异种脱蛋白骨管复合不同移植材料修复大段骨缺损的实验研究

目的 应用异种脱蛋白骨管复合骨基质明胶(BMG)、骨形态发生蛋白(BMP)、骨膜细胞与复合脱钙骨基质(DBM)、自体红骨髓(RBM)作比较,观察修复猪大段骨缺损的成骨差异.方法 建立猪双侧胫骨大段骨缺损模型.24只成年猪,随机分成2组,实验组A:异种骨管+BMG、BMP、骨膜细胞;实验组B:异种骨管+DBMLRBM;两组均采用锁定钢板及异种皮质骨板固定.分别于术后4、8、12、24周时通过大体标本观察.放射学及组织学观察评估其修复猪大段骨缺损的成骨情况.结果 植入后24周,异种骨管复合BMG、BMP、骨膜细胞完全修复.成骨活跃程度,骨再生量和重建髓腔结构等方面均显著;复合脱钙骨基质、自体红骨髓成骨能力较弱.结论 异种脱蛋白皮质骨管复合骨基质明胶、骨形态发生蛋白及骨膜细胞能够有效修复大段骨缺损.     

自体脂肪干细胞复合珊瑚修复犬颅骨标准缺损的初步研究

目的 应用自体脂肪干细胞(adipose-derived stem cells,ADSCs)复合珊瑚构建组织工程化骨,修复犬颅骨标准缺损.方法 体外扩增培养、成骨诱导Beagle犬ADSCs,将第2代细胞接种在珊瑚支架上共同培养.制造实验犬双侧颅骨全层标准缺损(20 mm×20 mm),一侧以细胞材料复合物修复作为实验组(n=7),另一侧以单纯珊瑚材料修复作为对照组(n=7).术后24周分别通过影像学、大体形态观察、生物力学检测、组织学方法检测颅骨缺损的修复效果.结果 成骨诱导的犬ADSCs体外呈现成骨特性,在珊瑚支架上生长良好.3D-CT重建显示术后12周实验组有新生骨痂形成,对照组材料大部分降解;24周时实验组为骨性愈合,对照组为骨不连.24周时实验组缺损修复百分比为(84.19±6.45)%,显著高于对照组的(25.04 ±18.82)%(P<0.01).大体观察见实验组由新生骨痂修复缺损,对照组缺损边缘可见少量骨痂形成,主要为软组织充填;24周生物力学检测修复组织能耐受的最大压力载荷,实验组为(73.45±17.26)N,为犬顶骨最大压力负荷(104.27±22.71)N的70%,两者比较差异有统计学意义(P<0.01),对照组为软组织无法完成上述检测.HE染色见实验组有较多成熟骨呈骨性愈合,对照组为纤维性愈合.结论 自体成骨诱导的ADSCs复合珊瑚形成的组织工程化骨可修复犬颅骨标准缺损.     

应用BMP与FGF修复兔桡骨缺损的实验研究

目的 联合应用成骨及成血管的生长,探索治疗骨缺损的有效方法。方法 联合应用BMP+bFGF及单纯BMP复合去抗原异种松质骨,采用X线摄片及组织学方法检测两种方法修复兔桡骨2 cm缺损效果。结果 联合应用BMP+bFGF修复兔桡骨2 cm缺损在12周时缺损完全骨性连接,骨髓腔完全再通,明显优于单纯BMP组的修复效果(P<0.01)。结论 联合应用BMP+bFGF修复兔桡骨2 cm缺损在12周时缺损完全骨性连接,骨髓腔完全再通,能成为修复缺损的新的有效方法。     

BMP/PCC人工骨修复长骨大段缺损的实验研究

目的 研究理想的、能较快修复大段骨缺损的人工骨材料。方法 将骨形态发生蛋白（BMP）和多孔复合陶瓷（PCC）结合研制成BMP/PCC人工骨，并将BMP/PCC和PCC人工骨进行兔桡骨大段骨缺损修复的对比研究。术后2、4、8和12周时取材，分别作大体、组织形态学、新骨形成定量分析及生物力学测试。结果 BMP/PCC人工骨内新骨形成量明显多于PCC人工骨。术后12周时，BMP/PCC侧植入部位的抗折强度明显高于PCC侧。结论 BMP/PCC人工骨能更快促进长骨大段骨缺损的修复，是一种较理想的人工骨材料。     

