重组人骨形态发生蛋白-7与脱钙骨基质复合物双重骨诱导作用的生物学评价

将不同剂量的重组人骨形态发生蛋白-7(rhBMP-7)与脱钙骨基质(DBM)分别复合后,植入小鼠股部内侧肌间隙,三周后取材,通过组织学检查、碱性磷酸酶(ALP)及钙含量的测定比较各组的骨诱导活性。结果显示,三组复合物均有骨组织生成,中、高剂量组可见骨小梁、板层骨和原始骨髓腔,血管和骨髓丰富;低剂量组的成骨量明显少于中、高剂量组,且新骨的成熟度低于其他两组,DBM少部分吸收;单独植入rhBMP-7组有编织骨形成;而DBM组可见成骨细胞的聚集。rhBMP-7/DBM复合组在ALP和Ca含量水平上与同等剂量的两对照组相比均有显著性差异(P<0.01),而rhBMP-7三种剂量之间均有显著性差异(P<0.01)。这充分说明DBM作为rhBMP-7的合适载体,具有缓释作用,且二者复合可起到双重骨诱导活性;而且rhBMP-7的骨诱导活性具有一定的剂量依赖性。     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

国产多孔钽材料修复兔胫骨缺损的实验研究

摘要 目的：研究国产多孔钽材料能否在兔胫骨缺损模型中顺利实现骨长入,用于修复胫骨缺损。方法：在36只新西兰大白兔双侧胫骨骨干处建立骨缺损模型,每只动物左右侧缺损随机分组,分别进入实验组(植入多孔坦材料)和对照组（不植入多孔坦材料）。植入后4周、8周和12周取材,通过X线检测以及硬组织切片苏木精伊红染色,检测多孔钽材料与骨界面的骨整合情况。采用推出实验检测多孔钽材料与骨界面的结合强度。结果：将术后不同时间点取得的胫骨标本作X射线拍片分析,4周时,骨缺损端与材料结合部位有骨质生成,在8周时材料表面有骨形成现象,逐渐完全覆盖材料表面,在12周时骨量继续增加,形成覆盖材料并桥接骨缺损断端的骨痂。样本行硬组织切片并行HE染色后检测,植入4周后实验组材料两端被新生骨所覆盖,材料深部的孔隙中也可见少量骨组织长入;植入8周后发现实验组材料与骨组织生长良好,多孔钽材料表面和两端材料孔隙内均有骨组织长入,材料孔隙与组织紧密连接,有骨小梁长入;植入12周时两端骨组织长入深度没有明显变化,但材料表面骨组织继续长入,并完全嵌入圆柱体材料内。材料植入后4周与8周比较差异无统计学意义（P＞0.05）,材料植入后8周与12周比较差异有统计学意义（P<0.05）。将植入4周、8周和12周后含材料样本置于动态疲劳试验机上进行推出实验,随时间延长所需推出力明显增加,植入后4周和8周相比,虽然后者所需推力较大,但两者比较差异无统计学意义（P＞0.05）,而8周和12周比较则差异有统计学意义（P<0.05）。结论：国产多孔坦材料能在胫骨缺损中实现与骨整合,能用于皮质骨缺损修复。     

多孔n-HA/PA66支架复合rhBMP-2修复兔桡骨缺损的实验研究

目的 ：研究多孔纳米羟基磷灰石/聚酰胺66（nHA/PA66）骨修复材料作为骨组织工程支架复合基因重组人骨形态发生蛋白2（rhBMP2）后的成骨能力的变化,探讨加速nHA/PA66人工骨与受体骨愈合的方法。方法：选用新西兰大白兔双侧桡骨制作骨缺损模型,将nHA/PA66/rhBMP2复合材料植入左侧骨缺损处,右侧骨缺损以nHA/PA66植入作为实验对照,另做不植入任何材料的骨缺损空白对照。在1、2、4、8、12周各时相点分别进行大体观察、X线照片、组织学切片、免疫组化原位杂交进行检测图象分析。结果：nHA/PA66/BMP2与nHA/PA66组骨缺损均完全修复,而空白对照组骨缺损未见修复;2周时nHA/PA66与nHA/PA66/rhBMP2两组间原位杂交阳性细胞表达有统计学意义（ P<0.05）, 4周时nHA/PA66与nHA/PA66/rhBMP2两组间原位杂交阳性细胞表达无统计学意义（ P>0.05）,2周及4周实验和实验对照两组分别与空白对照组比较均无统计学意义（ P>0.05）,nHA/PA66/rhBMP2组较nHA/PA66组可加速人工骨/植入体/受体界面骨愈合。 结论：多孔nHA/PA66作为骨组织工程支架复合具有诱导成骨活性的rhBMP2后,增强了早期成骨能力,加速了其与受体骨的愈合。     

