软骨单位力学—生物学分析及其在关节软骨损伤修复中的作用

Large cartilage defects are a well-known problem for orthopedic surgeons because the self-healing capacity of cartilage is limited. The repair tissues after tissue engineering treatments have been reported to be hyaline-like cartilage, but it is still difficult to maintain the quality for a long time. One of the major issues limiting its use clinically is the availability of a cell source that will form sufficient amounts of tissue comparable to in vivo cartilage both in composition and in mechanical properties. The pericellular matrix (PCM) characterized by the presence of type VI collagen is a narrow tissue region surrounding chondrocytes in the articular cartilage, which together with the enclosed cell(s), has been termed the "chondron." As the local mechanical environment of the cell, the PCM is thought to play an important role in transmitting biomechanical cues from the extracellular matrix (ECM) to the cell through ''outside-in" signaling mechanisms. In our previous studies, we had established the enzymatic methods to isolate the chondrons from normal rabbit knee articular cartilage. And shown that the viscoelastic properties (including equilibrium modulus, the instantaneous modulus, and the apparent viscosity) of the isolated chondrons were signi?cantly greater than the chondrocytes under micropipette aspiration, the glycosaminoglycans(GAG), and collagen-typeⅡ(Col-2) mRNA expression in chondrons were increased than cells in vitro by real time PCR, indicated that the chondrons were preponderant in tissue-engineered cartilage. In this study, we will first to analysis the biological properties for chondrons as the seeding cells in tissue-engineered cartilage compared with the chondrocytes, including proliferation, metabolic activity, and viscoelastic properties. And then focus on the co-culture of chondrons and chondrocytes in building the functional engineering cartilage and repairing osteochondral defects of joints.

组织工程技术为关节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形不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其发展的主要问题。软骨单位（Chondron）作为关节软骨最基本的功能结构，由细胞周基质与包裹在内的一个或几个软骨细胞共同构成，其在软骨细胞体内代谢及力学环境维持方面发挥了重要作用。我们前期研究建立了兔膝关节软骨单位体外酶解消化实验方法，且证实软骨单位体外力学及糖胺多糖、Ⅱ型胶原基因表达较软骨细胞明显提高。但软骨单位作为种子细胞力学-生物学特点及其在关节软骨损伤修复中的作用，尚不清楚。本课题将进一步与软骨细胞比较，分析软骨单位体外培养过程中增殖、代谢及生物力学特点，明确软骨单位作为种子细胞的力学-生物学优势。同时，将软骨单位体外稳定的基因表达与软骨细胞早期快速增殖的特点相结合，通过体内外实验，分析软骨单位和软骨细胞共培养在组织工程软骨构建和软骨损伤修复中的作用。

关节软骨由于无血管、神经及淋巴通过，其一旦损伤则很难自行修复，已知组织工程技术促进软骨的损伤修复，研究证实：在体外，软骨细胞作为种子细胞在体外培养极易发生去分化；在体内，经由其发育而来的修复组织的表型及力学特性均较正常关节软骨差，而且随着体内修复时间的推移，修复组织显现出现快速退变的特征，制约其临床应用的前景。软骨单位（chondron）被作为关节软骨的基本功能结构，其主要由软骨细胞与细胞周基质(PCM)组成，其中PCM对软骨细胞代谢和力学传导起着重要作用。.本课题进一步与软骨细胞比较，分析软骨单位体外培养过程中增殖、代谢及生物力学特点，明确软骨单位作为种子细胞的力学-生物学优势。同时，将软骨单位体外稳定的基因表达与软骨细胞早起快速增殖的特点相结合，通过体内外实验，分析软骨单位和软骨细胞共培养在组织工程软骨构建和软骨损伤修复中的作用。.体外实验证实：软骨单位与软骨细胞以1:1在海藻酸钠凝胶球中共培养，其生物学特性优于其他比例；软骨细胞与软骨单位立体共培养能使细胞增殖和相关基因表达维持较长的时间，亦能保持较好的力学特性。使用海藻酸钠立体培养的软骨单位、立体共培养的软骨单位与软骨细胞（1:1）移植对兔膝关节软骨缺损修复的作用，并对比分析两者修复效果。结果表明，软骨单位具有延缓软骨细胞凋亡的作用。软骨单位与软骨细胞（1:1）共培养作为种子细胞，早期对缺损组织有好的修复作用，晚期可延缓修复组织退化。