水凝胶支架中大孔结构的可控构筑及其对软骨细胞行为的影响

Hydrogel scaffold is able to mimic the extracellular matrix (ECM) of native cartilage and favor chondrocytes retaining their native morphology and function, however, its submicron/nano-scale mesh structure severely inhibits cellular growth and metabolism. Due to the presence of hundreds of micron-sized cavities, macro-porous hydrogel can provide spaces for chondrocytes' growth and secretion of cartilage-specific ECM, and has exhibited great theoretical value and good application prospect. However, the creation of macro-pores within current macro-porous hydrogels is rapid and uncontrollable, which makes it impossible to study the effect of the formation process of macro-pores on cell behaviors. Besides, it also easily induces the collapse of macro-porous hydrogels, leading to the decrease of the volume of engineered cartilage. Aiming at these problems, this project aims to prepare novel biodegradable polycarbonate-based micro-gel spheres, serving as porogens, and then which are encapsulated into hyaluronic acid hydrogel. The chemical structure, size, and dosage of the micro-gel spheres is tailored to set its degradation rate, and hence to control the formation of macro-pores in hydrogel. Subsequently, the project investigates the effect of macro-pore formation rate, pore size, and porosity on the proliferation and ECM secretion of chondrocytes in vitro. Finally, the project preliminarily explores the repair of articular cartilage defects in vivo, by establishing the constructs containing the micro-gel spheres and chondrocytes in situ. All the anticipated research results of this project provide a reliable base both in theory and experiment for deep understanding macro-porous hydrogel, promoting its practical applications.

水凝胶组织工程支架能够模拟天然软骨细胞外基质环境，维持软骨细胞表型及功能，但其亚微米/纳米级网格严重限制了细胞生长代谢。大孔水凝胶因其特有的百微米级大孔结构，可以为软骨细胞的增殖及基质分泌提供空间，已展现出重要的理论价值和应用前景。但目前大孔水凝胶中大孔的构筑速率较快且不可控制，导致大孔构筑过程对软骨细胞行为的影响机制尚不清楚，同时支架易于坍塌，难以获得特定体积的工程化软骨。针对上述问题，本项目拟制备新型可生物降解聚碳酸酯基水凝胶微球致孔剂，并负载于透明质酸水凝胶中，通过控制微球的化学结构、尺寸和用量，调控其降解速率，实现水凝胶中大孔的可控构筑；通过对关节软骨细胞的体外三维培养，研究大孔水凝胶的大孔构筑速率、孔径及孔隙率对细胞增殖及软骨基质分泌的影响及其机制，初步考察其对关节软骨缺损的修复。本项目的研究成果为深入理解大孔水凝胶支架提供理论和实验依据，对大孔水凝胶的实际应用具有重大意义。

软骨与骨缺损发生率很高，但临床治疗效果不佳，组织工程的兴起为其修复带来了新希望。组织工程支架不仅为细胞生长提供物理支撑，其微环境还能够影响细胞命运与组织形成。本项目设计合成了新型的组织工程支架并考察了其生物功能，具体包括三个方面的研究内容：首先，我们设计制备了新型的可用于细胞三维包覆的冷冻大孔水凝胶（cell encapsulatable cryogel, CECG），而且其具备贯通“孔洞”，克服了目前水凝胶支架的重大缺陷；CECG支架的高渗透性与大孔结构显著促进了软骨细胞增殖与软骨组织形成，显示了其作为软骨组织工程支架的巨大潜能。其次，我们利用高通量测序（Next-generation sequencing, NGS）技术研究了海藻酸钠水凝胶密度对软骨细胞转录组的影响；发现了大量差异表达基因，其中，低密度水凝胶有利于软骨细胞增殖及细胞外基质相关基因的表达，高密度水凝胶促进软骨基质降解相关基因表达；这一研究可以为开发更加理想的软骨组织工程支架提供参考。第三，将化学交联明胶微球（ccG-MSs, 直径100-300 μm）用作小鼠胚胎成骨细胞（MC3T3-E1 cell）载体与组织工程支架材料，构建新型的微球-细胞复合体，显著促进了细胞增殖以及组织工程化骨的形成，为骨组织工程研究提供了新思路。以上研究结果有助于我们更深层次的理解支架材料对细胞增殖代谢的促进作用及机制，也为设计制备具有实际应用价值的组织工程支架提供依据。