半月板细胞外基质-海藻酸水凝胶的制备及其对半月板细胞的影响

The preparation of AMECM-Alg hydrogel scaffolds and theirs effect on meniscal fibrochondrocytes

目的制备脱细胞半月板细胞外基质-海藻酸(AMECM-Alg)水凝胶,并研究AMECM-Alg水凝胶对半月板细胞增殖、再分化的影响。方法联合物理化学方法制备AMECM,以AMECM和海藻酸为原料制备5种不同浓度的AMECM-Alg水凝胶(0%、0.5%、1%、2%和4%);复合P_3代半月板细胞,通过细胞死活染色、CCK8实验、DNA定量评估水凝胶促进细胞增殖的能力,通过胶原、糖胺多糖定量,RT-PCR评估水凝胶促进细胞再分化的能力。结果死活细胞染色、CCK8实验和DNA定量实验表明AMECM-Alg水凝胶均能促进细胞的增殖,其中以2%浓度组作用最强;胶原和糖胺多糖定量结果表明AMECM-Alg水凝胶均能促进半月板细胞分泌细胞外基质,其中2%浓度组明显优于其他组。RT-PCR结果显示半月板细胞在2%浓度组水凝胶中collagen Ia2、collagen IIa1、SOX9和aggrecan基因表达量明显高于其他组。结论 AMECM-Alg水凝胶具有良好的细胞相容性,既能促进细胞增殖,又能促进细胞再分化,其中以2%浓度组的作用最强,在未来组织工程半月板生物材料研究方面具有很好的应用前景。

Objective To manufacture accellular meniscus extracellular matrix-alginate（AMECM-Alg） hydrogel scaffolds and investigate their biological effect on the cell viability, proliferation, and re-differentiation of the meniscal fibrochondrocytes.Methods Combined physical and chemical methods were used to prepare accellular meniscus extracellular matrix. Five concentrations of AMECM-Alg hydrogel were manufactured by AMECM and alginate with different ratios（0%, 0.5%, 1%, 2%, 4%）. The microstructrue of AMECM-Alg hydrogel was investigated by scanning electron microscopy（SEM）. Passaged meniscus fibrochondrocytes were seeded in the five different hydrogel scaffolds, and then cultured for 1, 4, 7 and 14 d. Live/dead staining was used to evaluate the cell viability. The cell proliferation on different hydrogel scaffolds was studied by CCK8 test and DNA quantification. Re-differentiation of the passaged meniscal fibrochondrocytes was determined by biochemical assays for GAG and collagen as well as RT-PCR. Results SEM results showed that the porosity of the hydrogel scaffolds became smaller as the AMECM concentration increased. Live/dead staining results indicated that meniscal fibrochondrocytes could maintain good viability in the different hydrogel scaffolds. The results of CCK8 test and DNA quantification revealed that AMECM-Alg hydrogels could promote cell proliferation, with 2% group having the strongest effect. GAG and collagen content assessment results demonstrated that AMECM-Alg hydrogels enhanced collagen and GAGs production, with 2% group inducing the greatest effect. RT-PCR results revealed that AMECM-Alg hydrogels up-regulated the expression of collagen Ia2, collagen IIa1, SOX9, aggrecan, with 2% group exhibiting the robustest effect. Conclusion AMECM-Alg hydrogel provides a suitable microenvironment for seed cells which may maintain cell viability and promote the cell proliferation and re-differentiation, with 2% group having the strongest effect. In conclusion, 2% AMECM-Alg hydrogel scaffold is a promising candidate biomaterial for meniscus tissue engineering.