Osteogenic differentiation of pluripotent stem cells induced by akermanite extracts

doi:10.3969/j.issn.2095-4344.2015.14.018

Abstract

Abstract:

BACKGROUND: Akermanite belongs to silicate bioactive ceramics, with degradation in vivo, and its ionic products can induce osteogenic differentiation, which is a good choice for bone tissue engineering scaffold.

OBJECTIVE: By studying the different concentrations of akermanite extracts on the proliferation and osteogenic differentiation of pluripotent stem cells, to determine the optimum concentration of akermanite extract to inducing the osteogenic differentiation of pluripotent stem cells.

METHODS: MTT assay was used to detect the proliferation of pluripotent stem cells cultured in different concentrations of akermanite extracts (1/2, 1/4, 1/8, 1/16, 1/32). After cultured with the extracts for 7, 14, 21 days, the culture supernatants were collected to detect the levels of alkaline phosphatase, osteocalcin, type I collagen.

RESULTS AND CONCLUSION: The MTT assay showed that the proliferation of pluripotent stem cells was increased in a concentration-dependent manner after induction with akermanite extracts. The pluripotent stem cells proliferated obviously at 3 days after induction, and then weaken at 7 days, but there was no difference at 3 and 5 days after induction. At 7 days after induction, the 1/4 extract had the best effect on promoting osteogenic differentiation of pluripotent stem cells; the levels of alkaline phosphatase and osteocalcin were increased with time, especially after induction with the 1/4 extract; but there was no expression of type I collagen. At 14 and 21 days after induction, the levels of alkaline phosphatase, osteocalcin, type I collagen were highest in the 1/4 extract group. These findings indicate that the 1/4 akermanite extract (Ca 2.37 mmol/L, Mg 1.12 mmol/L, Si 1.05 mmol/L) is the optimum to promote the osteogenic differentiation of pluripotent stem cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Silicates, Induced Pluripotent Stem Cells, Cell Differentiation, Alkaline Phosphatase, Osteocalcin, Collagen Type I

CLC Number:

R394.2

Du Qing-hua, Cao Jun-kai, Dong Xi-xi, E Ling-ling, Wei Li-jun. Osteogenic differentiation of pluripotent stem cells induced by akermanite extracts[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(14): 2236-2242.

Figures/Tables 2

2.1 浸提液对诱导性多潜能干细胞增殖的影响浸提液对诱导性多潜能干细胞的增殖具有时间依赖性。MTT结果显示，细胞接种后第1天，各组细胞均有增殖，各浸提液组和矿化液组的细胞增殖明显强于阴性对照组(P < 0.05)，在各浸提液组中，1/2和1/4浓度浸提液之间差异无显著性意义(P > 0.05)，二者促进细胞增殖的作用明显强于1/8、1/16、1/32浓度(P < 0.05)。第3天时，各组细胞均明显增殖，各浸提液组增殖明显，与第1天相比差异有显著性意义(P < 0.01)，其中1/4浓度对细胞增殖作用最强。第5天时，各组与第3天相比差异无显著性意义(P > 0.05)，仍然是1/4浓度增殖最强。第7天时，各组较第3天和第5天时细胞增殖明显减弱，1/4浓度组对细胞增殖作用仍然最强，见图1。 2.2 浸提液对诱导性多潜能干细胞成骨过程中碱性磷酸酶、骨钙素、Ⅰ型胶原蛋白的影响 2.2.1 浸提液对诱导性多潜能干细胞成骨过程中碱性磷酸酶活性的影响浸提液呈时间依赖性提高诱导性多潜能干细胞成骨过程中碱性磷酸酶的活性。各浓度浸提液在促进诱导性多潜能干细胞成骨过程中，碱性磷酸酶的活性随作用时间逐渐增强，第21天时最强。在实验组中，1/4浓度时促进诱导性多潜能干细胞成骨作用最强，与阴性对照组和矿化液组比较，3个时间段差异均有显著性意义(P < 0.01)，见图2。 2.2.2 浸提液对诱导性多潜能干细胞成骨过程中骨钙素的影响在促进诱导性多潜能干细胞成骨过程中，随作用时间的增加，各实验组骨钙素的质量浓度也逐渐增加，第21天时各组均达到最大值，其中矿化液组作用最强，与其他组比较差异有显著性意义(P < 0.01)。在各浸提液组中，1/4浓度组作用最强，第7天和14天时，1/4浓度组与其他各组比较差异有显著性意义(P < 0.01)；第21天时，1/4浓度组虽然高于1/2浓度组，但二者间差异无显著性意义，见图3。 2.2.3 浸提液对诱导性多潜能干细胞成骨过程中Ⅰ型胶原蛋白的影响细胞培养至第7天时，各实验组培养液中均未检出Ⅰ型胶原蛋白。至第14天和第21天时，除阴性对照组外，其他培养液中检测出Ⅰ型胶原蛋白，第21天Ⅰ型胶原蛋白水平高于第14天。矿化液组明显高于各浸提液组 (P < 0.01)；在各浓度浸提液组中，1/4浓度组高于其他浓度组(P < 0.05)，见图4。 2.3 诱导性多潜能干细胞形态及成骨诱导第21天各组细胞碱性磷酸酶和茜素红染色结果见图5-7。培养于饲养层上的诱导性多潜能干细胞呈圆形或椭圆形细胞簇。体外培养第21天，各组细胞碱性磷酸酶染色及茜素红染色均为阳性，矿化液组明显强于阴性对照组。不同浓度浸提液对诱导性多潜能干细胞的成骨分化均有诱导作用，在1/4浓度时诱导作用最强。 "

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