多孔自凝固磷酸钙/纤维蛋白1∶1比例血管化的分析研究

目的:观察复合纤维蛋白的多孔自凝固磷酸钙的理学性能及体内血管化情况,探讨其各组份对材料生物学特性的影响,为其临床应用提供实验数据.方法:1)采用凝固时间测定和电镜观察材料表面和断面等方法对复合支架材料构造和理学性能进行分析;2)将24只新西兰白兔随机分为3组,每组8只,分别在每只实验兔腰背筋膜下植入1:1、CPC两种比例材料各一枚.术后2、4、8周进行取材,对材料及其周围组织进行组织学观察、微血管情况定量分析.结果:1)CPC/FG复合支架材料以1∶1(g/ml)混合后,与单纯的CPC相比,初凝时间延长,而终凝时间没有明显的统计学差异;电镜观察发现多孔自凝固磷酸钙复合了纤维蛋白胶之后,纤维蛋白胶分布均匀贯穿多孔自凝固磷酸钙晶体之间,并将其紧密相连;2)术后2周材料外周可见幼稚的微血管.1∶1组高于CPC组(P＜0.05).4周微血管密度达到高峰,相对于2周时有明显差异.1∶1组高于CPC组(P＜0.05).8周时微血管密度相对4周没有显著差异.各组微血管密度与4周时没有明显变化(P＞0.05).结论:复合支架材料具有合适的凝固时间和较好的结构,纤维蛋白胶及其降解产物影响复合材料在体内的微血管密度,使复合材料血管化能力提高,其可作为细胞载体和骨缺损修复支架材料应用于临床和实验中.     

骨髓基质干细胞在中空多孔金属试件内诱导成骨的研究

目的:观察体外培养犬骨髓基质干细胞(BMSCs)在中空多孔金属试件与脱钙骨基质内、外的分化生长情况以及成骨的可能性.方法:按中空多孔金属试件的中空部分是否填充DBM,以及所选择成骨诱导培养液和rhBMP-2培养液的不同进行分组,共四组,每组四枚.体外培养BMSCs,诱导分化,借助相差显微镜、免疫染色、扫描电镜观察中空多孔金属试件内BMSCs的分化、增殖及成骨情况,并隔天测定各组的骨特异性ALP浓度.结果:体外BMSCs经诱导向成骨细胞分化,并可在中空多孔金属试件内的空间结构中形成组织块.以骨特异性ALP为指标比较成骨诱导培养液和rhBMP-2培养液的诱导成骨功效,证明成骨诱导液更为明显,但两者无显著差异(P＞0.05).结论:中空多孔金属试件中BMSCs可经诱导成骨.     

微弧氧化和碱处理技术在多孔钽修复兔颅骨缺损中的应用

目的观察微弧氧化和碱处理对多孔钽表面性状、生物相容性和成骨能力的影响。方法微弧氧化和碱处理多孔钽片后,扫描电镜观察表面微孔数量、表面钙磷沉积和接触角。植入钽片修复兔颅骨缺损模型,在4周和12周观察骨愈合情况。结果扫描电镜显示处理组表面有更多的微孔和钙磷沉积以及更小的接触角(P<0.05)。植入多孔钽片后,所有动物均生长良好,伤口愈合佳。CT观察多孔钽片和周围骨组织耦合良好;钙黄绿素标记检测显示12周时有新生骨长入多空钽材料内部;扫描电镜观察发现4周时多空钽材料内部有新生血管,12周时有骨小梁长入材料内部。结论微弧氧化和碱处理能改变多孔钽材料表面形状,处理后多孔钽片具有良好的生物相容性和成骨能力。     

