Indian hedgehog基因敲除优化种子细胞生物力学及其在组织工程软骨构建中的作用

Cartilage repair is a challenge because the self-healing capacity of cartilage is limited. Tissue engineering cartilage has been used to repaire the cartilage defects but it can't maintain its quality and mechanical properties in vivo for long-term because the seeding cells used for the tissue engineering cartilage are quickly dedifferentiation and lose their mechanical properties. In our previous studies, we have shown that 1) the seeding cells lose quickly the viscoelastic properties (including equilibrium modulus, the instantaneous modulus, and the apparent viscosity) and the self-adapting capability under mechanical strains in vitro. The finding suggests the changed biomechanical characters of the seeding cells may be unable to generate the normal function tissue-engineered cartilage; 2) stress induces the expression of Indian hedgehog (Ihh), a mechanical response gene , which lets to an increase of MMP-13 and cartilage degeneration; 3) the inhibition of Ihh increases the synthesis of cartilage matrix, including type II and agrecan; 4) finally, our study in vivo further demonstrates conditional deleted Ihh not only prevents the aged cartilage degeneration but also attenuates the severity of surgery induced OA in our Col2a1-CreER; Ihhfl/fl mice. Our findings strong suggest that the change of the mechanical property of the seeding cells may relate to Ihh gene and knock out Ihh may improve the quality of the tissue engineering cartilage by enhancing its mechanics characteristics. To test the hypothesis, we will analize the mechanical-biological properties for the BMSCs and chondrocytes isolated from the deleted Ihh mice in vitro and test the tissue engineering cartilage generated using the deleted Ihh BMSCs and chondrocytes in vivo. In summary, Ihh may be related to the change of the mechanical property of the seeding cells, which lets to the decrease of quality of the tissue engineering cartilage. In this application, we propose to evaluate novel strategies for improvement of tissue engineering cartilage using deletion of Ihh seeding cells. This research will significantly increase our understanding of Ihh in the chondrocyte mechanical properties.

组织工程技术为软骨损伤修复提供了新的选择和希望。但种子细胞的失分化及无法形成正常力学特性的透明软骨、且修复组织的快速退变成为制约组织工程软骨发展的主要问题。我们前期研究发现，软骨细胞体外传代扩增过程中力学性能发生退变，且伴随其适应力学刺激基质合成能力的降低；同时，Indian hedgehog(Ihh)作为一种力学敏感基因，前期证实其表达与软骨细胞力学呈负相关，且Ihh基因敲除小鼠明显延缓了创伤和年龄导致的软骨退变。本项目在前期建立Ihh条件性基因敲除小鼠动物模型的基础上，与正常细胞比较，通过微管吸吮力学分析及PCR、Western-blotting等技术手段，分析Ihh基因敲除小鼠来源的软骨细胞和BMSCs体外培养力学-生物学优势，分析其适应力学刺激基质合成能力的变化特点，明确Ihh基因敲除优化种子细胞力学-生物学特性的作用。

Indian hedgehog(Ihh)作为一种力学敏感基因，我们前期研究证实其表达与软骨细胞力学呈负相关，且Ihh基因敲除小鼠明显延缓了创伤和年龄导致的软骨退变。本项目在前期建立Ihh条件性基因敲除小鼠动物模型的基础上，与正常细胞比较，通过微管吸吮力学分析及PCR、Western-blotting等技术手段，分析Ihh基因敲除小鼠来源的软骨细胞体外培养生物学优势，分析其适应力学刺激基质合成能力的变化特点，明确Ihh基因敲除优化种子细胞生物学特性的作用。.1.通过与美国布朗大学骨科研究中心合作，在我们实验室成功繁殖、培育Ihh条件性基因敲除小鼠，解决了非特异性敲除Ihh基因后，基因工程小鼠早期死亡的问题。同时证实Ihh基因条件性敲除小鼠来源间充质干细胞和软骨细胞体外Ihh基因敲除的有效性和稳定性。.2.建立条件基因敲除未成熟小鼠肋软骨细胞培养方法，完成其表型鉴定，为后续研究奠定基础。.3.通过实验证实，敲除Ihh基因，能够有效提高软骨细胞体外II型胶原和糖胺多糖的表达，抑制X 型胶原和基质金属蛋白酶-13的表达，提示Ihh基因敲除优化种子细胞生物学功能的作用和意义。.4.以第一代未成熟小鼠肋软骨细胞作为实验对象，发现周期性张应变加载条件下，Ihh基因敲除促进软骨细胞体外II型胶原和糖胺多糖mRNA表达与分泌，抑制X 型胶原和基质金属蛋白酶-13 mRNA表达与分泌，提示Ihh基因敲除抑制了力学加载导致软骨细胞的肥大与分化，促进种子细胞适应力学刺激基质合成的能力。