fig. 3. mCT scans 28 days postscaffold implantation in MF-1 nu/nu mouse femur segmental defect model. PLA scaffold only (A), PLA scaffold seeded with

HBMSC (B), and VEGF encapsulated PLA scaffold seeded with HBMSC (C). High resolution images of the defects region of interest (ROI) (D). Scale

bars¼ 2 mm.

1896 J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

(Fig. 4F). Examination of bone surface to bone volume (BS/BV) (Fig. 4B) and trabecular thickness (Tb.Th.) (Fig. 4C)showed no significant differences between groups.

3.3. Immunocytochemical analysis of new boneformation in segmental bone defects

Extensive cell viability was observed in HBMSC cellsseeded on VEGF encapsulated PLA scaffolds within the femurdefect region, evidenced by immunofluorescence for Vybrant(CFDA SE) and negligible ethidium homodimer-1 expressioncompared to defects implanted with PLA alone (Fig. 5A, B).Segmental defects containing VEGF/PLAeHBMSC showedextensive collagen matrix deposition as evidenced by AlcianBlue/Sirius Red (proteoglycans and collagen, respectively)with enhanced immunostaining of Type 1 collagen in thescaffold/defect region of the VEGF/PLAeHBMSC group(Fig. 5F). In addition, histological staining with Goldner’strichrome confirmed new osteoid formation within the defectregion of the VEGF/PLAeHBMSC group (Fig. 5H) compared

to defects containing PLA scaffolds only (Fig. 5G). Blood ves-sel expression of vWF (Fig. 5J) was detected in the scaffolddefect region of the VEGF/PLAeHBMSC group and en-hanced alkaline phosphatase activity (red/purple) was also de-tected in the defect region at the bone/scaffold interface in thisgroup (Fig. 5L).

4. Discussion

These studies demonstrate the ability to deliver, temporally,a combination of VEGF released from porous biocompatiblescaffolds seeded with HBMSC, to sites of bone defects, en-hances the repair and formation of new bone tissue. The devel-opment of three-dimensional engineered skeletal tissue willdepend upon the ability to organise appropriate signal(s) intoa coordinated, temporal regenerative vehicle and, critically,an appropriate vascular network. Thus, the ability to deliverspecific growth factors, using tailored controlled release scaf-folds, to cells within the regenerating tissue structure todevelop vascular networks in vivo, offers significant

Fig. 4. mCT analysis of MF-1 nu/nu mouse femur segmental defect model 28 days postscaffold implantation. In the defect ROI the bone volume (A), bone volume/

total volume (C) and trabecular number (Tb.N)/mm (E) were significantly increased in the VEGF/PLAeHBMSC group compared to the PLA only and

PLAeHBMSC groups. Trabecular spacing (Tb.Sp.) (F) in the VEGF/PLAeHBMSC scaffold defect group were significantly reduced compared to the other groups

(*P< 0.05; ***P< 0.001). No differences were observed in bone surface/bone volume (B) and trabecular thickness (Tb.Th) (D) between the groups.

1897J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

therapeutic potential for a number of musculoskeletal situa-tions. It is estimated that within bone, cells are no more than100 mm away from a vascular supply, providing oxygen andnutrients together with the removal of waste products andtransport of biochemical signals [29]. Against this tissue de-mand, the infiltration of blood vessels into porous scaffoldsis slow, thus illustrating the need for the development of ap-propriate vascularisation strategies for regenerative medicine.

In bone, angiogenesis plays an integral part in osteogenicformation and repair [16], critically driving endochondraland intramembranous ossification. Hence, the failure to de-velop an adequate blood supply is one of the critical factorsin implant failure. Therapeutic strategies to enhance angiogen-esis will ultimately affect the bone repair mechanism [30]. Anumber of approaches have been developed to address this

problem of the lack of vascularisation in bone grafts, including(i) coating of angiogenic extracellular matrices on the surfaceof the implant by a tumorigenic cell line [31], (ii) vector deliv-ery of genes expressing angiogenic growth factors [32], (iii)the encapsulation of bioactive angiogenic factors in biodegrad-able scaffolds seeded with endothelial or osteoprogenitor cellsto stimulate in situ an endogenous angiogenic response[12,33,34].

