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Biomaterials 28 (2007) 1005–1013

www.elsevier.com/locate/biomaterials

Repair of canine mandibular bone defects with bone marrowstromal cells and porous b-tricalcium phosphate

Jie Yuana,b,1, Lei Cuia,b,1, Wen Jie Zhanga,b, Wei Liua,b, Yilin Caoa,b,�

aShanghai Key Laboratory of Tissue Engineering, Department of Plastic and Reconstructive Surgery, Shanghai 9th People’s Hospital,

Shanghai Jiao Tong University School of Medicine, 639 Zhi Zao Ju Road, Shanghai 200011, ChinabNational Tissue Engineering Center of China, Shanghai, China

Received 15 August 2006; accepted 9 October 2006

Available online 7 November 2006

Abstract

Tissue engineering has become a new approach for repairing bone defects. Previous studies have been limited to the use of slow-

degradable scaffolds with bone marrow stromal cells (BMSCs) in mandibular reconstruction. In this study, a 30mm long mandibular

segmental defect was repaired by engineered bone graft using osteogenically induced autologouse BMSCs seeded on porous b-tricalciumphosphate (b-TCP, n ¼ 5). The repair of defects was compared with those treated with b-TCP alone (n ¼ 6) or with autologous

mandibular segment (n ¼ 4). In the BMSCs/b-TCP group, new bone formation was observed from 4 weeks post-operation, and bony-

union was achieved after 32 weeks, which was detected by radiographic and histological examination. In contrast, minimal bone

formation with almost fibrous connection was observed in the group treated with b-TCP alone. More importantly, the engineered bone

with BMSCs/b-TCP achieved a satisfactory biomechanical property in terms of bending load strength, bending displacement, bending

stress and Young’s modulus at 32 weeks post-operation, which was very close to those of contralateral edentulous mandible and

autograft bone (p40.05). Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and

biodegradable b-TCP can well repair the critical-sized segmental mandibular defects in canines.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Tissue engineering; Bone marrow stromal cells; b-tricalcium phosphate; Mandibular reconstruction

1. Introduction

Mandible keeps the integrity of maxillofacial complexfor mastication and speech in oral-maxillofacial region.Repair of large mandibular bone defects caused by trauma,infection, cancer or congenital malformation remains a bigchallenge in reconstructive surgery [1]. Autogenous bonetransplantation is the ‘‘gold standard’’ approach forrepairing the bone defects at present [2]. The grafts canprovide three elements, which are critical for boneregeneration: scaffolds for osteo-conduction, growth fac-tors for osteo-induction, and cells for osteogenesis [3].

e front matter r 2006 Elsevier Ltd. All rights reserved.

omaterials.2006.10.015

ing author. Department of Plastic Surgery, Shanghai 9th

tal, 639 Zhi Zao Ju Road, Shanghai 200011, PR China.

138341x5192; fax: +8621 53078128.

ess: yilincao@yahoo.com (Y. Cao).

rs contributed equally to this work.

However, the major problem of this approach is thatpatients need to sacrifice their healthy tissue from donorsite which could result in donor site morbidity [4].Recently, tissue engineering approach has been shown tobe very effective in bone regeneration. The successful repairof bone defects has been demonstrated in immuno-deficientanimals, such as athymic mice and rats [5,6], as well as inimmuno-competent animals like canine and sheep [7–9].Bone marrow stromal cells (BMSCs) have been found to

be a major seed cell source for bone engineering. Theosteogenic potential of BMSCs has been demonstratedboth in vitro and in vivo [5–8,10]. In addition, many studieshave succeeded in the repairing of bone defects by usingBMSCs in animal model as well as in human beings[7–9,11,12]. Regarding scaffold, both nature and syntheticmaterials have been utilized for bone engineering[7–9,12–17]. In mandibular reconstruction, Abukawaet al. [18] have used poly-DL-lactic-co-glycolic acid (PLGA)

ARTICLE IN PRESSJ. Yuan et al. / Biomaterials 28 (2007) 1005–10131006

scaffold seeded with osteogenic induced autologousBMSCs to repair a 2� 2 cm partial mandibular defect ina porcine model, while Schliephake et al. [19] have repaireda 35mm length segmental mandibular defect with pyrolizedbovine bone as scaffold in a sheep model. It is known thatPLGA scaffold is difficult to shape for repairing large bonedefects, while pyrolized bovine bone is nearly undegradablein vivo. In addition, as a natural material, pyrolized bovinebone still remains the risk of pathogen transmission [20].Thus, other potential synthetic materials should be testedfor mandibular bone engineering.