应用BMP与FGF修复兔桡骨缺损的实验研究

目的 联合应用成骨及成血管的生长，探索治疗骨缺损的有效方法。方法 联合应用BMP bFGF及单纯BMP复合去抗原异种松质骨，采用X线摄片及组织学方法检测两种方法修复兔挠骨2cm缺损效果。结果 联合应用BMP bFGF修复兔挠骨2cm缺损在12周时缺损完全骨性连接，骨髓腔完全再通，明显优于单纯BMP组的修复效果(P＜0．01)。结论 联合应用BMP bFGF修复兔挠骨2cm缺损在12周时缺损完全骨性连接，骨髓腔完全再通，能成为修复缺损的新的有效方法。     

自固化磷酸钙复合BMP及同种异体骨修复兔股骨大段骨缺损

[目的] 观察一种新型自固化磷酸钙（CPC）复合BMP与同种异体骨修复兔股骨节段性骨缺损的效果，为临床复合应用大段同种异体骨移植提供参考。[方法] 54只新西兰大白兔随机分成3组，于一侧股骨中上段造成2cm长皮质骨缺损模型，分别进行：A组复合BMP与CPC的新鲜冷冻同种异体骨移植；B组单纯新鲜冷冻同种异体骨移植；C组自体大段骨移植。移植骨均用直径3mm三棱髓内针固定。于术后4、8、12周，进行影像学、组织学检查，对比各组移植骨愈合过程与修复效果。[结果] CPC复合BMP大段同种异体骨移植早期骨修复效果优于单纯异体骨移植（P〈0．01），与自体骨移植修复效果相似，至12周3组均达骨性愈合，以A组及C组骨修复塑形较好。CPC复合BMP组骨痂量较多，分布于移植骨与宿主骨结合部及移植骨周围，形成皮质骨外骨桥，并较早在异体骨外表面形成破骨与成骨，异体骨内哈佛氏管扩大，衬垫细胞、成骨细胞、破骨细胞及血细胞较其它组多。CPC随着新骨的形成及改建塑形逐步缓慢降解。[结论] CPC复合BMP对大段同种异体骨移植的愈合及替代有增强和促进作用。     

VEGF165基因修饰脂肪干细胞对糖尿病大鼠骨缺损的修复研究

目的 :探讨采用基因转染大鼠脂肪干细胞构建血管化组织工程的方法对糖尿病骨质疏松性骨缺损的修复效果。方法:选取雄性Wistar大鼠78只,体重180~220 g,其中72只通过化学药物(STZ)诱导法建立糖尿病动物模型,成模大鼠血糖值均≥16.7 mmol/L。将实验动物随机分为5组,正常对照组6只,其他实验组各18只。正常对照组:在正常大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞;糖尿病组:单纯糖尿病骨缺损大鼠;生长因子组:在糖尿病大鼠骨缺损内单纯植入VEGF生长因子;干细胞组:在糖尿病大鼠骨缺损内单纯植入脂肪干细胞;实验组:在糖尿病大鼠骨缺损内植入经VEGF165基因修饰的脂肪干细胞。将5×106个VEGF165-ADSCs细胞与凝胶海绵结合后,植入到糖尿病大鼠骨缺损模型中,在植入后第4周时,采用光学显微镜观察缺损修复组织大体形态;采用免疫组化SP法测定骨缺损区修复后局部微血管密度;应用美国IRIS IntrepidⅡXSP电感耦合等离子体发射光谱仪对修复骨痂内钙/磷含量和碱性磷酸酶(ALP)含量测定;统计分析上述测量结果验证VEGF165-ADSCs对糖尿病大鼠骨缺损的修复作用。结果:荧光染色结果显示,VEGF165表达定位于ADSCs的细胞浆,表达率在87﹪以上;大体组织学观察结果显示:实验组修复区内骨痂生成范围和质量接近正常组,糖尿病组、生长因子组、干细胞组修复效果欠佳。植入后第4周,实验组单位体积的修复组织钙、磷含量和ALP含量明显高于生长因子组、干细胞组(P0.05),与正常对照组组比较差异无统计学意义(P0.05);第4周时,实验组修复局部的血管密度低于正常对照组(P0.05),而显著高于其他组(P0.05)。结论 :VEGF165基因修饰的脂肪干细胞在糖尿病大鼠体内具有良好的成骨及成血管作用,有望成为修复糖尿病特定骨质条件下骨缺损的一种有效手段。     