多孔自凝固磷酸钙/纤维蛋白1：1比例血管化的分析研究

目的：观察复合纤维蛋白的多孔自凝固磷酸钙的理学性能及体内血管化情况,探讨其各组份对材料生物学特性的影响,为其临床应用提供实验数据。方法：1）采用凝固时间测定和电镜观察材料表面和断面等方法对复合支架材料构造和理学性能进行分析;2）将24只新西兰白兔随机分为3组,每组8只,分别在每只实验兔腰背筋膜下植入1：1、CPC两种比例材料各一枚。术后2、4、8周进行取材,对材料及其周围组织进行组织学观察、微血管情况定量分析。结果：1）CPC/FG复合支架材料以1：1（g/ml）混合后,与单纯的CPC相比,初凝时间延长,而终凝时间没有明显的统计学差异;电镜观察发现多孔自凝固磷酸钙复合了纤维蛋白胶之后,纤维蛋白胶分布均匀贯穿多孔自凝固磷酸钙晶体之间,并将其紧密相连;2）术后2周材料外周可见幼稚的微血管。1：1组高于CPC组（P〈0.05）。4周微血管密度达到高峰,相对于2周时有明显差异。1：1组高于CPC组（P〈0.05）。8周时微血管密度相对4周没有显著差异。各组微血管密度与4周时没有明显变化（P〉0.05）。结论：复合支架材料具有合适的凝固时间和较好的结构,纤维蛋白胶及其降解产物影响复合材料在体内的微血管密度,使复合材料血管化能力提高,其可作为细胞载体和骨缺损修复支架材料应用于临床和实验中。     

新型多孔生物活性玻璃修复羊腔隙性骨缺损的实验研究

目的:评价新型多孔生物活性玻璃修复羊腔隙性骨缺损的能力.方法:12只成年绵羊L3-L5,共36个椎体均建立8mm× 15mm腔隙性骨缺损模型,实验组为Macro Porous Putty(MPS)和Injectable Putty(INJ),对照组为Nova Bone Putty(NBP).按照随机区组设计,随机分配每只羊的三个椎体缺损分别填充NBP、MPS、INJ材料.每种材料均填充12个椎体.术后6周、12周取材,通过术后一般状况、标本大体观察、X线平片以及组织病理学结果评价新型多孔生物活性玻璃的成骨作用.结果:①术后各组动物进食及粪便均正常,对外界刺激反应性良好.术后切口均一期愈合.②大体观察未发现缺损处有异常纤维组织包块及材料漏出.③术后6周和12周,MPS组和INJ组Lane-Sandhu X射线评分大于NBP组(P＜0.05).④组织学切片VG染色结果显示,术后6周和12周,MPS组和INJ组新生骨量多于NBP组(P＜0.05).MPS组和INJ组成骨性能相当(P＞0.05).术后12周和术后6周比较,NBP、MPS、INJ三种材料新生骨量均明显增多(P＜0.01).结论:新型多孔生物活性玻璃材料MPS和INJ具有可靠的骨缺损修复能力,比传统的NBP材料成骨能力更佳,具有良好的应用前景.     

Bone neogenesis in domes made of expanded polytetrafluoroethylene: efficacy of rhBMP-2 to enhance the amount of achievable bone in rats

For bone reconstructive purposes, it would be a great advantage to be able to gain bone without grafting. In experimental studies, barrier membranes have been used to accomplish this, however, with limited efficacy. In this study, the effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) on the early onset of bone formation, as well as on the final amount of achievable bone, was investigated in an experimental osteo-neogenesis model. In 60 adult rats, dome-shaped barrier membranes made of expanded polytetrafluoroethylene (Gore-Tex membrane), with an inside volume of approximately 60 mm3, were placed on the left parietal bone. The domes were pretreated according to four different alternatives: (1) filled with autogenous blood only (n = 15); (2) filled with 5 microg of rhBMP-2 in an absorbable collagen sponge carrier (n = 15); (3) filled with 15 microg of rhBMP-2 in absorbable collagen sponge carrier (n = 15); or (4) filled with the absorbable collagen sponge carrier only (n = 15). The animals treated according to each alternative were then divided into three equal groups with five rats in each, and subsequently killed after 3, 6, or 12 weeks. The amount of bone formed within the domes was evaluated by light microscopy and computer-assisted image analysis. It was found that the amount of newly formed bone could be enhanced by approximately 100 percent by simultaneous implantation of rhBMP-2, irrespective of dose. The early onset of bone formation was, however, not affected by the rhBMP-2 supplementation. This finding was interpreted as being due to the delivery system used, because as long as the carrier was still present, no significant difference between the treatment groups was observed. The bone formed in domes with carrier implantation, with or without rhBMP-2, displayed more marrow spaces in comparison to controls. The combined treatment with barrier membranes and local delivery of rhBMP-2 may be a useful tool in reconstructive surgery, for instance replacing onlay grafting, especially when a more delicate anatomy is necessary, because membranes can be shaped in multiple ways.     