Porous b-tricalcium phosphate (b-TCP) is a syntheticand biodegradable ceramic material that has been com-monly used in various applications in oral and maxilo-facial surgery, such as alveolar ridge augmentation, sinusfloor augmentation [21,22], and has also been widely usedin benign bone tumor filling [23]. In a sheep model offemoral condyle implantation, it has been reported that55% of the b-TCP could be degraded after 24 weeks ofimplantation [24]. Porous b-TCP is also known as amaterial with good osteo-conductive capacity due to it’shigh porosity and interconnectivity that can provideintercellular communication among osteogeneic cells restedin lacunae [25,26]. However, porous b-TCP is very fragile,the scaffold solely made of b-TCP cannot bear strongmechanical forces [27]. Whether pure b-TCP scaffold couldbe used to repair load-bearing bone defect in large animalmodel become an attractive issue.

In this study, porous b-TCP seeded with in vitro cultureexpanded, osteogenically induced BMSCs had been im-planted to repair a 30mm length critical-sized segmentalmandibular defect in a canine model. A satisfactory repairwas achieved at 32 weeks post-implantation, suggestingthat autologous BMSCs, along with porous b-TCP, couldbe potentially used for the clinical repair of mandibularbone defects.

2. Materials and methods

2.1. Animals

A total of 16 adult mongrel dogs in healthy condition, aged 18 months

old with an average weight of 20.9 kg were used in this study. All dogs

were prepared by extraction of the upper and lower premolar and first

molar teeth at right sides 8 weeks before segmental mandibular resection.

The dogs were kept on a soft diet during the study and were allowed

complete gingival healing before mandibular resection surgery. The

experimental protocol was approved by the Animal Care and Experiment

Committee of Shanghai Jiao Tong University School of Medicine.

2.2. Cell culture and induction

After intravenous anesthesia with 5% sodium pentobarbital (0.5mL/kg),

3mL of bone marrow aspirates were harvested from the iliac crests of each

dog and transferred into a pre-heparinized centrifuge tube. Mononuclear

cells were separated by percoll (1.073 g/mL, Sigma, St Louise, MO) gradient

centrifugation [28], and were plated in 100-mm dishes (Falcon, Franklin

Lakes, NJ) at a density of 1� 105 cells/cm2. Cells were cultured in osteogenic

induction media (Dulbecco’s Modified Eagle Medium (DMEM, Gibco,

Grand Island, NY) supplemented with 10% fetal bovine serum (FBS,

Gibco), 10�8mol/L dexamethasone, 10mmol/L b-phosphoglycerol and

50mmol/L L-2-ascorbic acid (all from Sigma)) at 37 1C with 5% CO2. The

culture medium was changed after 48h and then every 3 days. When BMSCs

reached 80–90% confluence, cells were detached with 0.25% trypsin/EDTA

(Gibco), subcultured at a density of 1� 105 cells/cm2 in 100-mm dishes.

2.3. Preparation of BMSCs/b-TCP construct

b-TCP scaffolds (purchased from Shanghai Bio-Lu Biomaterials Co.,

Ltd., Shanghai, China) were molded into cuboids (30� 15� 10mm) with

a 3mm diameter tunnel which mimicking a normal structure of mandible

(Fig. 1), and were sterilized by 60Co irradiation before use. The scaffolds

have volume porosity of 70% with pore diameter of 450750mm. Scanning

electron microscope showed that all pores were interconnected with inter-

pore diameter of 150750 mm (Fig. 2A). The initial compression strength

was 2–4MPa [29]. For cell seeding, osteogenically induced BMSCs at

passage 2 were detached from culture dishes, centrifuged to remove

supernatant, and then resuspended in the culture media at a density of

2� 107 cells/mL. Cells in suspension were slowly injected into the b-TCPcuboids using a syringe (3mL/cuboid). After being incubated for 4 h to

allow cell attachment, 30mL of osteogenic induction medium were then

added to cover the constructs. The BMSCs/b-TCP constructs were

subsequently cultured for 7 days in vitro before implantation.