It is in the last category whereby recent successful develop-ments in scaffold design have led to bioactive factors being in-corporated onto or entrapped within the biodegradablescaffolds [5,11,12,33e39]. A significant step forward hasbeen the development of scCO2-based techniques for prepara-tion of novel scaffolds for tissue engineering. scCO2 is em-ployed in a one step process to both encapsulate bioactives

Fig. 5. Histological representative sections of the PLA scaffolds and the VEGF encapsulated PLA scaffolds seeded with HBMSC ROI in the mouse critical-sized

femur defect 28 days postimplantation. Fluorescent imaging for Vybrant (CFDA SE) labelled HBMSC, control PLA scaffold (A), VEGF/PLAeHBMSC (B). Al-

cian blue and Sirius red staining in control PLA scaffold (C) with enhanced Sirius red staining in the ROI of the VEGF/PLAeHBMSC scaffold (D) and increased

Type 1 collagen expression in VEGF/PLAeHBMSC scaffold/defect ROI (F) compared to control PLA (E). Regions of osteoid staining (arrows) by Goldner’s

trichrome were observed in the ROI of the VEGF/PLAeHBMSC scaffolds ROI (H) compared to the control PLA (G). vWF immunostaining shows the presence

of increased blood vessel formation in the VEGF/PLAeHBMSC scaffold/defect ROI (arrows) (J) than in the PLA scaffold group (I). Elevated activity of alkaline

phosphatase activity (arrows) was found in the VEGF/PLAeHBMSC scaffold defect group (L) compared to the control PLA group (K). *Scaffold region. Scale

bar¼ 100 mm.

1899J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

without loss of activity, and to introduce controlled and inter-connected porosity [24e26,33,40,41]. Potentially this bringsadvantages over the salt leaching approach which involvesmore steps and requires the use of organic solvents, and canresult in loss of some of the growth hormone during the wash-ing phase. However, successful encapsulation has beenachieved by using the salt leaching technique [42].

As these scaffolds are biodegradable, bioactive factors canbe released locally over a period of time. We have previouslydemonstrated that VEGF can be incorporated into PLA scaf-folds by the technique of scCO2 technology and releasedover time to induce an angiogenic effect [26]. We have ex-tended this work in this study to examine the effect of thesescaffolds in the repairing of a critical-sized femoral defect.We demonstrated that the PLA scaffolds encapsulated withVEGF and seeded with HBMSC showed significant bone re-generation in the femur segmental defect in contrast to thePLA and PLAþHBMSC groups, respectively. Interestingly,the seeding of HBMSC on the PLA scaffold showed no signif-icant enhancement of bone repair. This maybe due to a numberof issues including the need for specific growth factors such asBMP-2 or Osteogenic Protein-1 (OP-1) and/or specific mesen-chymal Stro-1 þve osteoprogenitors to enhance the osteogenicresponse [34,43,44]. Co-implantation of osteoprogenitor celltypes with endothelial cells may play a critical role in enhanc-ing the repair of the defect. Recently, induction of endothelialvessel networks in engineered skeletal muscle tissue con-structs using a three-dimensional multi-culture system consist-ing of myoblasts, embryonic fibroblasts and endothelial cellsco-seeded on highly porous, biodegradable polymer scaffoldshave been developed [6]. This multi-cell type therapy could beapplied to enhance the osteogenic repair of a bone defect. In-deed, a co-culture of endothelial cells and mesenchymal cellshas already been shown to increase the osteogenic response[45]. VEGF plays an important role in protecting cells [46]and the close proximity release of VEGF to the HBMSCmay not only keep the HBMSC viable sufficiently to enabledifferentiation and proliferation but may also allow endothelialcell recruitment.

Recent studies have shown that the combination of angio-genic and osteogenic factors can stimulate bone healing andregeneration [12,32,47]. Kaigler et al. [12] demonstratedVEGF scaffolds to enhance neovascularization and bone re-generation in irradiated osseous defects, whereas load-bearingBMP-2 scaffolds can maintain bone length and enhance regen-eration of bone in a critical-sized defect [4]. BMP-2 and TGF-b3, combined with RGDealginate hydrogel co-delivered tofemoral defects within polymer scaffolds showed an increasein bone formation in a critical-sized defect (8 mm) [36].

