Regeneration of Bone and Cartilage by a Novel 3-D Culture System Based on Formation of Vascutar Network

为了建立一种临床有用的骨和软骨再生方法，我们获得了以下结果：1。在新型支架方面，我们开发了一种由PLGA和胶原蛋白组成的混合网状材料。混合网的厚度可调节，混合网具有良好的强度和生物相容性。将牛软骨细胞植入混合网后，细胞能有效附着，生成丰富的软骨基质，并抑制其去分化。我们得出结论，混合网格比其他支架有优势。至于新的培养系统，我们开发了一种旋转培养方法，其中培养皿水平旋转，细胞簇形成球体。有了这个系统，我们能够在更生理的环境中评估分化和增殖刺激对成骨细胞和软骨细胞的影响。我们还能够大规模地生成成骨细胞和软骨细胞球体，我们计划将它们植入支架中，以发育人工骨和软骨。在诱导成骨细胞和软骨细胞分化的新方法方面，我们明确了IINOS和细胞内钙在成骨细胞分化中的作用。利用这一发现，我们可以通过rnai下调iNOS mRNA的表达，逆转iNOS对成骨细胞分化的抑制作用。在诱导血管再生方面，我们发现bFGF的诱导效果优于VEGF，两者联合使用效果更好。我们认为，该项目已经成功建立了基于血管网络形成的三维立体培养系统实现骨和软骨再生的基础技术。

To establish a clinically useful method to regenerate bone and cartilage, we have obtained the following results :1.As for new scaffolds, we developed a hybrid mesh composed of PLGA and collagen. The thickness of the hybrid mesh was adjustable, and the hybrid mesh had both good strength and biocompatibility. When we seeded bovine chondrocytes into the hybrid mesh, the cells attached to it efficiently, producing rich cartilaginous matrices, and their dedifferentiation was suppressed. We conclude that the hybrid mesh has advantages over other scaffblds.2.As for new culture systems, we developed a rotational, culture method, in which culture dishes were rotated horizontally, and spheroids were formed by clusters of cells. With this system, we were able to assess effects of differentiation and proliferation stimuli on osteoblasts and chondroeytes in a more physiologic setting. We were also able to generate osteoblast and chondrocyte spheroids on, a large scale, which we plan to seed into the scaffold to develop artificial bone and cartilage.3.As for new methods to induce osteogenie and chondrogenic differentiation, we clarified the role of IINOS and intracellular calcium in osteoblastic differentiation. By applying this finding, we could reverse the suppression of osteoblastic differentiation by iNOS through down-regulating mRNA expression of iNOS with RNAi.4.As for regeneration of blood vessels, we found that the bFGF was superior to VEGF in induction, and that the combination of both proved even better.We conclude that this project has successfully established the basal technologies that will help realize regeneration of bone and cartilage by a hovel 3-D culture system based on formation of vascular network.