In a parallel experiment, 3� 3� 3mm cuboids were prepared and

seeded with BMSCs at an identical cell density. After 7 days of incubation,

the constructs were fixed in 2% formalin for 2 h, cut into two halves, and

then subjected for scanning electron microscopy (Philips SEM XL-30,

Amsterdam, Netherlands) examination.

2.4. Surgical procedure

The canines were anesthetized through intramuscular injection of

ketamine (10mg/kg) and were placed in a supine position. The body of

right mandible was exposed through a submental midline skin incision.

After adjustment of the titanium plate, an osteoperiosteal segmental defect

of 30mm length was made at the mid-portion of mandible using

an oscillating bone saw that was cooled continuously by saline irrigation

(Fig. 3A). The neurovascular bundle was cut off and stanched by filling

bone wax into mandibular canal. Then, the titanium plate was secured in

place with six screws (three at the mesial side and three at the distal side).

The defects were filled either with osteogenically induced BMSCs/b-TCPconstructs (experimental group, n ¼ 6), resected autologous mandibular

segments (autograft group, n ¼ 4), or with b-TCP cuboids alone (scaffold

group, n ¼ 6) (Fig. 3B). The incisions were closed with 3-0 silk suture.

Meanwhile, the lower premolar and first molar teeth at left side were

extracted and the edentulous mandibles were served as normal control

(n ¼ 16). All animals were kept on a soft diet during the study. In order to

give newly formed tissue a mechanical loading, the titanium plates were

removed from all dogs at 22 weeks post-operation.

2.5. Radiographic and dual energy X-ray absorptiometry analyses

Mandibular radiograms were obtained under general anesthesia at 4,

12, 26 and 32 weeks post-operation. Maxillofacial CT images were

obtained and the three-dimensional images were reconstructed using a

multi-slice spiral CT (GE Lightspeed Ultra 16, Milwaukee, WI) at 32

weeks post-operation. For dual energy X-ray absorptiometry (DXA)

analysis, animals were sacrificed at 32 weeks post-operation, and the

mandibles were harvested. The local bone mineral densities (BMD) were

measured on a DXA system (Hologic Discovery A, Bedford, MA).

2.6. Biomechanical analysis

For biomechanical analysis, mandibles of all groups were harvested at

32 weeks post-operation. The left side mandible of each animal was also

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Fig. 1. Gross view of b-TCP cuboids. The cuboids are 30mm long, 15mm high and 10mm thick, with a tunnel of 3mm in diameter through each cuboid.

Fig. 2. Scanning electron microscopic evaluation of the scaffold microstructure and biocompatibility. (A) Three-dimensional porous structures of b-TCP.(B) BMSCs attach on b-TCP with abundant matrix after 7 days of culture.

Fig. 3. Surgical procedure. (A) After adjustment of the titanium plate, an osteoperiosteal segmental mandibular defect of 30mm length was made at right

side. (B) The defect was filled with BMSCs/b-TCP construct.

J. Yuan et al. / Biomaterials 28 (2007) 1005–1013 1007

collected as normal control. Each mandible was placed in a three-point

bending system, providing an unsupported length of 30mm. Load was

applied in the occlusal-to-inferior direction to the midpoint of the

unsupported length, approximately in the middle of the repaired defect.

The measurement was conducted in a hydraulic materials-testing machine

(Shimadzu AG-5KN, Kyoto, Japan) and stopped following bone fracture.

Deformation was measured using the strain gauge at a loading rate of

0.5mm/min. The bending stress was calculated with the equation: bending

stress¼ 3LF=ð2WT2Þ, where L, F, W and T are the test span (mm), the

bending strength load (KN), the width (mm), and the thickness of the

specimen (mm), respectively. Young’s modulus was achieved from stress-

strain curve as the slope in the linear region.

2.7. Histological examination

After the bending tests, all the samples were processed for histological

examination. Since we could not perform undecalcified tissue section

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due to the limited condition, tissues from original defect area of all groups

were fixed in buffered 10% formalin in PBS for 72 h, and decalcified in

15% formic acid in PBS for 2–10 weeks. The decalcified tissues of

interfacial portion were then embedded in paraffin. Tissue sections in 5 mmthickness were obtained for hematoxylin and eosin (H&E) staining.