5. Conclusions

Our current studies suggest the ability to deliver growthfactors locally from biodegradable scaffolds in close proximityto seeded HBMSC can enhance the reparative mechanism ofcritical-sized bone defects; thus, mimicking the in vivo bonerepair environment. Clearly, strategies to enable the multiple

release of growth factors such as VEGF and BMP-2 may infact be able to mimic, more appropriately, the conditions pres-ent in bone fracture repair. Hence, a scaffold that can tempo-rally release an active angiogenic factor to promote earlyvascularisation and attract osteogenic precursor cells followedby the slow release of an osteogenic factor would promote pro-liferation, differentiation of mesenchymal precursor cells intoan osteogenic outcome. Such studies are currently the focus ofon-going studies in our laboratories. In conclusion, these stud-ies demonstrate the ability to deliver, temporally, a combina-tion of bone marrow stromal cells and VEGF released frombiocompatible scaffolds to sites of bone defects in a regulatedmanner, resulting in the enhancement of the repair and forma-tion of new osteoid tissue. Such cell and tissue engineeringstrategies offer tremendous therapeutic opportunities in ortho-pedics and the wider reparative arena.

Acknowledgements

The authors would like to thank members of the Bone andJoint Research Group for useful discussions, in particular MrBen Bolland MRCS. S.M.H. is a Royal Society Wolfson Re-search Merit Award Holder. We would like to thank Dr LarryFisher for provision of Type I collagen antibody. Janos Kanc-zler and Patrick Ginty are supported by grants from theBBSRC and EPSRC.

References

[1] Kneser U, Schaefer DJ, Polykandriotis E, Horch RE. Tissue engineering

of bone: the reconstructive surgeon’s point of view. J Cell Mol Med

2006;10:7e19.

[2] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920e6.

[3] Bolland BJ, Tilley S, New AM, Dunlop DG, Oreffo RO. Adult mesen-

chymal stem cells and impaction grafting: a new clinical paradigm shift.

Expert Rev Med Devices 2007;4:393e404.

[4] Chu TM, Warden SJ, Turner CH, Stewart RL. Segmental bone regener-

ation using a load-bearing biodegradable carrier of bone morphogenetic

protein-2. Biomaterials 2007;28:459e67.

[5] Murphy WL, Simmons CA, Kaigler D, Mooney DJ. Bone regeneration

via a mineral substrate and induced angiogenesis. J Dent Res 2004;

83:204e10.

[6] Levenberg S, Rouwkema J, Macdonald M, Garfein ES, Kohane DS,

Darland DC, et al. Engineering vascularized skeletal muscle tissue. Nat

Biotechnol 2005;23:879e84.

[7] Leach JK, Mooney DJ. Bone engineering by controlled delivery of

osteoinductive molecules and cells. Expert Opin Biol Ther 2004;

4:1015e27.

[8] Wei G, Ma PX. Structure and properties of nano-hydroxyapatite/polymer

composite scaffolds for bone tissue engineering. Biomaterials 2004;

25:4749e57.

[9] Rose FR, Hou Q, Oreffo RO. Delivery systems for bone growth factors e

the new players in skeletal regeneration. J Pharm Pharmacol

2004;56:415e27.

[10] Yang XB, Whitaker MJ, Sebald W, Clarke N, Howdle SM,

Shakesheff KM, et al. Human osteoprogenitor bone formation using en-

capsulated bone morphogenetic protein 2 in porous polymer scaffolds.

Tissue Eng 2004;10:1037e45.

[11] Huang YC, Kaigler D, Rice KG, Krebsbach PH, Mooney DJ. Combined

angiogenic and osteogenic factor delivery enhances bone marrow stromal

cell-driven bone regeneration. J Bone Miner Res 2005;20:848e57.

1900 J.M. Kanczler et al. / Biomaterials 29 (2008) 1892e1900

[12] Kaigler D, Wang Z, Horger K, Mooney DJ, Krebsbach PH. VEGF scaf-

folds enhance angiogenesis and bone regeneration in irradiated osseous

defects. J Bone Miner Res 2006;21:735e44.

[13] Brey EM, King TW, Johnston C, McIntire LV, Reece GP, Patrick Jr CW.