Ectopic bone formation in titanium mesh loaded with bone morphogenetic protein and coated with calcium phosphate

The osteoinductive properties of porous titanium fiber mesh, with or without a calcium phosphate coating and loaded with recombinant human bone morphogenic protein-2 (rhBMP-2) or rhBMP-2 and native bovine BMP (S-300) were investigated in a rat ectopic assay model. A total of 112 calcium phosphate-coated and 112 noncoated porous titanium implants, either loaded with rhBMP-2 and S-300 or loaded with rhBMP-2 alone, were subcutaneously placed in 56 Wistar-King rats. The rats were killed 5, 10, 20, and 40 days postoperatively, and the implants were retrieved.Histologic analysis demonstrated that all growth factor and carrier combinations induced ectopic cartilage and bone formation at 5 and 10 days, respectively. At 20 days, bone formation increased and was characterized by trabecular bone and bone marrow-like tissue. At 40 days, more lamellar bone and hemopoietic bone marrow-like tissue were present. At both times, more bone had been formed in calcium phosphate-coated implants than in noncoated samples. Further, in rhBMP-2 and S-300-loaded specimens, bone formation was higher than in rhBMP-2 only-loaded specimens. In rhBMP-2 only-loaded specimens, bone formation was mainly localized inside the mesh material, whereas in specimens loaded with both rhBMP-2 and S-300, the bone was localized inside and surrounding the titanium mesh. The histological findings were confirmed by calcium content and alkaline phosphatase activity measurements. In addition, all specimens showed osteocalcin expression as early as 5 days postoperatively.Our results show that the combination of titanium mesh with BMPs can induce ectopic bone formation and that this bone formation seems to be similar to "enchondral" ossification. In addition, a thin calcium phosphate coating can have a beneficial effect on the bone-inducing properties of a scaffold material. Finally, rhBMP-2 and native BMP act synergistically in ectopic bone induction.     

重组人骨形态发生蛋白-2生物学活性测定新方法的建立

目的:采用小鼠异位成骨技术及甲基麝香草酚蓝比色法检测重组人骨形态发生蛋白-2(rhBMP-2)的生物学活性。方法:将rhBMP-2埋入小鼠肌间隙内,14d后取出新生组织,采用血清钙试剂盒检测其钙含量。结果:随着给药组剂量递增,相应地钙含量也增加,二者具有较强的量效关系。结论:此方法为本实验室独创,较传统的血清碱性磷酸酶方法更为方便、快捷,是一种能够定量检测rhBMP-2活性的新方法。     

Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2

BACKGROUND: Adipose tissue has been demonstrated to contain a population of progenitor cells that can differentiate into bone and cartilage. Studies have suggested that adipose-derived adult stem (ADAS) cells can be induced to differentiate into chondrocytes by transforming growth factor-beta (TGF-beta). In this study, we examined whether bone morphogenetic protein-2 (BMP-2), as a member of the TGF-beta superfamily, could regulate ADAS cells to differentiate into a chondrolineage. METHODS: ADAS cells were isolated and induced by rhBMP-2. These cells were cultured in pellets for 2 weeks, and the chondrogenic phenotype was observed in vitro and in vivo. ADAS cells cultured without BMP-2 were used as controls. RESULTS: After 2 weeks of culture, the differentiated ADAS cells reacted positively to Alcian blue and collagen II, and the content of collagen II protein was obviously up-regulated at day 14. Glycosaminoglycan (GAG) content gradually increased from day 2 to day 14 (P < 0.05). However, H&E staining and collagen II expression were weak, and there was a little collagen II protein and GAG detected in the control group. Additionally, the pellets of ADAS cells induced by rhBMP-2 were transplanted into BALB/C nude mice and formed cartilage lacuna at week 8 in vivo. DISCUSSION: These data demonstrate that rhBMP-2 induce ADAS cells to differentiate into chondrocytes in vitro and in vivo. This is useful for basic and clinical studies aimed at repairing cartilage damage. But in a control group, ADAS cells tended towards differentiation into chondrocytes, which was affected by ITS. We will be exploring the mechanism further.     