2.8. Statistical analysis

Data from DXA and biomechanical examination were analyzed by

one-way ANOVA test. The differences between experimental group

(n ¼ 5), scaffold group (n ¼ 6), autograft group (n ¼ 4), and normal

group (n ¼ 5) were assessed with Student-Newman-Kewls test. A p value

less than 0.05 was considered statistically significant.

3. Results

3.1. Growth of osteogenically induced BMSCs on b-TCP

After being cultivated in the presence of dexamethasoneand b-phosphoglycerol for 2 passages, BMSCs showed atypical osteogenic phenotype as previously described,including calcium deposition and positive alkaline phos-phatase (AKP) staining (data not shown).

To evaluate the compatibility of the scaffold, BMSCswere seed on the b-TCP scaffolds, incubated in vitro for 7days, and cell-scaffold complexes were subjected for SEMexamination. As shown in Fig. 2B, the pores were filled bycells together with abundant extracellular matrices after7 days of incubation, indicating that the scaffold has good

Fig. 4. Radiographs of treated defects taken at different time points post-opera

and more calluses are observed at 12 weeks (D). At 26 weeks, the radiopacity is

to that of contralateral normal mandible (K) with smoothly remodeled bone con

weeks (B) nor 12 weeks (data not shown). Radiolucency and minimal callus

complete radiolucency is observed at 32 weeks (H). In the autograft group, som

radiopacity decreased at 12 weeks (F). At 26 weeks, bony-union is achieved wi

new bone with insular bone pieces are coexisting in the graft after 32 weeks (L

compatibility. Accordingly, day 7 of in vitro co-culture waschosen as the time point to implant the cell-scaffoldconstructs in vivo. In addition, cells grew on the scaffoldsmaintained their osteogenic phenotype even after 28 daysof in vitro culture, which were evaluated by AKP andosteocalcin expression (data not shown).

3.2. Radiographic and DXA analyses

After a critical-sized segmental defect was created, thedefect was filled with either osteogenically induced BMSCs/b-TCP construct (experimental group, n ¼ 6), autologousmandibular segment (autograft group, n ¼ 4), or b-TCPcuboid alone (scaffold group, n ¼ 6). All animals survivedthe surgical procedure, but one dog in the experimentalgroup got infection at the site of implantation and wasexcluded from the study.To follow up the new bone formation and the develop-

ment of bone union within the defects, X-ray images weretaken at 4, 12, 26 and 32 weeks post-operation. Repre-sentative photographs of each group were shown in Fig. 4.In the experiment group that implanted with BMSCs/b-TCP constructs, little calluses were observed beneath theimplants at 4 weeks post-operation (Fig. 4A), and morecalluses were observed at 12 weeks (Fig. 4D); at 26 weekspost-operation, the volume and radiopacity of newlyformed bone was highly increased and bony-union wasachieved (Fig. 4G); at 32 weeks, the bone contour was

tion. In the BMSCs/b-TCP group, little calluses are formed at 4 weeks (A),

highly increased and bony-union is achieved (G). The radiopacity is close

tour at 32 weeks (J). In the b-TCP group, no callus is observed at neither 4

formation at the cutting ends are observed at 26 weeks (E), while nearly

e callus formation is observed at the junction sites at 4 weeks (C), and the

th some bone pieces in the middle portion of the graft (I). High density of

).

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remodeled smoothly (Fig. 4J) and the radiopacity was closeto that of contralateral normal mandible (Fig. 4K). On thecontrary, no callus could be observed in the groupimplanted with b-TCP alone, neither at 4 weeks (Fig. 4B)nor at 12 weeks (data not shown). At 26 weeks, obviousradiolucency and minimal callus formation at the cuttingends was observed in this group (Fig. 4E), and nearlycomplete radiolucency was observed at 32 weeks post-operation (Fig. 4H). As a positive control, the autograftgroup showed some callus formation at the junction sites at4 weeks post-operation (Fig. 4C), and the radiopacity wasdecrease in the middle portion of the graft at 12 weeks(Fig. 4F); at 26 weeks post-operation, bony-union was alsoachieved with some insular bone pieces observed in themiddle portion of the grafts (Fig. 4I). The bone densitycontinuously increased with newly formed bone and insularbone pieces coexisting in the graft after 32 weeks (Fig. 4L).Radiographic union was observed in all of the experimentgroup (5/5) and autograft group (4/4), but not in thescaffold group (0/6).

