Repair of segmental bone-defect of goat's tibia using a dynamic perfusion culture tissue engineering...

Repair of segmental bone-defect of goat’s tibia usinga dynamic perfusion culture tissue engineering bone

Chaofeng Wang,1 Zhen Wang,1 Aimin Li,1 Feng Bai,1 Jianxi Lu,2 Shanglong Xu,3 Dichen Li31Institute of Orthopedics and Traumatology, Xijing Hospital, The Fourth Military Medical University,Xi’an 710032, China2Laboratoire de Recherche sur les Biomateriaux et les Biotechnologies, Universite du Littoral Cote d’Opale,62608 Berck sur Mer, France3State Key Lab Manufacturing Systems Engineering, Xi’an Jiaotong University, Xi’an 710049, China

Received 16 December 2007; revised 30 July 2008; accepted 7 October 2008Published online 25 March 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32347

Abstract: Segmental bone defect resulted from trauma orexcision of bone tumor or pathology represents a commonand significant clinical problem. In an attempt to solve thisdilemma, we use b-TCP combined with autologous bonemarrow mesenchymal stem cells (auto-BMSC) and cul-tured by dynamic perfusion to repair the segmental bonedefects of goat’s tibia. The b-TCP scaffolds combined withauto-BMSC by dynamic perfusion bioreactor or in staticstate were respectively transplanted into the defect(30 mm) of the goat tibias. The X-ray films were gatheredand analyzed at the different time points. At 24 weekspost-operation, tissue engineering bones implanted wereanalyzed by histology and Micro-CT. Results show that

the capacity of osteogenesis in experimental group washigher than that of control group by X-ray, histological,and micro-CT analysis (p < 0.05). According to the study,we found that repair for segmental bone defects of tissueengineering bone cultured by dynamic perfusion culturebioreactor outweigh those cultured in static state. And wecan conclude that this technology of tissue engineeringbone will become a clinical method to the segmental bone-defects repair in the future. � 2009 Wiley Periodicals, Inc.J Biomed Mater Res 92A: 1145–1153, 2010

Key words: tissue engineering bone; dynamic perfusionculture; mesenchymal stem cells; segmental bone defect

INTRODUCTION

Segmental bone defect resulted from trauma orexcision of bone tumor or pathology represents acommon and significant clinical problem. In thepast, amputation was the only option for thesedefects as they typically do not heal spontaneously.1

With advances in medicine, alternative treatmentoptions have developed such as the use of bone-grafting techniques including autologous, allogeneic,and tissue engineering bone. Autologous bone graftsare preferred as they possess inherent osteoconduc-tivity, osteogenicity, and osteoinductivity. However,there is often limited supply of suitable bone for au-tologous grafting, and its collection is frequentlyassociated with donor-site morbidity. Another alter-native is to use allogeneic bone grafts from donors

or cadavers. Although allogeneic bone grafts haveovercome the difficulty of few sources, they are notextensively used in clinic because of limited osteoin-ductivity and a potential risk of disease transmis-sion. These limitations necessitate the pursuit ofalternatives for the management of segmental bonedefects, with the latest approach being to use tissue-engineering techniques.

One of the strategies to construct tissue engineer-ing bone is to use bone marrow mesenchymal stemcells associated with the three-dimensional scaffold.The MSCs are multipotent but are present in strik-ingly low quantity in bone marrow (account for0.001% of nucleated bone marrow stromal cells inthe adult).2 Therefore, the MSCs need to beexpanded in vitro before implantation into the body.Although sufficient quantity of MSCs can beobtained by two-dimensional culture, the quantity ofMSCs adhering to and growing in three-dimensionalscaffold is limited when MSCs cultured by two-dimension are digested and seeded. Therefore, thecomposite of MSCs-scaffold need to be cultured afew days in vitro so that sufficient quantity of MSCscan be proliferated in three-dimensional scaffold.Although the three-dimensional culture technology