Prefabricated vascularized bone flap: a tissue transformation technique for bone reconstruction

In this study, an attempt was made to transform a muscle vascularized pedicle raised on host vessels into a vascularized bone flap, using recombinant human bone morphogenetic protein 2 (rhBMP-2). The purpose of this study was to produce new bone vascularized in nature to increase the survival rate of the subsequently grafted bone and to fabricate the newly formed bone into the desired shape. Silicone molds in the shape of a rat mandible were used to deliver rat bone matrix impregnated with or without rhBMP-2. A muscle pedicle the same size as the mold was raised on the saphenous vessels in the rat thigh and then sandwiched in the center of the silicone molds. The molds were sliced in half and each section was filled with rat bone matrix that was impregnated either with 25 microg of rhBMP-2 for the experimental group or with diluting material alone for the control group. The sandwiched flaps were then secured by tying them to the adjacent muscles and were harvested at 2 and 4 weeks after surgery. Three and six rats were used in the control and experimental groups at each time point, respectively. Bone formation was assessed in the ex vivo specimens by macroscopic, radiologic, and histologic evaluation. Macroscopically, the continuation of the vascular pedicle was clearly visible for both the control and experimental muscle flaps. However, no evidence of muscle-tissue transformation was observed in the control flaps, whereas all the flaps treated with rhBMP-2 produced new bone that replicated the shape of the mold exactly and had saphenous vessels supplying the newly formed bone. This study demonstrates that this experimental model has the potential to be therapeutically applied for effective bone reconstruction.     

Recombinant human bone morphogenetic protein-2 enhances expression of interleukin-6 and transforming growth factor-β1 genes in normal human osteoblast-like cells

The process of recombinant human bone morphogenetic protein-2 (rhBMP-2)-induced endochondral ossification involves (1) the proliferation and differentiation of mesenchymal cells into chondroblasts and osteoblasts; (2) the production and maturation of cartilage and bone matrix; and (3) the differentiation of circulating osteoclast precursor cells into osteoclasts. Currently the molecular mechanisms of these complex sequential events are unknown. It seemed reasonable to us to assume that communication between cells through soluble mediators during bone induction by rhBMP-2 may play an important role in the sequential differentiation of chondroblasts, osteoblasts, and osteoclasts. We have therefore used a human osteoblast-like initial transfectant cell line (HOBIT) to study the effect of rhBMP-2 on gene expression of interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1), both of which affect osteogenesis and ostoeclastogenesis. Our results have demonstrated that rhBMP-2 acts on HOBIT cells to stimulate expression of IL-6 and TGF-β1 genes and the production of IL-6. Enhancement of gene expression of IL-6 and TGF-β1 by rhBMP-2 was both sensitive (half maximal effect at approximately 10 ng/ml) and potent (maximum induction was approximately four and threefold greater than controls, respectively). Time course studies showed that the induction of TGF-β1 and IL-6 mRNA occurs within short periods—4 and 8 hours after exposure to rhBMP-2, respectively. Interestingly, these effects, however, were not accompanied by the mitogenic action of rhBMP-2. It suggests that rhBMP-2 enhances IL-6 and TGF-β1 production during osteogenesis and at least in part mediates the complex sequential differentiation of chondroblasts, osteoblasts, and osteoclasts during rhBMP-2-induced endochondral ossification. © 1994 wiley-Liss, Inc.     