To detect the three-dimensional structure of repairedmandibles, 3D-CT images were taken at 32 weeks post-operation. Representative images from each group werepresent in Fig. 5. In the experimental group which wastreated with BMSCs/b-TCP constructs, bony-union and anice mandibular body shape were observed (Fig. 5A).

Fig. 5. 3D-CT images of treated mandibles at 32 weeks post-operation. (A) I

observed. (B) In the b-TCP group, non-union and minimal callus are formed. (C

at buccal side.

However, in the group treated with b-TCP alone, non-union was observed and only minimal calluses weredetected (Fig. 5B). In the autograft group, bony-unionwas achieved while insular bone pieces were also observedat the buccal side (Fig. 5C).To quantify the calcification of repaired mandibles, local

bone mineral densities (BMD) of all animals weremeasured on DXA at 32 weeks post-operation (Fig. 6).The BMSCs/b-TCP grafts showed relatively high BMD of0.5570.06 g/cm2, which was similar to the contralateralnormal group (0.6070.06 g/cm2, p40.05). Whereas in thescaffold group, the local BMD was 0.1970.05 g/cm2,which was significantly lower than that of the BMSCs/b-TCP grafts (po0.01). Meanwhile, the BMD of autograftgroup was significantly higher than other three groups(0.8770.17 g/cm2, po0.05).

3.3. Gross view and biomechanical analysis

Correlated with radiographic analyses, gross view of themandibles repaired with either BMSCs/b-TCP constructsor autologous bones achieved bony-union at 32 weekspost-operation (Fig. 7A, C). The shapes of repairmandibles were very close to that of normal edentulousmandibles (Fig. 7D). On the contrary, none bony-unionwas observed when implanted with b-TCP alone, obvious

n the BMSCs/b-TCP group, bony-union with mandibular body shape is

) In the autograft group, bony-union is observed together with bone piece

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soft tissues were detected in the original defect area in thisgroup (Fig. 7B).

To evaluate the biomechanical property of repairedmandibles, three-point bending test was performed at 32weeks post-operation. As shown in Table 1, the bendingload strength and bending stress in the BMSCs/b-TCPgroup were 1.8070.42 kN and 44.73710.12MPa, reached65.16721.41% and 81.63712.88% of the contralateralnormal edentulous mandible, respectively. Furthermore,comparing with the normal mandible, tissue-engineeredsegmental bone revealed similarities with respects tobending load strength, bending displacement, bendingstress and Young’s modulus (p40.05, Table 1). Thebending load strength and bending stress of the autograftgroup were 2.7170.30 kN and 43.12711.98MPa, respec-tively. Again, no significant differences were found betweenthe experimental and autograft group in terms of all the

Fig. 7. Gross view of repaired mandibles at 32 weeks post-operation. (A) In t

notches are observed. (B) In the b-TCP group, soft tissues are detected in the de

is observed. (D) Contralateral edentulous native mandible. Arrows indicate th

Fig. 6. Dual energy X-ray absorptiometry analysis at 32 weeks post-

operation. The local bone mineral density (BMD) of BMSCs/b-TCPimplants (n ¼ 5) was similar to that of normal control group (n ¼ 5,

p40.05), but significantly higher than that of b-TCP implants (n ¼ 6,

po0.01), and lower than that of bone autografts (n ¼ 4, po0.05).

biomechanical parameters tested (p40.05, Table 1). Sincethere is no bony-union in the scaffold group, which wastreated with b-TCP alone, three-point bending test couldnot be applied in this group.

3.4. Histological examination

To further confirm the above findings, the histology ofrepaired defects at 32 weeks post-operation was detectedafter bending tests by decalcified tissue section with H&Estaining. Representative photographs from each groupwere present in Fig. 8. In the experimental group, irregularosteon formation was observed together with some‘‘ghost’’ structures of undegraded b-TCP particles in therepaired area (Fig. 8A1), which has been previouslydescribed by Dong et al. [27]. In addition, a bony-unionwas observed without clear boundary between newlyformed bone and native bone in this group (Fig. 8A2). Inthe autograft group, a bony-union was also observed withintrinsic dead bone and newly formed bone coexisting inthe repaired area (Fig. 8C1 and C2). However, in thescaffold group, only fibrous tissues were observed in thedefect area with minimal new bone formation at cuttingend (Fig. 8B1 and B2). Similar results were observed withAzan-Mallory staining (data not shown).

