明胶海绵–海藻酸钠水凝胶制备关节软骨组织工程化支架

Construction of Tissue-Engineered Scaffolds in Vitro by Using Medical Gelatin Sponge and Sodium Alginate Hydrogel

摘要: 正常人的关节软骨具有优秀的生物力学性能，不仅可以在关节间起缓冲作用，而且也在促进人体身体运动方面起着至关重要的作用。因此关节软骨的完整性至关重要。但是由于关节软骨缺乏血管和血供，因此自我修复极为困难，所以一旦关节软骨损伤，其损伤很难自我修复。同时关节软骨的损伤还常常伴有关节下骨的损伤，如若不做处理，进一步发展可进发为骨关节炎，对患者生活造成巨大影响。但是目前临床上对软骨损伤的治疗，如软骨钻磨、骨膜和软骨膜移植、软骨下骨钻孔、人工关节置换等治疗效果并不理想。近年来组织工程的迅速发展，为软骨及软骨下骨的修复提供了新途径及新方法。支架作为组织工程的核心内容之一起着至关重要的作用。传统的软骨组织工程修复关节软骨缺损，是在生物支架上种植软骨细胞，这种方法一方面细胞的种植率较低；另一方面，从修复的角度来看，软骨损伤通常伴随软骨下骨的损伤，单纯的运用软骨细胞修复软骨而忽略了软骨下骨的修复，其修复效率有限。因此本研究设想能否构建一种复合支架，在支架上层种植软骨细胞，支架下层种植成骨细胞，通过这种方式可以同时修复关节软骨及软骨下骨的损伤。复合支架的难点在于如何选择合适的材料。因此在深入认识关节软骨及关节下骨的基础上，我们选用医用明胶海绵与海藻酸钠水凝胶来构建复合支架。以往多项研究表明医用明胶海绵及海藻酸钠的生物相容性、孔径率及力学特性均可以支持其作为关节软骨组织工程的支架材料。本研究阐明了在关节软骨组织工程中构建明胶海绵–海藻酸钠水凝胶复合支架的可行性，为修复关节软骨缺损提供依据及技术支持。方法：1) 构建含成骨细胞的底层支架。选取适量明胶海绵，切成小块后灭菌；成骨细胞MC3T3-E1扩增后种植在明胶海绵上，体外培养诱导成骨细胞分化，监测成骨细胞在支架表面的贴附、增殖和分化状况。2) 制备并培养含细胞的复合支架。将含有软骨细胞的海藻酸钠溶液利用与CaCl₂溶液交联在明胶海绵支架上形成海藻酸钠水凝胶；培养形成的复合支架并检测支架下层成骨细胞在支架表面的贴附、增殖和分化状况及水凝胶中软骨细胞的增殖情况。3) 对含细胞的复合支架进行检测。将不同时间提取的复合支架标本进行检测，明确复合支架中碱性磷酸酶及糖胺聚糖的表达情况。结果：1) MC3T3-E1在明胶海绵支架上扩增生长良好，活/死细胞染色示随时间推移活细胞在支架上数量增多。2) 海藻酸钠水凝胶与明胶海绵支架接合良好，猪膝软骨细胞及MC3T3-E1分别在海藻酸钠水凝胶与明胶海绵支架上生长良好，活/死细胞染色示随时间推移两种活细胞在支架上数量增多。3)复合支架上的细胞与单纯明胶海绵及单纯海藻酸钠水凝胶上的细胞相比碱性磷酸酶及糖胺聚糖表达提升。结论：通过海藻酸钠溶液可以与CaCl₂溶液交联形成水凝胶的方法成功构建了组织工程复合支架，明胶海绵–海藻酸钠水凝胶复合支架较单纯的明胶海绵支架及单纯的海藻酸支架可以更好地促进MC3T3-E1细胞及猪膝软骨细胞的增殖和分化。明胶海绵–海藻酸钠水凝胶复合支架在关节软骨组织工程中具有良好的应用前景。

