丝素蛋白-胶原骨软骨多孔支架的制备及对其生物相容性的检测

Preparation and test of biocompatibility on the silk fibroin-collagen osteochondral porous scaffold

目的制备丝蛋白-胶原骨软骨细胞支架并评价其生物相容性。方法利用透析和差速离心等物理方法以及三元法等化学方法制备丝素蛋白水溶液;利用乙酸法和酶降解法制备胶原蛋白水溶液;利用灌注和冷冻干燥等方法分别制备丝素蛋白支架、胶原支架和丝素蛋白-胶原支架等3种不同的细胞支架。通过大体观察、扫描电镜观察和生物力学检测等方法,对丝素蛋白-胶原细胞支架的物理化学性质和力学性能进行评价。原代培养小鼠软骨细胞,并将软骨细胞分别种植于上述3种支架,通过对支架材料的细胞毒和死(活)细胞进行检测,评价细胞生长情况,测定支架的生物相容性。采用噻唑蓝(MTT)法检测支架的细胞毒性;应用荧光显微镜观察支架中细胞的生长状况。结果大体观察见支架内孔隙结构相互连通且均具有立体多维性。胶原支架、丝素蛋白支架和丝素蛋白-胶原支架内的孔径分别为(121.6±8.7)、(98.4±5.6)和(103.2±3.9)μm,压缩模量分别为(67.6±2.9)、(140.7±2.5)和(94.3±5.2)kPa,弹性模量分别为(13.3±1.6)、(29.9±2.2)和(22.9±3.0)kPa。丝素蛋白-胶原支架无细胞毒性;支架纵切薄片上细胞生长分布均匀,表明支架具有好的生物相容性。结论丝素蛋白-胶原骨软骨多孔支架的制备可行;该支架具有较好的生物力学特性和良好的生物相容性,可促进软骨细胞的增殖。

Objective To prepare silk fibroin/collagen porous tissue scaffolds and to evaluate its biocompatibility.Methods Silk fibroin was prepared by physical methods such as dialysis,differential centrifugation and chemical methods such as ternary method.Collagen was prepared by acetic acid and enzymatic degradation method.Three different scaffolds including silk fibroin,collagen and silk fibroin/collagen scaffold were prepared by perfusion,freeze‐drying and other methods.The physicochemical and mechanical properties of silk fibroin/collagen scaffold were evaluated through gross observation,scanning electron microscope observation and biomechanical tests.The primary culture of mouse chondrocytes was carried out,and the chondrocytes were planted on the silk fibroin/collagen scaffold.The cell growth was observed by cytotoxicity and live/dead cell staining.Results Gross observation showed that the pore structure of scaffold was interconnected and multidimensional,and the pore size of collagen,silk fibroin and silk fibroin/collagen scaffolds were(121.6±8.7),(98.4±5.6)and(103.2±3.9)μm,respectively.At the same time,the compression modulus of 3 kinds of scaffolds were(67.6±2.9),(140.7±2.5)and(94.3±5.2)kPa,and the elastic modulus were(13.3±1.6),(29.9±2.2)and(22.9±3.0)kPa,respectively.The MTT assay showed that the silk fibroin/collagen scaffold was non‐toxic.Fluorescence microscope observation showed that the growth and distribution on the longitudinal slice of the scaffold were uniform,indicating that the scaffold had good biocompatibility.Conclusion Silk fibroin/collagen porous scaffold can be prepared and has good biomechanical properties and biocompatibility.It can promote the proliferation of chondrocytes.