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

为了建立一种具有临床实用价值的骨和软骨再生方法，我们获得了以下结果：1.对于新型支架，我们研制了一种由PLGA和胶原组成的杂化网状物。杂化网片厚度可调，具有良好的强度和生物相容性。当我们将牛软骨细胞接种到杂交网中时，细胞有效地附着在杂交网上，产生丰富的软骨基质，并抑制了它们的去分化。对于新的培养体系，我们开发了一种旋转培养方法，即培养皿水平旋转，球体由细胞簇形成。有了这个系统，我们能够在更具生理学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，我们计划将其种植到支架中，以发展人工骨和软骨。3.对于诱导成骨和软骨分化的新方法，我们阐明了IINOS和细胞内钙在成骨细胞分化中的作用。应用这一发现，我们可以通过下调iNOS的mRNA表达来逆转iNOS对成骨细胞分化的抑制。4.在血管再生方面，我们发现碱性成纤维细胞生长因子在诱导血管再生方面优于血管内皮生长因子，两者的结合证明更好。我们认为，本项目成功地建立了有助于实现基于血管网络形成的棚屋三维培养体系的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.