脱细胞真皮基质水凝胶的制备及生物学评价

Preparation and biological evaluation of decellularized dermal matrix hydrogel

背景:最近脱细胞真皮基质水凝胶因其可注射性和能填充不规则空间,在组织填充与修复中有着广阔的应用前景,而细胞相容性和生物相容性是组织修复的关键,探究脱细胞真皮基质水凝胶的相容性具有重要意义。目的:构建一种脱细胞真皮基质水凝胶,用于组织填充和修复。方法:制备可注射型脱细胞真皮基质水凝胶,扫描电镜下观察其微观形貌。将质量浓度8,10 g/L的脱细胞真皮基质水凝胶分别与骨髓间充质干细胞共培养,检测细胞增殖与细胞相容性。将蛋白质量浓度0.25,0.5 g/L的脱细胞真皮基质水凝胶分别注射至同一SD大鼠背部皮下不同部位,术后2,4周,观察水凝胶降解情况。将蛋白质量浓度1,1.5,2 g/L的脱细胞真皮基质水凝胶分别注射至同一SD大鼠背部皮下不同部位,术后8,16周,观察水凝胶降解情况,并进行组织学分析。结果与结论:①扫描电镜下可见,脱细胞真皮基质水凝胶呈现一种不规则随机走向的多孔纤维结构,孔径大小不一。②共培养1,3,5 d后,两种浓度的水凝胶对骨髓间充质干细胞的增殖无影响;共培养1,2,3 d后的Live/Dead染色显示,两种浓度的水凝胶对骨髓间充质干细胞的增殖无影响,但水凝胶上的细胞形态发生了一定变化。③蛋白质量浓度0.25 g/L的水凝胶皮下注射2周后降解,0.5 g/L的水凝胶皮下注射4周后降解,1,1.5,2 g/L的水凝胶皮下注射16周均未降解,甚至可能保留更长时间。苏木精-伊红染色显示,皮下注射8周后,3种蛋白质量浓度水凝胶组织较为致密,出现少量的炎症浸润,2 g/L组有微血管生成;16周后,水凝胶组织变得松散,以1 g/L组较明显,炎症浸润减轻。免疫组化染色显示,随着注射时间的延长,水凝胶中的微血管数量增加;并且随着蛋白质量浓度的增加,水凝胶中的微血管数量增加。④结果表明,可注射脱细胞真皮基质水凝胶具有良好的细胞相容性和生物相容性。

BACKGROUND:Recently,decellularized dermal matrix hydrogel has broad application prospects in tissue filling and repair because of its injectable and filling irregular space.Cytocompatibility and biocompatibility are the key to tissue repair.It is significant to explore the compatibility of decellularized dermal matrix hydrogel.OBJECTIVE:To construct a decellularized dermal matrix hydrogel scaffold for tissue filling and repair.METHODS:An injectable decellularized dermal matrix hydrogel was prepared,and its microscopic morphology was observed under a scanning electron microscope.The decellularized dermal matrix hydrogels with a concentration of 8 and 10 g/L were co-cultured with bone marrow mesenchymal stem cells,separately,and the cell proliferation and cytocompatibility were tested.The decellularized dermal matrix hydrogels with a protein concentration of 0.25 and 0.5 g/L were injected into different subcutaneous parts of the back of the same SD rat separately,and the degradation of the hydrogels was observed 2 and 4 weeks after the operation.Decellularized dermal matrix hydrogels with protein concentrations of 1,1.5,and 2 g/L were injected into different subcutaneous parts of the back of the same SD rat separately.At 8 and 16 weeks after the operation,the degradation of the hydrogel was observed and histological analysis was performed.RESULTS AND CONCLUSION:(1)Scanning electron microscopy analysis showed that the decellularized dermal matrix hydrogel exhibited an irregular and randomly oriented porous fibrous structure with different pore sizes.(2)After 1,3,and 5 days of co-culture,the hydrogels with the two concentrations had no effect on the proliferation of bone marrow mesenchymal stem cells.Live/Dead staining after 1,2,and 3 days of co-culture showed that the hydrogels with the two concentrations had no effect on the proliferation of bone marrow mesenchymal stem cells,but the cell morphology on the hydrogel changed to some extent.(3)The hydrogel with a protein concentration of 0.25 g/L was degraded after subcutaneous injection for 2 weeks;the hydrogel with a protein concentration of 0.5 g/L was degraded after subcutaneous injection for 4 weeks;the hydrogels with a protein concentration of 1,1.5,and 2 g/L did not degrade after subcutaneous injection for 16 weeks,and possibly even longer.Hematoxylin-eosin staining showed that after subcutaneous injection for 8 weeks,the hydrogel tissue of the three protein mass concentrations was relatively dense,with a small amount of inflammatory infiltration,and the 2 g/L group had microangiogenesis.After 16 weeks,the hydrogel tissue became loose,and the 1 g/L group was more obvious,and the inflammatory infiltration was alleviated.Immunohistochemical staining showed that the number of microvessels in the hydrogel increased with the increase of injection time.The number of microvessels in the hydrogel increased with the increase of protein concentration.(4)The results confirmed that the injectable decellularized dermal matrix hydrogel had good cytocompatibility and biocompatibility.