4. Discussion

The current study demonstrates that the critical-sizedsegmental defects of the canine mandible can be repairedby osteogenically induced BMSCs with biodegradable b-TCP scaffold. Meanwhile, scaffold alone is not sufficientenough to repair such a big defect in this model.Biodegradable scaffold plays a pivotal role in bone

engineering. The scaffold can provide a skeletal support forthe growth of osteogenic cells at early stage and createenough space for new bone formation when they are

he BMSCs/b-TCP group, bony-union with white colored cortex and some

fect. (C) In the autograft group, bony-union with red-white colored cortex

e junction sites.

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Table 1

Biomechanical analyses at 32 weeks post-operation

Parameters Experimental group Autograft group Normal control group

Bending strength load (kN) 1.8070.42 2.7170.30 2.9871.1

Bending displacement (mm) 1.8870.76 2.6070.38 3.7171.88

Bending stress (MPa) 44.73710.12 43.12711.98 54.3574.31

Young’s modulus (MPa) 437.207199.49 250.03777.30 275.817107.3

Data present mean value7SD (experimental and normal control group n ¼ 5, autograft group n ¼ 4). No significant differences were observed among

BMSCs/b-TCP, autograft and normal control group in term of all the biomechanical parameters tested (p40.05).

Fig. 8. H&E staining of repaired mandibles at 32 weeks post-operation. The macroscopical view of repaired area in each group is shown: (A) BMSCs/b-TCP group; (B) b-TCP group; (C) autograft group. Detail view of inside area (solid frame, A1, B1, C1) and interfacial area (dot frame, A2, B2, C2) were

taken at high magnification (bar scales: 100mm). In the BMSCs/b-TCP group, irregular osteons are observed (arrow head) together with some non-

degraded b-TCP particles (arrow) at inside area (A1), and bony-union occurs at interfacial area (A2). In the b-TCP group, minimal new bone (arrow head)

with many fibrous tissues (arrow) are found at inside area (B1), and fibrous-union occurs at interfacial area (B2). In the autograft group, intrinsic dead

bone (arrow) and newly formed bone (arrow head) are coexisting at inside area (C1), and bony-union also occurs at interfacial area (C2).

J. Yuan et al. / Biomaterials 28 (2007) 1005–1013 1011

degraded at later time point. Thus, besides the basicrequirement for implantable biomaterial, an optimalscaffold for bone engineering should have a suitabledegradation rate that can well match the speed of newbone formation. A premature degradation of scaffold maydestroy the tissue structure if new bone has not generatedyet, while a lingering degradation of scaffold may alsoeffects the repairing process, such as tissue structureremodeling. Comparing with undegradable pyrolizedbovine bone that has been used in mandibular reconstruc-tion, porous b-TCP has showed a good degradation ratethat could well match the new bone formation rate in therepairing process. The new bone formation started around

4 weeks post-operation with b-TCP degradation started atsame time. In addition, bony-union was achieved withalmost complete degradation of b-TCP after 32 weeks.It is known that degradation rate of porous b-TCP

scaffold depends on its’ porosity and pore size [24,30].Scaffold with high porosity and big pore size is favorablefor degradation as well as for better nutrient exchange andcell penetration. On the other hand, the mechanicalproperty of the scaffold will be decreased accordingly. Itis the nature of the b-TCP scaffolds that they are fragile. Inthis study, we found one b-TCP scaffold without seedingcell was fractured by titanium plate fixation during surgery(data not shown). However, all the constructs seeded with

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BMSCs maintained their shape without broken. Inconsistent with our results, Dong et al. [27] found thatthe compressive strength of b-TCP scaffold could besignificantly improved by cell seeding. These resultsindicate that the mechanical property of this scaffold couldbe improved. Further studies of modifying b-TCP scaffoldswith extracellular matrix to improve the mechanicalproperty are under investigation.

