Acceleration of bone regeneration by overexpression of vascular endothelial growth factor gene in bone marrow mesenchymal stem cells

(1)将小鼠Sox9基因导入骨髓间充质干细胞，观察其向软骨细胞分化的情况。体外高密度微团培养结果显示，Alcian蓝和II型胶原染色阳性。接下来，将转Sox9基因的MSCs加载到扩散室内，并移植到无瘤小鼠体内。移植4周后，小室内可见大量组织形成。组织学检查显示，阿利新蓝和II型胶原在肿块的细胞外基质中均呈阳性染色，而X型胶原未见染色。(2)评价连续传代的大鼠MSCs的成骨能力。通过碱性磷酸酶和骨钙素的浓度和基因表达来评价体外成骨分化。为了体内成骨，将不同分化程度的骨髓间充质干细胞植入裸鼠体内。虽然e…成骨标记物水平在连续传代较少的MSCs中显著升高，随着传代次数的增加而降低。P0~P2细胞膜内可见丰富的骨和软骨形成。(3)对3例软骨发育不全和2例腿长不全患者的5例股骨和3例胫骨进行了骨髓间充质干细胞和富含血小板血浆(PRP)的牵张成骨的临床研究。取自髂骨的骨髓间充质干细胞在成骨补充剂中培养，分化为成骨细胞样细胞。PRP是在移植前准备的。用凝血酶-钙混合物将培养扩增的成骨细胞和自体PRP注射到牵张的骨痂中，使PRP凝胶在注射部位形成。平均愈合指数24.0d/cm，无严重并发症发生。较少

(1)Mouse sox9 cDNA was transfected into bone marrow derived mesenchymal stem cells(MSCs) and chondrogenic differentiation was evaluated. In vitro high density micromass culture of these sox9 transfected MSCs demonstrated that a matrix-rich aggregate was positively stained by Alcian blue and type II collagen. Next, sox9 transfected MSCs were loaded into the diffusion chamber and transplanted into athymic mice. A massive tissue formation was visible in the chamber after 4 weeks transplantation. Histological examinations demonstrated that both Alcian blue and type II collagen were positively stained in the extracellular matrix of the mass while type X collagen was not stained.(2)Osteogenic potential of serially passaged rat MSCs was evaluated. Osteogenic differentiation in vitro was evaluated by the concentration and mRNA expression of alkaline phosphatase and osteocalcin. For in vivo osteogenesis, MSCs in various degrees of differentiation were implanted into the athymic mice. Although e … More levated levels of osteogenic markers were prominent in the less passaged MSCs continuously cultured with osteogenic supplements, they decreased with passaging. Abundant bone and cartilage formations inside the membrane were observed in the P0 through P2 cells. In the P3 cells, however, the chambers were filled with fibrous tissues.(3)Clinical results of distraction osteogenesis with transplantation of MSCs and platelet rich plasma(PRP) were reviewed in five femora and three tibiae of the three patients with achondroplasia and two patients with leg length discrepancy. MSCs derived from the iliac crest were cultured with osteogenic supplements and differentiated into osteoblast-like cells. PRP was prepared just before transplantation. Culture expanded osteoblast-like cells and autologous PRP were injected into the distracted callus with the thrombin-calcium mixture so that the PRP gel might develop within the injected site. The target lengths were obtained in every leg without major complications and the average healing index was 24.0 day/cm. Less