Correspondence to: Z. Wang; e-mail: [email protected] grant sponsor: Science and Technology Com-

mission of Shanghai Municipality; contract grant numbers:05DJ14005, 07XD14215

� 2009 Wiley Periodicals, Inc.

is used a few years, researchers can only constructthe smaller tissue engineering bone at present.3–5

One main reason in favor of constructing the smallertissue engineering bone is that the difficulty of bring-ing about cell proliferation in the center of the largerscaffold isn’t solved at present. The conventionalstatic culture of the cell-scaffold composite results incell survival and proliferation in the peripheral partrather than in the central part.6,7 The flow perfusionculture has demonstrated efficiency in transportingculture media to the center of the cell composite.8–10

Thus, the flow perfusion culture may provide ameans to solve this problem. Unfortunately, the cur-rent perfusion culture system is designed for engi-neering small cell composites measuring only severalmillimeters. And the size of tissue engineering bonein their studies is usually smaller than that of in clini-cal need. Moreover, the problem of the BMSCs’ nutri-tion supply in the center of the critical-size scaffoldhas not yet been solved. Thus, the previous tissue en-gineering bones have not yet met the requisites forrepair of the large-scale bone defects. Therefore, inthis article we present a new strategy for the cultureof tissue engineering bone by perfusion culture bio-reactor. Then the cultured tissue engineering boneswere implanted into 30-mm-sized defects in an estab-lished goat model. Thereby to explored the effects oftissue engineering bone cultured by our newly estab-lished approaches on segmental defect repairing.

METHODS AND MATERIALS

Animals

Six goats, 1-year-old female, with an average weight of25 kg (ranged, 23–27.5 kg) were provided by LaboratoryAnimal Research Centre of The Fourth Military MedicalUniversity (Xi’an, China) and fed in separate cages withfree access to food and water.

Porous b-TCP cylinder

Porous b-TCP cylinder (porosity rate, 70%; pore size,450 lm; diameter, 16 mm; height, 30 mm) were manufac-tured by Shanghai Bio-Lu Biomaterials Co., Ltd. (Shanghai,China) and provided to us as a sterile material for thisstudy. The center of b-TCP cylinder have a 3-mm-diametercaecus. The initial compression strength was 2–4 MPa.

BMSC isolation and culture

All plastic ware, cell culture media, and serum werepurchased from Invitrogen. After each goat was anesthe-tized, 0.5 mL sample of bone marrow was obtained fromthe dorsal iliac crest and mixed with 100 IU/mL heparin.

A single-cell suspension was prepared by repeatedly aspi-rating the cells through 18- and then 21-gauge needles.The number of nucleated cells was determined by count-ing using a hemocytometer. The cell suspension was cul-tured in DMEM with 15% fetal calf serum. Cells were cul-tured at 378C in a 5% CO2-95% air atmosphere at 95%humidity. On reaching confluence, cells were passaged at1:3 ratios with 0.05% trypsin. The BMSCs of each of sixgoats were gathered in the third passage.

The design of dynamic perfusion culture bioreactorand the culture of tissue engineering bone

The tissue-engineering bone culture bioreactor wasdesigned by us. The bioreactor was composed of threeparts: the driving part, the reservoir, and the tubing part.The driving part consists of up to eight 3-stop tubes(PharMed, Akron, OH), which are fixed in an 8-channelpump head, and driven by a peristaltic pump (ISMATECIP 65, Switzerland). The reservoir is a 75-cm2 flask (Cell-Star, Greiner, Germany) containing the perfusion medium.In the tubing part, there are two silicone tubes with oneend penetrating through the plug cap into the flask as theinlet and outlet. The other end is connected with the cen-tral tunnel of the b-TCP scaffold by a tube connector. Thebasic circuit and design drawing of perfusion culture bio-reactor are presented in Figure 1. The total 24 scaffoldswere needed for six goats. The third passage cell suspen-sion (2 3 106 cells/mL) of every goat was respectivelycombined with four a-TCP scaffolds under the same nega-tive pressure. Four scaffolds for every goat were dividedequally into two groups: the dynamic culture group andthe static culture group. Both groups were put into the bio-reactor. The dynamic culture groups were perfused by cy-cling flow with opening of the tubing compression pump,while the static culture groups were not perfused. Theflow rate of culture fluids is 3 mL/min.11 The culture flu-ids of both groups were changed every 3 days for 2 weeks.After 2 weeks, one scaffold in every culture group of everygoat was analyzed in histology. The other scaffold in everyculture group of every goat was implanted into the rightor left tibia of the same goat.

