三维生物打印梯度软骨支架植入。(A) (A)人尺度软骨支架和(b和c)兔尺度软骨支架的大体外观(b, NG, 150 μm间距;c, NG，间距为750 μm)。兔软骨支架的俯视图(d, NG, 150 μm间距;e，间距为750 μm的NG;f，间距为150 ~ 750 μm的梯度支架)位于SEM图像顶部(g，水平切面;h，垂直切面)，用于150 μm NG支架，以演示PCL纤维在打印支架中的精确对齐。(B)梯度支架的解构。梯度支架的结构被解构为四层。各层水凝胶- pcl复合结构的微观形貌表明，各层结构相互连通良好，排列有序。 (C and D) Good cell viability is shown respectively for superficial and deep layers after printing with live/dead assay (green, live cells; red, dead cells) (C) under a microscope and (D) under a confocal microscope. DAPI, 4′,6-diamidino-2-phenylindole. (E) Cell spreading in superficial and deep layers with cytoskeleton staining. (F) Immunostaining for cartilage markers in superficial and deep layers. Expression of COL2A1 and PRG4, the lubrication markers, was significantly higher in the superficial layers with small pore size (a and b), while the chondrogenic cells in the deep layers (c and d) mostly presented with hypertrophic phenotype (COL10A1 expression). Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University. Credit: Science Advances, doi: 10.1126/sciadv.aay1422
3d生物打印软骨结构
该团队使用3d生物打印技术开发了不同的关节组织结构，用于关节重建。他们通过包含不同生长因子释放结构的生化刺激(BCS)和小孔径的生物力学刺激(BMS)来模拟原生软骨软骨形成．然后，他们创建了第三个软骨结构，作为双刺激(DS)组，包括两个版本的刺激。对于生长因子，研究小组选择了骨形态发生蛋白(BMP4)和转化生长因子β3(TGFβ3)在软骨结构中再生复杂的不均匀关节组织。Sun等人随后开发了一种水凝胶来传递生长因子并使用保利(lactic-co-glycolic酸)(PLGA)微球作为载体/载体。研究组在BMS(生物力学刺激)组和BCS(生化刺激)组中保持恒定的纤维间距，形成无梯度支架，而在DS(双刺激)组中引入逐渐变化的纤维间距的支架。科学家们还使用了聚(ε-己内酯)(PCL)聚合物，并将其集成到仿生支架结构中。通过这种方式，他们开发了使用4 x 4 x 4 mm支架的兔软骨结构和使用14 x 14 x 14 mm支架的人类软骨结构。
细胞活力和在打印的各向异性支架中的锚定。(A)各向异性软骨支架结构示意图，左图为梯度支架结构的制作，右图为排列的蛋白释放bmscs负载水凝胶的大规模打印。鳞片条，1mm。(B)显微镜下PLGA μ s包封的载bmscs水凝胶的大体外观(上)。打印的细胞负载水凝胶使细胞沿打印路径的纵向排列，形成细胞相互作用的网状网络(底部)。(C)活细胞/死细胞试验表明，4层梯度间距(第4行，150 μm间距;第3行，间距350 μm;第二行，间距550 μm;第一行，间距750 μm)。细胞骨架免疫染色(最右一列)显示细胞在水凝胶和PCL纤维中扩散，遍及四层结构。比例尺，500 μm。 (D and E) Quantified cell viability and proliferation in the printed scaffolds. (F) Cell anchoring in the scaffolds. (a to c) At day 21, good 3D anchoring to the PCL fiber cylinder was observed for the MSC cells released from the hydrogel. (d to f) Similar cell anchoring was observed for PCL fibers in adjacent layers. (b), (c), (e), and (f) are 3D demonstration of cell anchoring in (a) and (d), respectively. Scale bars, 100 μm. Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University. Credit: Science Advances, doi: 10.1126/sciadv.aay1422
双因子释放、梯度结构软骨支架对兔膝关节软骨缺损模型的体内修复效果较好。(A) 8周、12周和24周时支架植入过程和修复软骨大体外观。手术膝关节(第五排)MRI显示，DS支架移植关节软骨下水肿和关节面(白色箭头)愈合明显改善。(B到F)通过(B)在体内植入时修复软骨组织的组织学评分评估，比较支架的软骨保护效果。(C)支架植入两组股骨髁(FC)和胫骨平台(TP)关节软骨的Mankin评分和(D) ICRS(国际软骨修复学会)组织学评分。*本族组与其他组比较P < 0.05。BCS组与DS组#P < 0.05。数据以平均值±标准差表示(N = 6)。(A) 24周时新软骨组织的组织形态学分析。PR，苦天狼星红。左下面板是在彩色方框中形成的新软骨轮廓的高分辨率图片。 (a to e) Sections were stained with (a) H&E, (b) Safranin O, (c) TB, and (d) AB staining to indicate the presence of proteoglycans in different groups compared with native cartilage. (e) Picrosirius red was used to stain collagens I and III. The brown irregular area at the interface under the formed neocartilage was undegraded PCL material as supporting structure for the scaffolds. Photo credit: Ye Sun, First Affiliated Hospital of Nanjing Medical University. Credit: Science Advances, doi: 10.1126/sciadv.aay1422