Abstract: Normal human articular cartilage has excellent biomechanical properties and can play a buffering role between joints. The integrity of cartilage plays an important role in promoting body movement. Therefore, the integrity of articular cartilage is very important. However, articular cartilage injury is a common type of injury in orthopedics. Due to the lack of blood vessels and blood supply of articular cartilage, it is very difficult to self repair. The injury of articular cartilage is usually accompanied by the injury of the infraarticular bone, which can further develop into osteoarthritis, which has a huge impact on the life of patients. At present, the clinical treatment of cartilage injury, such as cartilage drilling, periosteum and perichondrium transplantation, subchondral bone drilling, artificial joint replacement and so on, is not ideal. In recent years, the rapid development of tissue engineering provides a new way and method for the repair of cartilage and subchondral bone. As one of the core contents of tissue engineering, scaffold plays an important role. The traditional cartilage tissue engineering is to implant chondrocytes on the scaffold, on the one hand, the implantation rate of cells is relatively low; on the other hand, from the perspective of repair, cartilage damage is usually accompanied by the injury of subchondral bone, and the repair efficiency of subchondral bone is limited simply by using chondrocytes to repair cartilage and ignoring the repair of subchondral bone. Therefore, we consider whether we can construct a composite scaffold, and implant chondrocytes in the upper part of the scaffold and osteoblasts in the lower part of the scaffold. The difficulty of composite support lies in how to choose the right material. Therefore, based on the in-depth understanding of articular cartilage and articular subchondral bone, we choose medical gelatin sponge and sodium alginate hydrogel to build composite scaffolds. Previous studies have shown that the biocompatibility, pore size ratio and mechanical properties of medical gelatin sponge and sodium alginate can support their use as scaffold materials for cartilage tissue engineering. The feasibility of gelatin sponge alginate hydrogel composite scaffolds in cartilage tissue engineering is elucidated, which provides theoretical basis and technical support for the regeneration and repair of articular cartilage defects with new ideas. Method: 1) To construct the underlying scaffold containing osteoblasts. The osteoblasts MC3T3-E1 were expanded and planted on the gelatin sponge. The osteoblasts were cultured in vitro to induce the differentiation of osteoblasts. The adherence, proliferation and differentiation of osteoblasts on the scaffold surface were monitored. 2) Preparation and culture of composite scaffolds containing cells. Sodium alginate solution containing chondrocytes was crosslinked into CaCl₂ gelatin solution to form sodium alginate hydrogel on the gelatin sponge scaffold. The scaffold was cultured and the attachment, proliferation and differentiation of osteoblasts on the scaffold surface and the proliferation of chondrocytes in the hydrogel were detected. 3) Detection of composite scaffolds containing cells. The expression of alkaline phosphatase and glycosaminoglycan in the composite scaffolds was determined. Result: 1) MC3T3-E1 grew well on the gelatin sponge scaffold, and the number of living cells on the scaffold increased with time. 2) Sodium alginate hydrogel and gelatin sponge scaffold joined well. Pig knee cartilage cells and MC3T3-E1 grew well on alginate hydrogel and gelfoam sponge respectively. Living/dead cell staining showed that the number of two kinds of living cells on scaffolds increased with time. 3) The expression of alkaline phosphatase and glycosaminoglycan increased in cells on composite scaffolds compared with those in pure gelatin sponge and sodium alginatehydrogel alone. Conclusion: The scaffold constructed by sodium alginate solution can be successfully cross-linked with CaCl₂ solution. The gelatin sponge alginate hydrogel composite scaffold can promote the proliferation and differentiation of MC3T3-E1 cells and pig knee chondrocytes better than simple gelatin sponge scaffolds and simple alginate scaffolds. The application of gelatin sponge sodium alginate hydrogel composite scaffold in articular cartilage tissue engineering has a good application prospect.