含软骨钙化层的仿生支架体内构建组织工程骨软骨

摘要

Objective The biomimetic osteochondral scaffold contained calcified cartilage layer(CCL) was fabricated using slik fibroin (SF) and hydroxyapatite (HA) for materials.To investigate effects of biomimetic osteochondral scaffold contained CCL compounding with ADSCs on regeneration of the osteochondral defect on the rabbit knee,explore the feasibility of this design as a concept of osteochondral tissue engineering.Methods We fabricated a novel biomimetic osteochondral scaffold with CCL using SF and HA by the combination of paraffin-sphere leaching and modified temperature gradient-guided thermal-induced phase separation (TIPS) technique.The pore size,porosity,and compressive modulus of elasticity of the scaffold cartilage layer and the osteogenic layer were measured by scanning electron microscopy and microscopy CT.The osteochondral defect model on rabbit bilateral knees were established,and implanted with the non-CCL group (non-CCL scaffold compounding with ADSCs) and CCL group (CCL scaffold compounding with ADSCs).At 4,8 and 12 weeks after implantation,the rabbits were euthanized,respectively.Gross observation score,histological and immunohistochemical assessment,biochemical quantitative of new osteochondral tissue,micro-CT scans for new bone,were executed.We evaluated the regeneration of osteochondral defects in each group,and verified the role of CCL in vivo.Results The biomimetic osteochondral scaffold with CCL had a consecutively overlapping trilayer structure with different densities and pore structures,including a chondral layer (top layer),intermediate layer and bony layer (bottom layer).The cartilage layer had a well-oriented microporous structure with a uniform distribution with a pore size of (112.43± 12.65)μm and a porosity of 90.25％±2.05％.The subchondral bone layer had a good three-dimensional macroporous structure,good connectivity,pore size (362.23±26.52) μm,porosity of osteogenic layer was 85.30％± 1.80％.The cartilage regeneration in CCL+AD-SCs group was better than non-CCL+ADSCs group.The content of GAG and type Ⅱ collagen in new cartilage tissue in CCL+AD-SCs group was much more than non-CCL+ADSCs group.The new bone tissue analysis and biomechanical testing had no significant differences between the two groups.Conclusion The biomimetic osteochondral SF/HA scaffold contained CCL mimics the structure of normal osteochondral tissue with good 3-dimensional pore structure and biocompatibility.The scaffold complex autologous ADSCs successfully repair osteochondral defects in rabbit knee,and the presence of CCL accelerates the growth of cartilage.%目的 以丝素蛋白(silk fibroin,SF)和羟基磷灰石(hydroxyapatite,HA)为材料制备含软骨钙化层(calcified cartilage layer,CCL)的仿生骨软骨支架,接种兔脂肪干细胞(adipose-derived stem cells,ADSCs),评估CCL在体内修复中起到的作用,探讨这一设计组合作为骨软骨组织工程理念的可行性.方法 以SF/HA为材料,采用石蜡微球沥滤和定向结晶(thermally induced phase separption,TIPS)结合技术,制备含软骨钙化层的多相一体化仿生支架,通过扫描电镜、micro-CT进行观察,测定支架软骨层和成骨层的孔径、孔隙率和压缩弹性模量;ADSCs接种到支架上,建立兔双侧膝关节骨软骨缺损模型,分别植入CCL支架组、non-CCL+ADSCs组(不含CCL的支架复合自体ADSCs)和CCL+ADSCs组(含CCL的支架复合自体ADSCs),分别在术后4、8、12周取材,进行大体观察评分、组织学和免疫组化评估、新生骨组织Micro-CT扫描,评估各组兔膝关节骨软骨缺损修复效果,对比分析CCL在体内修复中起到的作用.结果 含CCL的仿生骨软骨支架由层次鲜明的“透明软骨-CCL-软骨下骨”组成,各层连接紧密.软骨层具有明显的取向微孔结构,分布均匀,孔径(112.43±12.65) μm,孔隙率为90.25％±2.05％;软骨下骨层具有良好的三维大孔结构,连通性好,孔径(362.23±26.52) μm,成骨层孔隙率为85.30％±1.80％;CCL+ADSCs组软骨再生情况优于non-CCL+ADSCs组,组织学和免疫组化染色可见GAG和Ⅱ型胶原表达较non-CCL+ADSCs组高;新生骨组织分析、生物力学性能测试在两组间未见明显差异.结论 含CCL的SF/HA仿生骨软骨支架模仿正常骨软骨组织结构,具有良好的三维孔隙结构和生物相容性.该支架复合自体来源的ADSCs成功修复兔膝关节骨软骨缺损,CCL的存在加速软骨组织的生长.