Ectopic study of tissue-engineered bone complex with enamel matrix proteins, bone marrow stromal cells in porous calcium phosphate cement scaffolds, in nude mice

Objective: This study aimed to investigate the potential of enamel matrix proteins (EMPs) on promoting osteogenic differentiation of porcine bone marrow stromal cells (pBMSCs), as well as new bone formation capabilities, in a tissue‐engineered bone complex scaffold of EMPs, pBMSCs and porous calcium phosphate cement (CPC). Materials and methods: Effects of EMPs on pBMSCs in vitro was first determined by alkaline phosphatase (ALP) activity, von Kossa staining assay and mRNA expression of ALP, bone sialoprotein (BSP) and osteocalcin (OCN) genes. Next, an ectopic new bone formation test was performed in a nude mouse model with four groups: CPC scaffold alone; CPC scaffold + EMPs; CPC scaffold + pBMSCs; and CPC scaffold + EMPs + pBMSCs, for 2 or 4 weeks. Results: ALP activity, von Kossa assay and mRNA expressions of ALP, BSP and OCN genes were all significantly higher with 150 μg/ml EMP treatment in vitro. In nude mice, new bone formation was detected only in the CPC scaffold + EMPs + pBMSCs group at 2 weeks. At 4 weeks, in the tissue‐engineered construct there was significantly higher bone formation ability than other groups. Conclusions: EMPs promoted osteogenic differentiation of pBMSCs, and the tissue‐engineered complex of EMPs, pBMSCs and CPC scaffold may be a valuable alternative to be used in periodontal bone tissue engineering and regeneration.     

Bone morphogenetic protein 2 promotes osteogenesis of bone marrow stromal cells in type 2 diabetic rats via the Wnt signaling pathway

Type 2 diabetes mellitus impairs osteogenesis in bone marrow stromal cells (BMSCs). Bone morphogenetic protein 2 (BMP2) has been extensively applied for bone defect restoration and has been shown to activate the Wnt signaling pathway. The objective of this study was to investigate the effects of BMP2 on the cell proliferation and osteogenesis of type 2 diabetic BMSCs in rats and explore whether BMP2 induced osteogenesis via the stimulation of Wnt signaling pathway. The cell experiments were divided into DM (diabetic BMSCs), BMP25 (induced with 25 ng/ml BMP2), BMP100 (induced with 100 ng/ml BMP2) and BMP25  + XAV groups. All cells with or without the different concentrations of BMP2 were cultured under the same experimental conditions. The in vitro results indicated that BMP2 enhanced cell proliferation by 130%–157% and osteogenic differentiation by approximately two-fold in type 2 diabetic BMSCs. The expression levels of β-catenin, cyclin D1, Runx2 and c-myc related to the Wnt signaling pathway were also upregulated from 180% to 212% in BMP2-induced type 2 diabetic rat BMSCs, while the level of GSK3β decreased to 43%. In BMP2-induced type 2 diabetic BMSCs with calcium phosphate cement (CPC) scaffolds for osteoblast study in vivo, the appearance of newly formed bone dramatically increased to 175% compared with type 2 diabetic BMSCs. These data demonstrated that BMP2 enhanced bone regeneration in diabetic BMSCs by stimulating the Wnt signaling pathway with the accumulation of β-catenin and the depressed expression of GSK3β. Diabetic BMSCs associated with BMP2 might be a potential tissue-engineered construct for bone defects in type 2 diabetes mellitus.     

Evidence of in vivo osteoinduction in adult rat bone by adeno-Runx2 intra-femoral delivery

The bone marrow microenvironment provides a unique opportunity in vivo to assess the role of genes in bone remodeling. The objective of this study was to determine whether Runx2 expression is regulated by rhBMP-2 in vivo and to examine the effect of Runx2 overexpression on bone in vivo. In the in vivo calvaria model we used, rhBMP-2 induced Runx2 protein expression in periosteal cells while in vitro, adenovirus-mediated Runx2 overexpression induced mineralization in mesenchymal stem cells. A single injection of adeno-Runx2 directly into the bone marrow of the right femur in mature rats, and subsequent analysis after 3 weeks, showed a significant bone mineral density (BMD) increase ( approximately 15%) as compared to the controls. The whole-femur mean BMD of the active virus-injected group was 0.193 (g/cm(2)) while that of the control virus-injected group was 0.175 (g/cm(2)) (P < 0.05). In addition, a significant increase (36%) in trabecular BMD at the distal end of the femur was observed. These data demonstrate that directly delivering adeno-Runx2 into bone marrow of adult rats induces osteogenesis and illustrates potential advantages of such approaches over ex vivo gene therapy protocols involving marrow cell isolation, gene transduction, and subsequent in vivo transfer.