Surgical procedures

Each of six goats was sedated with medetomidine(5 mg/kg) served as premedication and anaesthesia wasinduced with diazepam (0.1 mg/kg) and ketamin (2 mg/kg) was maintained under anesthesia with an intravenousinjection of pentobarbital (25 mg/kg). Cephazolin (10 mg/kg) was administered subcutaneously as prophylactic anti-biotics. The goat was fixed in the right lateral position onan operating table. After the skin was sterilized with povi-done–isodine solution and wrapped using sterile tech-nique, a skin incision of 10 cm length along the left tibiawas made and the tibia was exposed. The tibia was stabi-lized with a plate and screws, (8 3 141, Guke Renli, Tian-jin, China), A 30-mm segment of the tibia including theperiosteum was removed by wire saw. The tissue engi-neering bone by dynamic perfusion culture was placed

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Journal of Biomedical Materials Research Part A

into the tibia defects (experiment group). And the skinincision was closed with interrupted sutures (Fig. 2). Thetissue engineering bone by culture in static state (controlgroup) was implanted into the right tibia by the same pro-cedure. During the first week after surgery, cephazolin(10 mg/kg) was routinely administered intramusculalyonce a day and the physical states of all the animals werealso closely monitored. All the experimental protocolswere reviewed, approved, and performed in strict accord-ance with the guidelines of the Institutional Committee forAnimal Care and Experiments of the Fourth Military Med-ical University.

Radiographic analysis

In vivo, X-rays of all the goats were taken of at 1, 4, 12,and 24-weeks postoperation by X-ray machine (PHILIPS

Corp). Exposure was at 55 kVp for 2.0 mAs. The changesof radiopacity in and around the tissue engineering bonesat the defect and bone union at the cut-ends of the originaltibias were analyzed. Radiograghs of the tibia removedfrom the goats were taken by routine procedures.

Micro-CT reconstruction

Three-dimensional analysis was conducted on all tibiabone samples in both groups. Bone specimens were col-lected immediately after sacrifice and conserved frozen. Amicrofocus X-ray tube with a focal spot of 20 lm was usedas a source. To perform a measurement, the specimen wasmounted on a turntable that could be shifted automaticallyin the axial direction. All specimens were cut into 4-cmcylinder, which includes tissue engineering bone, andwere put into X-ray tube of Micro-CT (GE Corp, Ameri-can) to reconstruct.

Histology and histomorphometry analysis

After Micro-CT reconstruct, undecalcified histologicalsections from the tibias defects were prepared.12 The sam-ples were fixed in 10% buffered formaldehyde solution(pH7.2) for 14 days and then were rinsed under tap waterfor 12 h. After being dehydrated through gradient alco-hols, they were embedded in methylmethacrylate withoutdecalcification. The cross sections were cut to about200 mm thick with a Leitz Saw Microtome 1600 (Wetzlar,Germany) and were ground and polished to about 50-mmthick. All sections were stained with van Gieson’s picro-fuchsine. Observations were made on the tissue in oraround the a-TCP and change in ceramic shape. The follow-ing parameters were measured by image analysis software(Lieca M550): NB% (new bone volume inside total implantvolume) represents as the percentage of total volume occu-pied by new bone; b-TCP% (residual b-TCP material vol-ume/total implant volume) expresses as the percentage of