Regard to bone regeneration, some groups reported thatscaffold alone or scaffold modified with growth factorscould achieve bone reconstruction without seeding osteo-genic cells [31–34]. However, no bony-union could beobserved in our study with b-TCP alone. Comparing withthose studies, we found that only small defects could berepaired with scaffold alone but not big defects, which isconsistent with other groups reported [32,35]. In smalldefects, native cells could migrate into the scaffold togenerate new tissues and achieve bony-union. While in bigdefects, the migration rate of native cells was not fastenough to match the degradation of scaffold. Thus, onlyfibrous union was achieved. It is known that seededBMSCs can not only provide an osteogenic cell source fornew bone formation, but also secret growth factors torecruit native cells migrate into the defect site [36–40]. Inorder to clarify the repairing mechanism and to follow thelong-term fate of the implanted BMSCs, further study oftreating defects with green fluorescence protein genelabeled BMSCs is ongoing.

The degradation of calcium phosphate biomaterials invivo could be through two processes: first, throughextracellular liquid dissolution, and second, throughdisintegration into small particles that would be eitherintra-cellularly digested or be transported to neighboringtissues such as the lymph nodes finally [41]. In this study,the degradation of b-TCP might be also through bothprocesses [24]. The abundant blood circulation of oral-maxillofacial region could promote the dissolution process,which has been well shown in the experimental and scaffoldgroup at early stage (Fig. 4, 4 weeks data). When thescaffold broke into small pieces at late stage (Fig. 4, 12weeks data), the small particles could be either phagocy-tosed or be transported to neighboring tissues, although wehave not found typical osteoclasts in the experimentalgroup by histological analysis. One possible reason couldbe that the decalcification process could partially destroythe tissue structure and affect the results. More accuratehistology could be achieved by undecalcified tissue section.Handschel et al. [42] reported that b-TCP was hardlyresorbed in a non-loading calvarial model, while it could beeasily degraded in the load-bearing area as we and otherresearchers observed [34], indicating that mechanical forcescould also facilitate the degradation of b-TCP scaffold. Inaddition, mechanical forces are believed to be responsiblefor determining the structure of regenerated bone [43]. Togive newly formed bone a mechanical loading, we haveremoved the titanium plate at 22 weeks post-operation inall groups in the study. A mandibular contour remodeling

could be observed in the experiment group when compar-ing the radiographic data at 32 weeks with the data at 26weeks post-operation (Fig. 4G and J). Although thestructure of regenerated bone has not been completelyremodeled into the structure like normal mandible has,biomechanical analysis revealed that the engineered bonewas functionally similar to normal mandible in terms ofmechanical property. Long term follow up is required tosee if engineered bone can be remodeled into a normalmandibular structure with canal finally.Autogenous bone transplantation is the ‘‘gold standard’’

approach for repairing bone defects. The repairing processis called a ‘‘creeping substitution’’ which includes bothresorptive and appositional processes [2]. In this study, wefound that the BMD of the bone graft was significantlyhigher than that of the normal control mandibles at 32weeks after implantation. This is probably due to theexistence of both newly formed bone and intrinsic deadbone in the grafts, which was demonstrated by histologicalanalysis. Accordingly, the bending stress of the autolo-gouse bone grafts was lower than that of engineered bone.It has been reported that it would normally take 1–2 yearsto achieve normal biomechnical property after autologousbone transplantation [44].

5. Conclusions

This study demonstrates that bone substitutes con-structed by porous b-TCP scaffold loaded with osteogeni-cally induced BMSCs could repair the critical-sizedmandibular defects in large mammal. The satisfactoryrepair rate suggests that this might be a feasible approachfor the clinical reconstruction of segmental mandibulardefects.

Acknowledgements

This work was supported by Major State Basic ResearchDevelopment Program of China (G1999054300,2005CB522700), National High Technology Researchand Development Program of China (2002AA205011),and Shanghai Science and Technology DevelopmentFoundation.We thank Drs. Deli Liu, Yulai Weng, Guangdong Zhou,

Lian Zhu, Ming Wang, Li Zhao, Guangpeng Liu, GangCai, Wanyao Xia, and Jinjun Chen for their contributionsto this paper. We also appreciate the technical assistancefrom Drs. Demin Ying, Lijuan Zong, Juanjuan Wu, andBin Zhong.

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