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Title: [Effects of alginate/collagen scaffold on cell proliferation and differentiation of human adipose-derived mesenchymal stem cells].
Author: Cheng W, Han XP, Mou SL, Yang F, Liu LP.
Journal: Zhonghua Kou Qiang Yi Xue Za Zhi; 2017 Apr 09; 52(4):259-264. PubMed ID: 28412794.
Abstract:
Objective: To build scaffold materials with different concentrations of alginate and collagen, and to observe the effects of alginate/collagen ratio on the proliferation of human adipose-derived mesenchymal stem cells (hAMSC) and osteogenic differentiation. The optimal concentration of alginate/collagen will be chosen for constructing hydrogel that will be used for bone tissue engineering. Methods: Soluble hydrogel scaffold materials containing alginate/collagen were prepared, and the following groups were established based on different alginate/collagen ratio: 4∶1 (group A), 2∶1 (group B), and 1∶1 (group C). Cell proliferation on the material surface was observed using the cell counting kit-8 (CCK-8) assay, while cell viability in each material group were observed using live/dead staining. Quantitative real-time PCR(qPCR) was used to measure the differential expression of osteogenesis-related genes on and in the materials. Immunofluorescence staining was used to measure the differential gene expression of osteogenesis-related proteins in each group. Results: The results from the CCK-8 assay showed increasing cell proliferation rate on the lyophilized hydrogel material surface as the collagen concentration increased, and the highest cell proliferation was observed in group C. Live/dead staining assay indicated that cells were able to proliferate in all three types of hydrogel materials, and the highest cell viability was found in material from group B ([87.50±2.65]%). qPCR showed that the expression of osteogenesis-related genes in group C was the highest, among the three groups, while the expression of osteocalcin in group B was significantly higher than those in the other two groups (P<0.05). Immunofluorescence staining was carried out for osteocalcin on and in the hydrogel material and the results were consistent with that of qPCR. Conclusions: The alginate/collagen scaffold materials did not show adverse effects on the cell proliferation of hAMSC and osteogenenic differentiation. Bone tissue engineering can use 10% hydrogel material, and when the sodium alginate and collagen have a ratio of 2∶1, the hydrogel can be conducive to cell differentiation and proliferation. 目的： 观察人脂肪间充质干细胞（human adipose-derived mesenchymal stem cell，hAMSC）在不同比例海藻酸钠-胶原支架材料表面及内部的增殖、分化情况，探讨适合骨组织工程的材料比例，为骨组织缺损修复提供基础。 方法： 制备含10%海藻酸钠-胶原的支架材料，按海藻酸钠与胶原比例分为A（4∶1）、B（2∶1）和C组（1∶1）；各组分别制备冻干薄膜和凝胶小球两种形式；冻干薄膜为冻干后灭菌，将hAMSC覆于薄膜表面；凝胶小球制备时与hAMSC混合。细胞检测试剂盒检测hAMSC在各组冻干薄膜表面的增殖情况；活细胞-死细胞染色检测各组凝胶小球内部hAMSC的细胞存活率；实时荧光定量PCR检测各组冻干薄膜表面和凝胶小球内部hAMSC骨钙蛋白和Runt相关转录因子2（Runt-related transcription factor-2，RUNX2）基因的表达差异；免疫荧光染色观察各组hAMSC骨钙蛋白的表达差异。 结果： 随胶原比例增加，细胞增殖逐步增多，C组细胞增殖量最多。hAMSC在3组凝胶小球内部均可增殖，其中B组细胞存活率[（87.50±2.65）%]最高，与A组[（78.20±2.81）%]及C组[（74.76±1.98）%]相比差异均有统计学意义（P<0.05）。3组冻干薄膜表面hAMSC骨钙蛋白和RUNX2基因表达差异均有统计学意义（P<0.05），其中C组骨钙蛋白和RUNX2基因表达量最高；而B组凝胶小球内部骨钙蛋白基因表达量显著高于其他组（P<0.05）；骨钙蛋白免疫荧光染色结果与基因检测结果一致。 结论： 海藻酸钠-胶原支架材料hAMSC增殖、成骨向分化无不利影响，10%水凝胶材料海藻酸钠与胶原比例为2∶1时，可利于细胞分化和增殖。.

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