Figure 1. The basic circuit of perfusion dynamic culture bioreactor (A). Illustrated here is the design of the flow perfu-sion culture system used in this study (B). The cell-TCP composite is connected with a silicon inlet tube and is immersed inthe complete medium. The medium is pumped into the scaffold via a peristaltic pump. Perfusion dynamic culture bioreactoris working in incubator. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2. Surgical procedure. An osteoperiosteal segmentaltibia defect of 30 mm length was made at two sides. Thedefect were respectively filled with tissue engineering bonesconstructed using perfusion dynamic culture bioreactor orstatic culture. [Color figure can be viewed in the onlineissue, which is available at www.interscience.wiley.com.]

REPAIR OF SEGMENTAL BONE-DEFECT OF GOAT’S TIBIA 1147


residual material volume; FT% (fibrous tissue of BMSCs/total implant volume) expresses as the percentage of totalvolume occupied by fibrous tissue of BMSCs.

Statistical analysis

All quantitative results related to NB%, b-TCP%, andFT% were statistically compared using matched t-test anal-ysis. p values <0.05 were considered significant.

RESULTS

Histology analysis of tissue engineering bonescultured in vitro

After 2 weeks in vitro culturing, the cells in staticculture group mainly existed and proliferated on thesurface of material (three to five rows). In thedynamic culture group the cells survived and prolif-erated through the scaffolds. Moreover, there werethe smooth channels with no cell in the materialspores. We considered that these channels wereformed under the pressure of perfusion and pro-vided the nutrition of BMSCs metabolism throughculture fluid exchanging (Fig. 3). However, the evi-dence to support the hypothesis need be obtained infurther study.

Gross view

All the surgeries on the six goats were completedsuccessfully, and all the animals survived withoutany difficulty during the 24 postoperative weeksexcept one goat that died in the 13th postoperative

week. The goat was found to have diarrhea in the12th postoperative week. By autopsy, the septicemiainduced by the diarrhea was verified to cause thegoat death. Because the goat died over 12 posthu-mous hours, we did not analyze the tissue engineer-ing bones implanted into this goat’s tibias. At24 weeks post-operation, all the five remaining goatswere sacrificed. The cylinder implants placed indefects in experimental group were completely cov-ered with hard bony tissue layers and could not berecognized based on gross view. In control group,the cylinder implants were covered with uneven andthin layers of bony tissue and could be recognizedfrom outside (Fig. 4).

Radiological examinations

One week after operation, the tibia defects withboth the experimental and control implants showeda sand-like radiopacity in radiographs (Fig. 5). Thetibia defect in both groups showed a shell-like radio-pacity at 4 weeks postoperation. Furthermore, afuzzy sand-like radiopacity was observed in bothgroups. However, the difference between the twogroups is that there was low density shadow in themiddle of the implant and shell-like radiopacity inexperimental group. At 12 weeks, a sand-like radio-pacity is still observed and implants became fuzzierin experimental group, while a shell-like radiopacityof new bone became even more noticeable. However,in control group the implant became small becauseof biodegradation. Moreover, radiolucency at thecutting ends became clearer and shell-like radiopac-ity of new bone around the implants are observed.At 24 weeks postoperation, the implant showed thesame density as the remains of the tibias in experi-

Figure 3. Histological section of the cell-TCP composite stained with Von Gieson. After 14 days, in the static culture, theMSCs only proliferated sufficiently in those macropores just under the scaffold surface (A). While in perfusion dynamicculture, the MSCs survived and proliferated through the scaffolds. Moreover, there were the smooth channels with no cellin the materials. Arrows indicate the smooth channel (B). (A and B magnification 310). [Color figure can be viewed in theonline issue, which is available at www.interscience.wiley.com.]

