基于低温沉积打印技术构建分级多孔脱细胞华通胶支架

Construction of a hierarchical microporous decellularized Wharton′s jelly scaffold through low-temperature deposition manufacturing system

目的 制备分级多孔的脱细胞华通胶支架,评价其生物相容性。方法 从临床上获得人脐带,并分离出华通胶组织,通过组织学染色观察其生物学成分。采用Na OH溶液对华通胶进行脱细胞处理,采用苏木精-伊红(HE)及DAPI染色观察脱细胞效果,扫描电镜评估脱细胞前后胶原保留情况。通过低温沉积3D打印技术制备分级多孔脱细胞华通胶支架,采用扫描电镜观察其内部微观结构。采用CCK-8实验检测分级多孔支架的细胞毒性。将第三代大鼠骨髓间充质干细胞接种于支架上,培养7 d后分别进行活/死染色和细胞鬼笔环肽微丝骨架染色观察。将第三代大鼠骨髓间充质干细胞接种于支架上,加入成软骨诱导培养基培养14d后,通过q RT-PCR法检测成软骨分化相关基因(SOX9、ACAN、COLII、COLI)的表达水平。结果 (1)人脐带呈条索状,由外层的羊膜、华通胶、两条脐动脉和一条脐静脉组成,分离出的华通胶为乳白色,呈胶状;(2)HE染色显示,华通胶富含细胞外基质;番红-O染色显示,华通胶富含糖胺多糖成分;天狼猩红染色显示,华通胶富含胶原成分;甲苯胺蓝染色显示,华通胶富含糖胺多糖成分;(3)经脱细胞处理好,华通胶色泽变得透明。HE及DAPI染色显示,脱细胞华通胶无细胞核结构;扫描电镜显示,脱细胞后,华通胶保留了很好的胶原成分;(4)通过低温沉积3D打印的支架,扫描电镜显示,支架内部呈现分级多孔的微观结构;(5)CCK-8细胞毒性实验显示,打印的支架无明显的细胞毒性;(6)活/死细胞染色显示,骨髓间充质干细胞能够在分级多孔的支架上保持很高的活性(96.0±3.3)%;(7)鬼笔环肽微丝骨架染色显示,骨髓间充质干细胞能够很好地在分级多孔的支架铺展开;(8)qRT-PCR结果显示,相比于普通的平板培养,支架能够促进骨髓间充质干细胞成软骨分化相关的基因表达。结论 脱细胞华通胶富含胶原、糖胺多糖等成分,低温沉积3D打印的脱细胞华通胶支架具有分级多孔的微观结构,具有良好的细胞相容性,有利于细胞的生长和黏附,并且可以促进骨髓间充质干细胞的成软骨分化。

Objective To prepare a hierarchical microporous decellularized Wharton′s jelly scaffold and evaluate its biocompatibility.Methods Human umbilical cords were obtained from the hospital, and Wharton′s jelly was isolated and its biological composition was observed by histologically staining. Wharton′s jelly was decellularized with sodium hydroxide(NaOH) solution.Hematoxylin-eosin(HE) and DAPI staining were used to observe the decellularization effect, and the collagen before and after decellularization was evaluated by scanning electron microscopy. Scaffolds were prepared by low-temperature deposition 3D printing techniques, and their internal microstructures were observed by scanning electron microscopy. The cytotoxicity of the scaffold was detected using a CCK-8 assay. Third-generation bone marrow mesenchymal stem cells were seeded on the 3D-printed scaffold. After 7 days of culture, the scanning electron microscopy, dead/live staining and F-actin staining were used to observe the cytocompatibility of the 3D-printed scaffold. The P3 rat bone marrow mesenchymal stem cells were seeded on scaffolds and cultured with chondrogenic induction medium for 14 days. The expression levels of chondrogenic differentiation related genes(SOX9, ACAN,COLII, COLI) were measured by qRT-PCR.Results(1)The human umbilical cord was in the form of strips, consisting of an outer layer of amnion, Wharton′s jelly, two umbilical arteries and one umbilical vein, and the separated Wharton′s jelly was milky white and gelatinous;(2)The results from HE staining showed that Wharton′s jelly was rich in extracellular matrix;Wharton′s jelly was rich in GAG by safranin-O staining;the data form sirius red staining showed that Wharton′s jelly was rich in collagen;Wharton′s jelly was rich in glycosaminoglycan after toluidine blue staining;(3)After decellularization, the color of Wharton′s jelly became transparent;decellularized Wharton′s jelly had no nucleus through HE and DAPI staining;The results from scanning electron microscopy showed that after decellularization,Wharton′s jelly retained good collagen content;(4)The data from scanning electron microscopy of the 3D printed scaffold by low-temperature deposition manufacturing(LDM) showed that hierarchical microporous structure was inside the scaffold;(5)The data from CCK-8 cytotoxicity assay showed that the 3D-printed scaffolds were not significantly cytotoxic;(6)The results from live/dead cell staining showed that bone marrow mesenchymal stem cells were able to maintain a high level of activity on the graded porous scaffolds(96.0 ± 3.3)%;(7)Bone marrow mesenchymal stem cells spreaded well in the scaffold by F-actin microfilament backbone staining;(8)The results of qRT-PCR showed that the scaffold could promote the expression of genes related to chondrogenic differentiation of bone marrow mesenchymal stem cells as compared with the plate culture.Conclusion Decellularized Wharton′s jelly is rich in collagen, glycosaminoglycans and other components, and the 3D-printed decellularized Wharton′s jelly scaffold produced by low-temperature deposition manufacturing has a hierarchical microporous microstructure with good cytocompatibility, which is conducive to the growth and adhesion of cells. Furthermore, it can promote chondrogenic differentiation of bone marrow mesenchymal stem cells.