A technique for quantitative three-dimensional analysis of microvascular

structure. Microvasc Res 2002;63:279e94.

[14] Stahl A, Wenger A, Weber H, Stark GB, Augustin HG, Finkenzeller G.

Bi-directional cell contact-dependent regulation of gene expression be-

tween endothelial cells and osteoblasts in a three-dimensional spheroidal

coculture model. Biochem Biophys Res Commun 2004;322:684e92.

[15] Cassell OC, Hofer SO, Morrison WA, Knight KR. Vascularisation of tis-

sue-engineered grafts: the regulation of angiogenesis in reconstructive

surgery and in disease states. Br J Plast Surg 2002;55:603e10.

[16] Carano RA, Filvaroff EH. Angiogenesis and bone repair. Drug Discov

Today 2003;8:980e9.

[17] Gerber HP, Vu TH, Ryan AM, Kowalski J, Werb Z, Ferrara N. VEGF

couples hypertrophic cartilage remodeling, ossification and angiogenesis

during endochondral bone formation. Nat Med 1999;5:623e8.

[18] Mayr-Wohlfart U, Waltenberger J, Hausser H, Kessler S, Gunther KP,

Dehio C, et al. Vascular endothelial growth factor stimulates chemotactic

migration of primary human osteoblasts. Bone 2002;30:472e7.

[19] Wang Y, Wan C, Deng L, Liu X, Cao X, Gilbert SR, et al. The hypoxia-

inducible factor alpha pathway couples angiogenesis to osteogenesis dur-

ing skeletal development. J Clin Invest 2007;117:1616e26.

[20] Hausman MR, Schaffler MB, Majeska RJ. Prevention of fracture healing

in rats by an inhibitor of angiogenesis. Bone 2001;29:560e4.

[21] Street J, Bao M, deGuzman L, Bunting S, Peale Jr FV, Ferrara N, et al.

Vascular endothelial growth factor stimulates bone repair by promoting

angiogenesis and bone turnover. Proc Natl Acad Sci U S A 2002;

99:9656e61.

[22] Arosarena OA, Collins WL. Bone regeneration in the rat mandible with

bone morphogenetic protein-2: a comparison of two carriers. Otolaryngol

Head Neck Surg 2005;132:592e7.

[23] Betz OB, Betz VM, Nazarian A, Pilapil CG, Vrahas MS, Bouxsein ML,

et al. Direct percutaneous gene delivery to enhance healing of segmental

bone defects. J Bone Joint Surg Am 2006;88:355e65.

[24] Howdle SM, Watson MS, Whitaker MJ, Popov VK, Davies MC,

Mandel FS, et al. Supercritical fluid mixing: preparation of thermally

sensitive polymer composites containing bioactive materials. Chem

Commun 2001;01:109e10.

[25] Barry JJA, Silva MMCG, Popov VK, Shakesheff KM, Howdle SM. Su-

percritical carbon dioxide; putting the fizz into biomaterials. Philos

Transact A Math Phys Eng Sci 2006;364:249e61.

[26] Kanczler JM, Barry J, Ginty P, Howdle SM, Shakesheff KM, Oreffo RO.

Supercritical carbon dioxide generated vascular endothelial growth factor

encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro.

Biochem Biophys Res Commun 2007;352:135e41.

[27] Oreffo RO, Virdi AS, Triffitt JT. Modulation of osteogenesis and adipo-

genesis by human serum in human bone marrow cultures. Eur J Cell Biol

1997;74:251e61.

[28] Miao D, Scutt A. Histochemical localization of alkaline phosphatase ac-

tivity in decalcified bone and cartilage. J Histochem Cytochem 2002;

50:333e40.

[29] Santos MI, Fuchs S, Gomes ME, Unger RE, Reis RL, Kirkpatrick CJ.

Response of micro- and macrovascular endothelial cells to starch-based

fiber meshes for bone tissue engineering. Biomaterials 2007;28:240e8.

[30] Hsiong SX, Mooney DJ. Regeneration of vascularized bone. Periodontol

2000 2006;41:109e22.

[31] Kidd KR, Nagle RB, Williams SK. Angiogenesis and neovascularization

associated with extracellular matrix-modified porous implants. J Biomed

Mater Res 2002;59:366e77.