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mental group and it could hardly be distinguishedfrom the tibias. The defects of the tibias wererepaired thoroughly in radiographs. In controlgroup, radiolucency at the cutting ends and aroundthe implants became clearer. It easily recognized theimplants from the tibias at 24 weeks postoperation(Fig. 6).

Micro-CT assays

Twenty-four weeks later, the experimental groupshowed a higher capacity of ossification than that ofthe controls under the scanning of Micro-CT. Asseen in Figure 7, newly formed bones in experimen-tal group were closely conjunct to the fractured tibiabone, whereas in control group there was only athin layer of newly formed bone covering b-TCPand an obvious crevice was also found between thefractured bone and the materials, which is formedbecause the degradation rate of materials is higherthan the formation rate of the new bone. Moreover,particles of materials were found loosely scattered inthe newly formed bone in experimental group in 2Dgraph, whereas in control group the boundarybetween newly formed bone and materials was veryclear. Other than these, the ossification in oppositeside of the plate was found higher than that of theside adherence to the plate in both groups.

Histological analysis

With van Gieson’s picro-fuchsine stain, the tissuewere stained orange or red for newly formed bone,green for fibrous tissue, bright green for osteoid andblack for b-TCP scaffold range. At 24 weeks post-operation, newly formed bone along b-TCP wasfound in the undecalcified sections from experimentgroups. Mature bone tissue was found in the outerlayer. And residual b-TCP material was in the center.The mix of BMSC and fibrous tissue was betweenthem. Moreover, the structure of newly formed bonewas lined with the fibrous tissue of BMSCs, demon-strating that ossification might be on the base of fi-brous-tissue mineralization (Fig. 8). Although bothgroups showed the same mechanism of ossification,the control group showed higher content of b-TCPand a lower rate of ossification than that of experi-mental group, which was shown in Table I.

DISCUSSION

At present, tissue engineering bone focuses onconstructing segmental tissue engineering bonewhich is constituted by scaffold combined withosteogenesis seed cells and proper cell factor. Cultur-ing the composite of the scaffold combined withosteogenesis seed cells and cell factor in vitro, manyresearchers used them to repair the defects of bonein vivo. According to the methods mentioned above,many researchers concluded that the capacity repair-

Figure 5. At 1 week post-operation, the experimental X-ray film.

Figure 4. Gross view of repaired tibias at 24 weeks post-operation. (A) In the dynamic culture group, bony-unionwith cortex and notches are scarely observed. (B) In thestatic culture group, soft tissues are also detected in thedefect except of bony-union. Arrows indicate the junctionsites. [Color figure can be viewed in the online issue, whichis available at www.interscience.wiley.com.]



ing defects of the scaffolds combined with seed cellsor cell factors outweigh the simple scaffold.13,14

It is reported that smaller bone defect and themethod of static culture are the main objective inrecent years. In the b-TCP ceramics that we selected,pore size (450 6 50 lm) and interconnection poresize (150 6 50 lm), were found to be well biocom-patible, osteoconductive, degradationable, and tolead to a fast turnover to bone and to benefit bloodvessel to put into.15–17 We selected BMSC as seedcells because of general availability, high osteogenicpotential and high proliferation rate in vitro. More-over, previous evidences indicated that BMSC withina porous ceramic implant can lead to osteogene-

sis.13,18–20 We utilized an innovative perfusion cul-ture bioreactor designed by ourselves to culture thetissue engineering bone in vitro. The system of cul-ture can not only make BMSC well proliferation andwell-distributed into b-TCP ceramics, but also makeBMSC differentiate into the cell that have osteogene-sis.21 We did not induce the BMSC to osteoblastin vitro, to ensure the BMSC to proliferate largely inthe short term and to avoid the activity of the cellproliferation to decrease.22 The seeded cells can onlygrow on the edge of the scaffold under traditionalstatic culturing condition.11 Nevertheless, under thedynamic perfusion culturing condition, there isenough culture fluid to guarantee the seed cells to