复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙人工骨结合带血供骨膜修复羊大段骨缺损

目的 研究复合骨形态蛋白-2的聚乳酸-乙醇酸共聚物/磷酸三钙(PLGA-TCP-BMP-2)人工骨结合自体带血供尺骨骨膜移植修复大段骨缺损的效果. 方法 手术造成30 mm绵羊桡骨骨缺损,A组植入PLGA-TCP-BMP-2人工骨及带血运的尺骨骨膜,B组仅植入PLGA-TCP-BMP-2人工骨,C组不植入任何材料.3组均以钢板固定骨缺损区.术后24周拍摄X线片并处死动物,进行组织学观察评价. 结果 X线检查示A组桡骨缺损处完全成骨修复,皮质骨与髓腔的轮廓清晰;B组亦完全修复,但新生骨密度及髓腔轮廓清晰度均不如A组;C组无有效骨痂形成.组织学检查示A、B组骨痂外层形成为板层骨,C组缺损区可见大量纤维组织填充. 结论 PLGA-TCP-BMP-2人工骨结合自体带血供尺骨骨膜移植能够很好的修复绵羊桡骨30 mm的骨缺损.     

PLGA-TCP-BMP-2人工骨结合带血供自体骨修复骨缺损

[目的]研究PLGA-TCP-BMP-2人工骨结合自体带血供自体尺骨移植修复大段骨缺损的效果.[方法]手术造成30 mm绵羊桡骨骨缺损,A组置入PLGA-TCP-BMP-2人工骨及带血运的长段尺骨,B组仅置入PLGA-TCPBMP-2人工骨,C组不置人任何材料.3组均以钢板固定骨缺损区.术后24周拍摄X线片并处死动物,进行组织学观察评价.[结果]X线检查示A组桡骨缺损处完全成骨修复,皮质骨与髓腔的轮廓清晰;B组亦能完全修复,但新生骨密度及髓腔轮廓清晰度均不如A组;C组无有效骨痂形成.组织学检查示A、B组骨痂外层形成为板层骨,C组缺损区可见大量纤维组织填充.[结论]PLGA-TCP-BMP-2人工骨结合自体带血供长段尺骨移植能够很好的修复绵羊桡骨30 mm的骨缺损.     

骨形成蛋白增强自体骨修复股骨头骨缺损的研究

目的　对比评价应用骨形成蛋白 (BMP)增强自体骨与单纯自体骨重建股骨头的不同能力。方法　用健康成年杂种犬 9只 ,以环钻钻入股骨头造成股骨头内骨缺损模型。实验犬分为 A、B、C三组。A组为骨缺损模型组 ,B组取自体同侧大转子骨植入 ,C组取自体大转子骨加 BMP植入。术后 3、6和 9周行 X线片、CT、光镜及电镜观察 ,了解股骨头骨缺损的修复情况。结果　 A组术后 3周可见股骨头骨缺损内血肿机化 ,编织骨形成 ,但骨小梁改建慢 ,9周时编织骨小梁仍未被板层骨小梁取代。B组植入的自体骨坏死 ,9周时仍可见到大量的坏死骨 ,坏死骨主要起到成骨支架的作用。C组术后 3周可见股骨头骨缺损内软骨内成骨及膜内成骨活跃 ,9周时骨缺损已完全由致密的新生板层骨修复。结论　应用单纯自体骨修复股骨头骨缺损仅能发挥成骨支架的作用 ,而自体骨加BMP复合移植能在术后早期 (9周 )有效地重建股骨头内结构 ,是修复重建股骨头骨缺损的好方法     

人骨形态发生蛋白-2基因转染人脂肪源性基质细胞的异位成骨作用

目的探讨腺病毒介导的人骨形态发生蛋白-2(Adv-hBMP-2)基因转染人脂肪源性基质细胞(ADSCs)的可行性及其成骨作用。方法从人脂肪组织中提取间充质细胞，分别转染β-半乳糖苷酶(β-gal)基因和hBMP-2基因，通过X-gal染色明确转染效率．-免疫沉淀 Western blot法和ELISA法检测BMP-2表达，确定BMP-2表达量、表达时间及其相互关系，流式细胞仪观察基因转染对细胞凋亡的影响，并将转基因细胞注入裸鼠股部肌内行X线及组织学检查。结果X-gal染色显示转染效率达91％。免疫沉淀 Western blot法和ELISA法显示转染hBMP-2的ADSC具有较高且稳定的BMP-2的表达，20d内表达量无明显减退。流式细胞表明腺病毒转染后细胞凋亡率上升，但上升幅度不大。裸鼠肌内注射转基因细胞后2周即显示有异位骨形成，4周明显增多，对照组无骨组织形成。结论ADSC是较好的基因治疗载体细胞，转染hBMP-2后可以诱导裸鼠体内骨形成。．     