[32] Geiger F, Bertram H, Berger I, Lorenz H, Wall O, Eckhardt C, et al. Vas-

cular endothelial growth factor gene-activated matrix (VEGF165eGAM)

enhances osteogenesis and angiogenesis in large segmental bone defects.

J Bone Miner Res 2005;20:2028e35.

[33] Murphy WL, Peters MC, Kohn DH, Mooney DJ. Sustained release of

vascular endothelial growth factor from mineralized poly(lactide-co-gly-

colide) scaffolds for tissue engineering. Biomaterials 2000;21:2521e7.

[34] Yang XB, Green D, Roach HI, Anderson HC, Howdle SM,

Shakesheff KM, et al. The effect of an admix of bone morphogenetic pro-

teins on human osteoprogenitor activity in vitro and in vivo. Tissue Eng

2006;12:1002e3.

[35] Jansen JA, Vehof JW, Ruhe PQ, Kroeze-Deutman H, Kuboki Y, Takita H,

et al. Growth factor-loaded scaffolds for bone engineering. J Control Re-

lease 2005;101:127e36.

[36] Oest ME, Dupont KM, Kong HJ, Mooney DJ, Guldberg RE. Quantitative

assessment of scaffold and growth factor-mediated repair of critically

sized bone defects. J Orthop Res 2007;25:941e50.

[37] Jeon O, Song SJ, Kang SW, Putnam AJ, Kim BS. Enhancement of ec-

topic bone formation by bone morphogenetic protein-2 released from

a heparin-conjugated poly(L-lactic-co-glycolic acid) scaffold. Biomate-

rials 2007;28:2763e71.

[38] Hou LT, Liu CM, Liu BY, Chang PC, Chen MH, Ho MH, et al. Tissue

engineering bone formation in novel recombinant human bone morpho-

genic protein 2eatelocollagen composite scaffolds. J Periodontol

2007;78:335e43.

[39] Wei G, Jin Q, Giannobile WV, Ma PX. The enhancement of osteogenesis

by nano-fibrous scaffolds incorporating rhBMP-7 nanospheres. Biomate-

rials 2007;28:2087e96.

[40] Whitaker MJ, Quirk RA, Howdle SM, Shakesheff KM. Growth factor re-

lease from tissue engineering scaffolds. J Pharm Pharmacol 2001;

53:1427e37.

[41] Woods HM, Silva MMCG, Nouvel C, Shakesheff KM, Howdle SM. Ma-

terials processing in supercritical carbon dioxide: surfactants, polymers

and biomaterials. J Mater Chem 2004;14:1663e78.

[42] Sheridan MH, Shea LD, Peters MC, Mooney DJ. Bioabsorbable polymer

scaffolds for tissue engineering capable of sustained growth factor deliv-

ery. J Control Release 2000;64:91e102.

[43] Yang XB, Webb D, Blaker J, Boccaccini AR, Maquet V, Cooper C, et al.

Evaluation of human bone marrow stromal cell growth on biodegradable

polymer/bioglass composites. Biochem Biophys Res Commun 2006;

342:1098e107.

[44] den Boer FC, Wippermann BW, Blokhuis TJ, Patka P, Bakker FC,

Haarman HJ. Healing of segmental bone defects with granular porous hy-

droxyapatite augmented with recombinant human osteogenic protein-1 or

autologous bone marrow. J Orthop Res 2003;21:521e8.

[45] Kaigler D, Krebsbach PH, West ER, Horger K, Huang YC, Mooney DJ.

Endothelial cell modulation of bone marrow stromal cell osteogenic po-

tential. FASEB J 2005;19:665e7.

[46] Ferrari G, Pintucci G, Seghezzi G, Hyman K, Galloway AC, Mignatti P.

VEGF, a prosurvival factor, acts in concert with TGF-beta1 to induce en-

dothelial cell apoptosis. Proc Natl Acad Sci U S A 2006;103:17260e5.

[47] Peng H, Usas A, Olshanski A, Ho AM, Gearhart B, Cooper GM, et al.

VEGF improves, whereas sFlt1 inhibits, BMP-2-induced bone formation

and bone healing through modulation of angiogenesis. J Bone Miner Res

2005;20:2017e27.