Figure 6. Radiographs of treated defects taken at different time points post-operation. In the dynamic culture group,fuzzy sand-like radiopacity and little calluses with shell-like shadow are formed at 4 weeks (A1), and the implants becamefuzzier and shell-like radiopacity of new bone are observed at 12 weeks (A2). At 24 weeks, it is hardly recognized theimplants from the tibias and the radiopacity is close to that of normal remains of tibia in radiographs (A3). In the staticculture group, fuzzy sand-like radiopacity and no calluses is observed at 4 weeks (B1). Radiolucency at the cutting endsand shell-like radiopacity of new bone around the implants are observed at 12 weeks (B2), while nearly complete radiolu-cency at the cutting ends and the same shell-like radiopacity of new bone around the implants as that of at 12 weeks isobserved at 24 weeks, and the implants became smaller (B3).

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get well nourished and decrease the concentration ofthe cell metabolin in the scaffold.23,24 The experimentsurveys that there are a number of the BMSC andwell-distributed in the tissue engineering bone cul-tured by perfusion culture bioreactor, but the BMSCwere only distributed surface of the tissue engineer-ing bone cultured by static culture, none in inner ofthe material. The experimental result explains the rea-son in theory that the osteogenesis of the tissue engi-neering bone cultured by perfusion outweighs the tis-sue engineering bone cultured by static culture.

Many researchers13,25,26 found that the capacityrepairing defects of the scaffolds combined with

seeded cells by static culture or no culture obviouslyoutweigh the simple scaffold in vivo. We constructedthe tissue engineering bone by perfusing culture bio-reactor in vitro, and implanted into 30-mm-sizeddefects in an established goat tibia defect model. Wefound that tissue engineering bone by perfusion cul-ture presented better effect in the repair of bonedefect than that of static culture. At present, manyresearches focus on repairing small bone defectsinstead of in the region of segmental bone defects.On the other hand, the therapy of segmental bonedefects is urgent in clinic, which is also the factor ofrapid development of tissue engineering bone. The

Figure 7. Reconstructing 3D and 2D imaging by Micro-CT at 24 weeks post-operation. In the dynamic culture group,newly-formed bones are closely conjunct to the fractured tibia bone (A: 3D imaging), particles of materials are observedloosely scattered in the newly-formed bone (A1: longitudinal section 2D imaging and A2: cross section imaging). In thestatic culture group, a thin layer of newly-formed bone covering implants and an obvious crevice between implants andnewly-formed bone are observed (B), the boundary between newly-formed bone and materials was very clear (B1: longitu-dinal section 2D imaging and B2: cross section imaging).



defect of the goat tibia in our experiment can beseen as segmental bone defects, whose length is30 mm, average diameter is 16 mm. It is an attemptto repair segmental bone defects. As a result, wefound that tissue engineering bone by dynamic per-fusion culture presented better effect in the repair ofbone defect, whatever in gross view, radiographic,

histological analysis, and Micro-CT reconstruction.The superiority of tissue engineering bone by perfu-sion culture provides reference for study both ingroundwork and clinic in the future.

We observed in experiment that the way of repair-ing bone defect is that BMSC keep the spatial struc-ture, differentiate into osteoblasts under the condi-

TABLE IPercent of NB, FT, and b-TCP in Both Groups

Experimental Group Control Group

NB% FT% b-TCP% NB% FT% b-TCP%

Sample1 76.11 12.30 11.59 40.23 18.61 41.16Sample1 79.23 14.88 5.89 49.11 16.23 34.66Sample1 81.36 11.27 7.37 48.37 14.10 37.53Sample1 83.16 12.21 4.63 44.30 15.27 40.43Sample1 77.84 11.27 10.89 45.21 16.79 38.00

Data present that percent of new bone volume, residual b-TCP material volume and fibrous tissue of BMSCs in bothgroups were respectively significant difference, p is respectively 0.000, 0.014, and 0.000. (p < 0.05).