BMP2Gene Therapy on the Repair of Bone Defects of Aged Rats

Age-related decline in the number of mesenchymal stem cells (MSCs) and their reduced capability to differentiate osteogenically, along with diminished availability of growth factors, may be major factors accounting for reduced bone formation in the aging mammalian body. In the first part of the study, we compared the number of MSCs in bone marrow (BM) and the content of bone morphogenetic protein 2 (BMP2) in cortical bone tissue in juvenile, adult, and aged (1, 9, and 24 months, respectively) male rats. To assay the influence of aging on osteogenic differentiation ability, MSCs from the three age groups were transduced with the BMP2 gene. Following gene transduction, the production of BMP2 in culture media, expression of osteogenic proteins (e.g., alkaline phosphatase, type Iα1 collagen, osteopontin, and bone sialoprotein), as well as ectopic bone formation in athymic mice were compared. Results showed that the number of MSCs in BM as well as the content of BMP2 in cortical bone tissue decreased with age, but no significant differences between the three age groups were found with regard to production of BMP2 or capability of BMP2 gene-modified MSCs to differentiate osteogenically. The second part of the study applied BMP2 gene-modified autologous MSCs/β-tricalcium phosphate for repair of bone defects in aged rats with positive results. Our data indicate that the osteogenic potential of MSCs of aged rats can be restored following BMP2 gene transduction and that this technique may be a useful approach in the future planning of gene therapy for age-related osteoporotic fractures.     

骨髓间充质干细胞膜片复合磷酸三钙陶瓷构建工程化骨组织的体内成骨研究

目的：探讨骨髓间充质干细胞膜片复合磷酸三钙支架材料构建组织工程骨的可行性。方法：将兔骨髓间充质干细胞高密度接种于普通培养皿,在成骨诱导条件下连续培养2周,获得细胞膜片,修剪成长方形,并由一端卷起包裹圆柱状的磷酸三钙材料。静置孵育24h后将构建物移植到裸鼠背部皮下。术后6周取材,进行大体观察、组织学检查、组织定量学分析。结果：所获骨髓间充质干细胞膜片有多层细胞组成,保留了细胞外基质。实验组在材料表面及其孔隙内有较多的骨质形成;单一材料组空隙内为纤维组织,未见骨或软骨样组织;单一膜片组见片状编织骨形成。结论：骨髓间充质干细胞膜片在体内具有良好的成骨能力,可作为细胞释放载体与磷酸三钙支架复合构建骨组织。本研究为骨组织工程构建提供了新的方法。     

Comparison of Osteogenic Potentials of BMP4 Transduced Stem Cells from Autologous Bone Marrow and Fat Tissue in a Rabbit Model of Calvarial Defects

We compared bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) of adult rabbits under identical conditions in terms of their culture characteristics, proliferation capacity, osteogenic differentiation potentials induced by adenovirus-containing bone morphogenetic protein 4 (Ad-BMP4) in vitro, and capacity to repair calvarial defects in the rabbit model by autologous transplantation ex vivo. According to the results of growth curve, cell cycle, and telomerase activity analysis, ADSCs possess a higher proliferation potential. Both of the Ad-BMP4 transduced MSCs expressed BMP4 mRNA and protein and underwent osteogenic differentiation. Up-regulated mRNA expression of all osteogenic genes was observed in differentiated BMSCs and ADSCs, but with different patterns confirmed by real-time RT-PCR. Deposition of calcified extracellular matrix was significantly greater in differentiated ADSCs compared with differentiated BMSCs. X-ray and histological examination indicated significant bone regeneration in the calvarial defects transplanted with Ad-BMP4 transduced autologous MSCs compared to the control groups. There was no significant difference in new bone formation in Ad-BMP4 transduced MSCs based on quantitative digital analysis of histological sections. The use of ADSCs often resulted in the growth of fat tissue structures in the control groups, and the fat tissue structures were not seen with BMSC cells. Our data demonstrate that BMP4 can be potently osteoinductive in vivo, resulting in bone repair. ADSCs may be an attractive alternative to BMSCs for bone tissue engineering under appropriate stimuli. But the easy adipogenic differentiation needs to be considered when choosing adipose tissue for specific clinical application.     

