微流控芯片调控的双相应力作用下-软骨细胞/仿生化取向支架复合体生物学特性及其软骨组织工程的实验研究

Articular cartilage is a tissue with a limited capacity for self-repairs after lesion. The autologous chondrocyte transplantation (ACT) is a hopefully medical therapy to restore articular cartilage lesion.However, autologous chondrocyte has been used to repair articular cartilage defects, but limited cell supply and dedifferentiation remains an issue.We need to be research the scaffold with biomimetic composition and structure.To aim directly at above-mentioned difficulties and according to related researches of Microfluidic、decellularized cartilage and fabrication of oriented scaffold，our research team plan to make a exploratory study as follows: The ECM origined from decellularized cartilage was fabricated into tissue engineering cartilage scaffold with biomimetic composition and orientation structure. The microstructure and property of oriented scaffold was assessed biologically in vivo and in vitro; The amplified chondrocytes were inoculated into the oriented scaffold and to form complex.The complex of chondrocytes/oriented scaffold was loaded by diphasic compressive stress origined from Microfluidic device. The compressive stress was biomimetic with the microenvironment of articular cartilage. The optimal compressive stress intensity gradient was detected by the quantity of proteoglycans and collagen Ⅱ and Ⅰ.We investigate the results of diphasic compressive stress influence on the chondrocyte proliferation and phenotype; After being stimulation by optimal compressive stress intensity gradient, autologous chondrocyte/oriented scaffold complex was transplanted into the defect of rabbit knee joint. We investigate the results of chondrocyte/oriented scaffold complex being stimulation by diphasic compressive stress influence on the defect restore. We investigated the feasibility that the chondrocyte/oriented scaffold complex being stimulation by diphasic compressive stress could be applied as tissue engineering cartilage. The experimental results provided theoretical and technological base for clinical application.

关节软骨损伤后的自我修复能力低下。自体软骨细胞移植技术为软骨损伤治疗带来希望。然而，软骨细胞来源有限，扩增容易出现表型丢失（去分化）；成份和结构仿生化的支架材料需要进一步优化。课题组拟针对以上难题，结合前期微流控芯片、软骨脱细胞及支架制备技术，进行以下探索性研究：利用脱细胞软骨制备成份和结构双重仿生的取向支架，对其进行体内外生物学评价；扩增软骨细胞并接种到取向支架上形成复合体，利用微流控芯片技术对细胞/支架复合体加载仿生关节软骨生物学微环境的"双相"压应力，通过蛋白多糖及Ⅱ型/Ⅰ型胶原蛋白定量检测，来筛选最佳的压力刺激强度，探讨对软骨细胞增殖及其表型的影响；根据测定的最佳压力强度梯度，对细胞/支架复合体应力刺激后，行自体软骨细胞/取向支架复合体修复兔关节软骨缺损模型，评价应力刺激下该复合体对软骨缺损的修复效果。探讨其在软骨组织工程应用的可行性。为临床应用提供理论与技术基础。

项目执行期间，本课题组利用用改良的、联合物理和化学脱细胞方法，去除了软骨细胞，保留了关节软骨细胞外基质（ECM）的主要成份，获得了脱细胞关节软骨ECM、成功构建出具有定向结构的软骨组织工程取向支架，其具有良好的细胞亲和性，并能够引导种子细胞的排列取向。首次证实了微流控芯片技术在软骨组织工程应用的可行性，模拟体内力学刺激的微环境，为体外快速扩增软骨细胞提供方案，从而提高种子细胞质量，改善自体软骨细胞移植治疗效果。利用成份和排列结构双重仿生的脱细胞软骨ECM 取向支架提供软骨细胞三维培养平台，将细胞/支架复合体植入到微流控芯片培养室内，对培养室内的细胞/支架复合体产生周期性的强度梯度变化的压应力刺激，经过力学传导引起取向 ACECM支架形变，对软骨细胞产生压应力刺激信号；同时，压应力推动液体流动产生间隙流液体流动刺激产生液相力学刺激信号；并通过蛋白多糖及II型/I型胶原蛋白定量检测，筛选出最佳的力学刺激强度，促进了软骨细胞增殖及体外表型的维持。. 另外，本课题组首次研究了神经白细胞素(NLK)和自分泌运动因子(AMFR)在关节软骨细胞的表达，并且发现 NLK作为一种生理性因子存在于关节软骨细胞并被软骨细胞分泌表达NLK通过其受体AMFR介导，促进了关节软骨细胞的体外增殖和胶原基质的分泌。发现NLK作为一种新的治疗途径应用于自体软骨移植和软骨损伤细胞治疗的潜能。在种子细胞方面，不再将单一软骨细胞作为种子细胞来源，本课题组以海藻盐凝胶体外培养体系，用来研究不同层的软骨细胞和骨髓间充质干细胞共培养后，发现共培养体系中骨髓间充质干细胞刺激软骨细胞成软骨，而且不同层软骨细胞与骨髓间充质干细胞共培养后能够在体内早期维持相应层软骨的特性。尽管根据本课题研究，骨髓间充质干细胞与不同层软骨细胞共培养只在早期维持不同层软骨细胞相应的特性，我们仍认为在选择合适培养环境的情况下，将不同层软骨细胞与不同来源的充质干细胞共培养用来再生相应分层是较理想的方法，有望为软骨细胞组织工程提供稳定的种子细胞。