Figure 8. Histological analysis by Von Gieson in the dynamic culture group. Microscopic examination showed numerousnew bone formation (NB) on the edge of the tissue engineering bone. A few degradation particles and fibrous tissue pre-sented around the b-TCP. The residual b-TCP were observed in the center of tissue engineering bone (A). New bone for-mation in the pore of the residual b-TCP and the residual b-TCP incased in new bone were observed (B). From outer toinner, new bone, fibrous tissue and b-TCP were observed (C). (A: magnification 31.6; B: magnification 310, and C: magni-fication 35). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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tion of osteogenesis, and excrete bone matrix to formmineralization bone after the solution of b-TCP scaf-fold. Therefore, the phenomenon appeared in VanGieson staining that in implant zone, mature bonetissue is in the outer layer, transits ripple shaped la-mellar bone, BMSC and fibrous tissue gradually, b-TCP scaffold is in the center. If new bone tissuegrows faster than the degradation of scaffold, it willsurround the residuary of b-TCP scaffold.

CONCLUSIONS

In this study, investigating dynamic perfusion-cul-tured tissue engineering bone in segmental bone-defects repairing, we found that segmental defects inthe goat tibia have advanced radiological, histologi-cal, and mechanical healing using our tissue engi-neering strategy of dynamic perfusion-cultured.Radiographical and histological healing is enhancedwith tissue engineering bone by dynamic perfusion-cultured. The satisfactory repair suggests that thismight be a feasible approach for the clinical recon-struction of segmental bone-defect. Although morework will be done, we believe that dynamic perfu-sion culture will become a feasible method to repairsegmental bone-defect.

Authors thank Youzhuan Xie in Department of Ortho-paedic Surgery, Ninth People’s Hospital, Shanghai JiaoTong University School of Medicine for expert technicalassistance. Authors also thank Doctor Min Li for technicalassistance.

References

1. Giannoudis PV, Pountos I. Tissue regeneration. The past, thepresent and the future. Injury 2005;36 (Suppl 4):S2–S5.

2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R,Mosca JD, Moorman MA, Simonetti DW, Craig S, MarshakDR. Multilineage potential of adult human mesenchymalstem cells. Science 1999;284:143.

3. Kruyt MC, de Bruijn JD, Wilson CE, Oner FC, van Blitters-wijk CA, Verbout AJ Dhert WJ. Viable osteogenic cells areobligatory for tissue-engineered ectopic bone formation ingoats. Tissue Eng 2003;9:327.

4. Shin M, Yoshimoto H Vacanti JP. In vivo bone tissue engi-neering using mesenchymal stem cells on a novel electrospunnanofibrous scaffold. Tissue Eng 2004;10:33.

5. Mauney JR, Blumberg J, Pirun M, Volloch V, Vunjak-Nova-kovic G, Kaplan DL. Osteogenic differentiation of humanbone marrow stromal cells on partially demineralized bonescaffolds in vitro. Tissue Eng 2004;10:81.

6. Ishaug SL, Crane GM, Miller MJ, Yasko AW, Yaszemski MJ,Mikos AG. Bone formation by threedimensional stromalosteoblast culture in biodegradable polymer scaffolds.J Biomed Mater Res 1997;36:17.

7. Holy CE, Shoichet MS, Davies JE. Engineering three-dimen-sional bone tissue in vitro using biodegradable scaffolds:Investigating initial cell-seeding density and culture period.J Biomed Mater Res 2000;51:376.

8. Sittinger M, Bujia J, Minuth WW, Hammer C, Burmester GR.Engineering of cartilage tissue using bioresorbable polymercarriers in perfusion culture. Biomaterials 1994;15:451.

9. van den Dolder J, Bancroft GN, Sikavitsas VI, Spauwen PH,Jansen JA, Mikos AG. Flow perfusion culture of marrow stro-mal osteoblasts in titanium fiber mesh. J Biomed Mater Res A2003;64:235.