Autogeneic cancellous bone grafts in extensive segmental ulnar defects in dogs. Effects of xenogeneic bovine bone morphogenetic protein without and with interposition of soft tissues and interruption of blood supply

Xenogeneic (bovine) bone morphogenetic protein (bBMP) and associated insoluble noncollagenous proteins (NCP) were implanted in inbred adult beagle dogs with 3-4 cm diaphyseal defects in the ulna. Defects were stabilized with internal plate fixation, and the control defects were not stabilized. The defects were implanted with either autogeneic cancellous bone grafts (ACG), bBMP/NCP, or a composite of ACG and bBMP/NCP. Of the plated ulnae, 18 of 19 ACG controls restored bone continuity; six of seven defects healed under the influence of bBMP/NCP plus ACG. Two of four defects with bBMP/NCP plus ACG healed and two were filled with osseous tissue, but fibrous tissue developed at one or both bone ends. Eight of nine defects implanted with bBMP/NCP capsules alone were repaired with fibrous tissue only. Of the nonplated defects, four were implanted with bBMP/NCP plus ACG and only one regenerated; three of four showed hypertrophic bone growth around a pseudarthrosis. Of six nonplated defects implanted with bBMP/NCP without ACG, all developed atrophic bone ends and fibrous tissue repair. Thus, to restore continuity of large segmental defects three times greater than the critical size for spontaneous regeneration, xenogeneic bBMP/NCP failed to induce bone regeneration in dogs. To exclude cell-mediated immune reactions and soft-tissue ingrowth, one defect was bridged with a polytetrafluoroethylene semipermeable tube (pore size 0.45 micron) containing implants of bBMP/NCP. In response to bBMP/NCP, cells from the host bone ends produced ossicles of induced woven bone formation. The observation that bBMP/NCP induced bone formation across the defect inside of semipermeable cylindrical chambers suggests that the experiments on bone defects larger than the critical size for spontaneous repair should be repeated with: (1) allogeneic dog BMP/NCP; (2) semipermeable cylinders to protect against muscle interposition; (3) compartment angiograms to evaluate blood supply; (4) treatment of the recipient with immunosuppressants and immunostaining to observe the concentration gradient of BMP; and (5) histologic observations on the first three days after implantation to evaluate cell-mediated immune barriers to the response of BMP.     

Repair of bone defects in revision hip arthroplasty by implantation of a new bone-inducing material comprised of recombinant human BMP-2, Beta-TCP powder, and a biodegradable polymer: an experimental study in dogs.

A recombinant BMP-2-retaining putty-form implant in combination with a hip prosthesis was used to reconstruct a canine hip joint with defects similar to those encountered in revision total hip arthroplasty (THA). The bone defects were made by resecting the medial half of the proximal femur and the superior acetabular bone with inner iliac wall perforation in 10 dogs. In five dogs, hip prostheses were implanted with the putty material consisting of a synthetic polymer (poly D,L-lactic acid-polyethylene glycol block copolymer), beta-tricalcium phosphate powder, and recombinant human BMP-2 in each defect (BMP/Polymer/TCP group). In the remaining five dogs, the same material without rhBMP-2 (control group) was implanted. In the BMP/Polymer/TCP group, new radiopaque shadows began to appear 4 weeks after surgery at the defects around the hip prostheses on both the femoral and acetabular sides. At 12 weeks, the defects were completely filled with new bone in contact with the prosthesis. On histology, the rhBMP-2/Polymer/beta-TCP composite putty implants had been completely resorbed and replaced by new bone. Repair of the bone defects was not seen in the control group. The ability of this material to restore bone effectively eliminates the dependency on bone grafts of autogeneic or allogeneic origin for revision hip arthroplasty and thus opens up a potential new treatment approach in hip cases requiring this type of surgery.     

Lentiviral-mediated BMP-2 gene transfer enhances healing of segmental femoral defects in rats

The objective of the present study was to assess the ability of bone marrow cells expressing BMP-2 created via lentiviral gene transfer to heal a critical sized femoral defect in a rat model. Femoral defects in Lewis rats were implanted with 5x10(6) rat bone marrow stromal cells (RBMSC) transduced with a lentiviral vector containing either the BMP-2 gene (Group I), the enhanced green fluorescent protein (LV-GFP) gene (Group IV), or RBMSC alone (Group V). We also included femoral defects that were treated with BMP-2-producing RBMSC transduced with lentivirus, 8 weeks after infection (Group III), and a group with 1x10(6) RBMSC transduced with a lentiviral vector with the BMP-2 gene (Group II). All defects (10/10) treated in Group I healed at 8 weeks compared with none of the femora in the control groups (Groups IV and V). In Group II, only one out of 10 femora healed. In Group III, 5 out of 10 femora healed. Significantly higher amounts of in vitro BMP-2 protein production were detected in Groups I, II, and III when compared to that of the control groups (p<0.05). Histomorphometric analysis revealed significantly greater total bone volume in defects in Group I and III when compared to control specimens (p<0.003). Biomechanical testing revealed no significant differences in the healed defects in Groups I and III when compared to intact, nonoperated femora with respect to peak torque and torque to failure. Our results indicate that BMP-2-producing RBMSC created through lentiviral gene transfer have the capability of inducing long-term protein production in vitro and producing substantial new bone formation in vivo.     

