丝素蛋白-明胶-壳聚糖-羟基磷灰石多孔支架的制备及性能评估

Preparation and performance evaluation of silk protein-gelatin-chitosan-hydroxyapatiteporous scaffolds

文章以丝素蛋白(SF)、明胶(Gel)、壳聚糖(CS)和羟基磷灰石(Hap)为基础材料,通过真空冷冻干燥和化学交联制备了5组SF-CS-Gel-n Hap多孔支架(Hap-1%、Hap-2%、Hap-3%、Hap-4%、Hap-5%)。通过扫描电子显微镜、X射线衍射仪(XRD)、孔隙率、吸水膨胀率、生物降解率及力学性能研究,筛选出理化性能优越适合全层软骨缺损再生重建的复合支架。随后将其与骨关节炎患者分离提取的软骨细胞共培养,通过细胞黏附率测定、细胞活死染色和CCK-8细胞活性增殖实验等方法评估多孔支架的生物性能,探索其用于修复全层软骨缺损的可行性。结果显示,基于明胶、壳聚糖、丝素蛋白和纳米羟基磷灰石制备的SF-CS-Gel-2%Hap多孔复合支架不仅可以模拟天然骨软骨的细胞外基质(ECM),而且具有良好的生物相容性,能够为营养物质的转运和细胞黏附、增殖和立体生长提供良好的网状骨架,为全层软骨缺损的治疗提供新的选择。

Total articular cartilage defects are usually accompanied by subchondral bone damage,and tissue engineering,as an interdisciplinary technique combining engineering and cell biology,provides new ideas and approaches for the repair of total cartilage defects.In this study,we developed a new approach for the repair of total cartilage defects by using silk fibroin(SF),gelatin(Gel),chitosan(CS)and hydroxyapatite(Hap)as the base materials to meet the needs of osteochondral extracellular matrix(ECM)and osteogenic-chondrogenic bipartite differentiation requirements.Five groups of SF-CS-Gel-n Hap porous scaffolds(Hap-1%,Hap-2%,Hap-3%,Hap-4%and Hap-5%)without concentration gradient were prepared by vacuum freeze-drying and chemical cross-linking by adding different contents of hydroxyapatite to the co-blended solution of silk protein,chitosan and gelatin.The 3D porous composite scaffolds with superior physicochemical properties were screened by scanning electron microscopy,X-ray diffractometry(XRD),and the study of porosity,water absorption and swelling,biodegradation rates and mechanical properties.The scaffolds were then co-cultured with chondrocytes isolated and extracted from patients with osteoarthritis.The biological properties of the porous scaffolds were evaluated by cell adhesion rate determination,cell live-dead staining and CCK-8 cell activity proliferation assay to explore the feasibility of their use for repairing total cartilage defects.All five groups of scaffolds had porous structures,and the water absorption swelling and porosity of SF-CS-Gel-n Hap porous scaffold decreased with increasing Hap content in the scaffold,while the biodegradation rate gradually slowed down with increasing Hap content.The SF-CS-Gel-1%Hap scaffold had the highest water absorption and swelling rate and porosity of 1466%±179%and 87.25%±2.28%,respectively,but it had the fastest and least stable degradation and poor mechanical properties.SF-CS-Gel-2%Hap scaffold had a pore size structure of(212.89±62.22)μm,a porosity of 76.52%±2.31%,good water absorption and swelling rate of 1286%±114%,and a biodegradation rate of 37.09%±1.41%.The physicochemical properties of SF-CS-Gel-2%Hap scaffold were more in line with the requirements of full-layered cartilage defect repair.During its co-culture with chondrocytes isolated and extracted from osteoarthritis patients,the SF-CS-Gel-2%Hap scaffold showed better bioactivity,i.e.good cell adhesion and proliferation properties,and the results of CCK-8 proliferation assay and live/dead cell double-staining assay showed that the porous scaffold had good biocompatibility and low cytotoxicity.The results of this experiment show that the SF-CS-Gel-2%Hap porous composite scaffold prepared based on gelatin,chitosan,silk fibroin and nano-hydroxyapatite can not only mimic the ECM of natural bone cartilage,but also has good biocompatibility and can provide a good reticular skeleton for nutrient transport and cell adhesion,proliferation and stereogenesis,providing a new option for the treatment of total cartilage defects.