10. Bancroft GN, Sikavitsas VI, Mikos AG. Design of a flow per-fusion bioreactor system for bone tissue-engineering applica-tions. Tissue Eng 2003;9:549.

11. Xie Y-Z, Zhu Z-A, Tang T-T, Dai K-R, Lu J-X, Pierre H. Usingperfusion bioreactor for mesenchymal stem cell proliferationin large tricalcium phosphate scaffold. Natl Med J China2006;86:1633–1637.

12. Xie Y-Z, Chopin D, Morin C, Pierre H, Zhenan Z, Jian T,Jianxi L. Evaluation of the osteogenesis and biodegradationof porous biphasic ceramic in the human spine. Biomaterials2006;27:2761–2767.

13. Petite H, Viateau V, Bensaıd W, Meunier A, de Pollak C,Bourguignon M, Oudina K, Sedel L, Guillemin G. Tissue-engineered bone regeneration. Nat Biotechnol 2000;18:959–963.

14. Jie Y, Lei C, Wen JZ. Repair of canine mandibular bonedefects with bone marrow stromal cells and porous b-trical-cium phosphate’ Biomaterials 2007;28:1005–1013.

15. Hirokazu K, Takaaki T, Masaaki C, Takahiro K. Repair ofsegmental bone defects in rabbit tibiae using a complex of b-tricalcium phosphate, type I collagen, and fibroblast growthfactor-2. Biomaterials 2006;27:5118–5126.

16. Yuan H, Yang Z, De Bruij JD, De Bruij JD, De Groot K,Zhang X. Material-dependent bone induction by calciumphosphate ceramics: A 2.5-year study in dog. Biomaterials2001;22:2617–2623.

17. Jianxi L, Michel D, Jacques D, Gilles K, Pierre H, Jacques L,Jean-Pierre P. The biodegradation mechanism of calciumphosphate biomaterials in bone. J Biomed Mater Res 2002;63:408–412.

18. Ohgushi H, Goldberg VM, Caplan AI. Repair of bone defectswith marrow cells and porous ceramic. Experiments in rats.Acta Orthop Scand 1989;60:334–339.

19. Bruder SP, Kurth AA, Shea M, Hayes WC, Jaiswal N,Kadiyala S. Bone regeneration by implantation of purified,culture-expanded human mesenchymal stem cells. J OrthopRes 1998;16:155–162.

20. Bruder SP, Kraus KH, Goldberg VM, Kadiyala, S. The effectof implants loaded with autologous mesenchymal stem cellson the healing of canine segmental bone defects. J Bone JointSurg Am 1998;80:985–996.

21. Minuth WW, Schumacher K, Strehl R. Physiological and cellbiological aspects of perfusion culture technique employed togenerate differentiated tissues for long term biomaterialexperimentaling and tissue engineering. J Biomater Sci PolymEd 2000;11:495–522.

22. Prockop DJ. Marrow stromal cells as stem cells for nonhema-topoietic tissues. Science 1997;276:71–74.

23. Davisson T, Sah RL, Ratcliffe A. Perfusion increases cell con-tent and matrix synthesis in chondrocyte three-dimensionalcultures. Tissue Eng 2002;8:807–816.

24. Bancroft GN, Sikavitsas VI, van den Dolder J, Sheffield TL,Ambrose CG, Jansen JA, Mikos AG. Fluid flow increasesmineralized matrix deposition in 3D perfusion culture ofmarrow stromal osteoblasts in a dose-dependent manner.Proc Natl Acad Sci USA 2002;99:12600–12605.

25. Dorozhkin SV, Epple M. Biological and medical significanceof calcium phosphates. Angew Chem Int Ed 2002;41:3130–3146.

26. Hench LL, Wilson J. Surface-active biomaterials. Science 1984;226:630–636.



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