应用3D生物打印的软骨支架修复关节软骨缺损

Repair of articular cartilage defects with three-dimensional bio-printing and its properties

目的通过多喷头3D生物打印机制作软骨支架,按压配方式将支架植入关节软骨缺损区,修复动物模型的关节软骨缺损并观察效果。方法在软骨细胞外基质(extracellular matrix,ECM)中加入适量的丝素蛋白(silk fibrion,SF),并加入交联剂聚乙二醇(polyethylene glycol,PEG)和骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)来配制生物墨水;用流变仪评估生物墨水的流变性能;使用傅立叶红外变换光谱鉴定生物墨水的蛋白质二级结构;利用装载有软骨生物墨水的加压喷头,打印厚2 mm,直径6 mm的组织工程支架;使用拉力机测量了组织工程支架的压缩模量;通过干失重法评估各个支架降解速率;CCK8及活死细胞染色评价支架上细胞的活力及增殖情况;实时荧光定量PCR评估体外培养28 d后支架上细胞软骨分化情况;按照自体软骨移植术的方式通过压配原理将支架嵌入动物关节软骨缺损区修复关节软骨缺损;用组织学染色及生化检测鉴定3个月后软骨修复效果。结果生物墨水均表现出剪切稀化的流动特性。含有丝素蛋白的生物墨水酰胺Ⅰ区吸收峰移至1623~1627 cm-1处。随着丝素蛋白含量增加,生物墨水的机械强度和降解性能提高,10%和15%打印支架等压缩模量分别达到(19.96±5.66)kpa和(26.87±10.68)kpa。各个生物墨水均无细胞明显细胞毒性。实时定量PCR表明,当丝素蛋白的含量达到10%~15%时,组织块中的骨髓间充质干细胞成软骨分化能力更强。体内研究:3个月后10%和15%的丝素蛋白生物支架sGAG/DNA含量分别为(0.25±0.01)μg/ng和(0.24±0.02)μg/ng,胶原/DNA含量分别为(17.71±0.83)ng/ng和(16.69±2.39)ng/ng,高浓度丝素蛋白打印的组织工程软骨能更好地修复关节软骨缺损。结论在含有10%和15%丝素蛋白生物墨水的3D生物打印支架中,骨髓间充质干细胞的软骨分化和细胞外基质(胶原蛋白和糖胺聚糖)的分泌均优于其他两种支架。不同支架的干细胞成软骨分化能力和细胞外基质分泌的变化,以及对关节软骨缺损的修复效果是由于支架力学性能的差异所致,并可以通过改变丝素蛋白的浓度优化。

Objective To repair the articular cartilage defects of animal models with cartilage tissue block made by multi-nozzle three-dimensional bio-printer and observe its effect.Methods Bio-ink was made by adding silk fibroin,polyethylene glycol and bone mesenchymal stem cells into extracellular matrix(ECM)solution.Rheological properties of biological ink were evaluated by rheometer,the protein secondary structure of biological ink was identified by Fourier transform infrared spectroscopy,and a tissue engineering scaffold with thickness of 2mm and diameter of 6mm was printed by using a pressure sprinkler loaded with cartilage biological ink.The compression modulus of tissue engineering scaffold was measured by tension machine.The degradation rate of each scaffold was evaluated by dry weight loss method,and the viability and proliferation of cells on the scaffold were evaluated by CCK-8 and live&dead cell staining.The differentiation of cellular cartilage on the scaffold was evaluated by real-time fluorescence quantitative PCR.The scaffold was embedded into the defect area of animal articular cartilage to repair articular cartilage defect according to the principle of autogenous cartilage transplantation.The effect of cartilage repair after 3 months was evaluated by histological staining and biochemical detection.Results We found that all biological inks showed the flow characteristics of shear thinning.The absorption peak of biological ink amide I region containing silk fibroin moved to 1623 cm-1.With the increase of silk fibroin content,the mechanical strength and degradability of biological ink were improved,and the compression modulus of 10%and 15%printing stand reached 19.96±5.66 kpa and 26.87±10.68 kpa,respectively.All biological inks had no obvious cytotoxicity.Real-time quantitative PCR showed that when the content of silk fibroin reached 10%-15%,the bone marrow mesenchymal stem cells in the tissue mass had stronger ability to differentiate into cartilage.In vivo studies showed that after 3 months,the sGAG/DNA content of 10%and 15%silk fibroin scaffolds reached 0.25±0.01μg/ng and 0.24±0.02μg/ng,respectively,and the collagen/DNA content reached 17.71±0.83 ng/ng and 16.69±2.39 ng/ng,respectively.Tissue engineered cartilage printed with high concentration silk fibroin can better repair articular cartilage defects.Conclusion TThe chondrogenic differentiation and extracellular matrix(collagen and glycosaminoglycan)secretion of BMSCs were superior to those of the other two scaffolds when the content of silk fibroin reaches 10%-15%.The changes of chondrogenic differentiation ability and extracellular matrix secretion of stem cells from different scaffolds,as well as the repair effect on articular cartilage defects are caused by the differences of mechanical properties of scaffolds,which can be produced by the changes of silk fibroin concentration.