Autogeneic bone marrow and porous biphasic calcium phosphate ceramic for segmental bone defects in the canine ulna.

The purpose of this study was to evaluate a porous biphasic hydroxyapatite-calcium phosphate ceramic as a modifier and extender of an autogeneic marrow graft for filling a 2.5-cm segmental bony defect. Twenty adult mongrel dogs were surgically treated to create diaphyseal defects in the left ulnae. The defects were (1) filled with autogeneic bone marrow mixed with granular hydroxyapatite-tricalcium phosphate ceramic (granular ceramic); (2) grafted with a solid block of ceramic soaked in autogeneic bone marrow (block ceramic); (3) received no graft (no implant); or (4) were grafted with autogeneic bone marrow alone (bone marrow). All animals were followed clinically and roentgenographically for 24 weeks and then killed. Repair of diaphyseal defects with the block ceramic led to three solid unions and three fibrous unions; with the granular ceramic implants and marrow, the defects of five dogs formed solid unions, and one progressed to a fibrous union. Defects in all five dogs grafted with autogeneic bone marrow united. The three dogs with no implant formed nonunions. Histology showed normal marrow and only a light immune reaction. Complete bridging of the defect in the dogs treated with the granular ceramic occurred significantly earlier than bridging in the dogs grafted with bone marrow alone. Histomorphometry, performed on the block ceramic implants indicated active resorption of ceramic. Clinically, addition of ceramic to a marrow graft improved the handling characteristics of the graft material and accelerated healing according to roentgenographic evaluation.     

Differential effect of BMP4 on NIH/3T3 and C2C12 cells: implications for endochondral bone formation.

After intramuscular implantation, BMP4-expressing NIH/3T3 fibroblasts and BMP4-expressing C2C12 myoblasts can promote ectopic cartilage and bone formation. Fibroblasts tend to undergo chondrogenesis, whereas myoblasts primarily undergo osteogenesis. These results suggest that endochondral bone formation may involve different cell types, a finding that could have major implications for the tissue engineering of bone and cartilage. INTRODUCTION: The delivery of BMP4 through cell-based gene therapy can trigger ectopic endochondral bone formation in skeletal muscle. We hypothesized that, when stimulated with or transduced to express BMP4, different types of cells residing within skeletal muscle might participate in different stages of endochondral bone formation. MATERIALS AND METHODS: We compared the responses of a fibroblast cell line (NIH/3T3), a myoblast cell line (C2C12), primary fibroblasts, and primary myoblasts to BMP4 stimulation in vitro. We then transduced the four cell populations to express BMP4 and compared their ability to promote ectopic endochondral bone formation in skeletal muscle. RESULTS: Under the influence of BMP4 in vitro and in vivo, NIH/3T3 cells differentiated toward both chondrogenic and osteogenic lineages, whereas most C2C12 cells underwent primarily osteogenic differentiation. NIH/3T3 cells genetically modified to express BMP4 induced delayed but more robust cartilage formation than did genetically modified C2C12 cells, which promoted rapid ossification. These differences in terms of the timing and amount of cartilage and bone formation persisted even after we introduced a retrovirus encoding dominant negative Runx2 (DNRunx2) into the C2C12 cells, which interferes with the function of Runx2. Superior osteogenic potential was also displayed by the primary myoblasts in vitro and in vivo compared with the primary fibroblasts. The different proliferation abilities and differentiation potentials exhibited by these cells when influenced by BMP4 may at least partially explain the differing roles that BMP4-expressing myogenic cells and BMP4-expressing fibroblastic cells play in endochondral bone formation. CONCLUSIONS: Our findings suggest that the process of endochondral bone formation in skeletal muscle after delivery of BMP4 involves different cell types, including fibroblastic cells, which are more involved in the chondrogenic phases, and myoblastic cells, which are primarily involved in osteogenesis. These findings could have important implications for the development of tissue engineering applications focused on bone and cartilage repair.