明胶-羟基磷灰石-磷酸三钙支架对hDPSCs的成骨刺激作用

doi:10.19439/j.sjos.2020.05.009

摘要/Abstract

摘要： 目的：探讨明胶-羟基磷灰石-磷酸三钙支架（明胶-HA-TCP支架）对人牙髓干细胞（human dental pulp stem cells,hDPSCs）的成骨刺激作用。方法：通过溶剂浇铸/粒子沥滤法制备明胶支架及明胶-HA-TCP支架,利用场发射扫描电镜观察支架微观特征,立式电子万能试验机检测支架应力强度（stress intensity,SS）及弹性模量（E）,制备成骨培养基,根据介质分为对照组（单纯成骨培养基）、实验1组（明胶支架提取物+成骨培养基）和实验2组（明胶-HA-TCP支架+成骨培养基）,采用CCK8法评估支架对hDPSCs增殖的影响,测定碱性磷酸酶（alkaline phosphatase,ALP）活性并进行ALP染色,通过茜素红染色和反转录-聚合酶链反(RT-PCR)应检测hDPSCs中成骨因子的表达。采用SPSS 20.0软件包对数据进行统计学分析。结果：与明胶支架相比,明胶-HA-TCP支架SS显著提高,E显著降低（P<0.05）;CD146和CD105在hDPSCs呈阳性表达,CD34和CD45在hDPSCs呈阴性表达;实验1组3、5和7 d细胞数量显著多于对照组,实验2组1、3、5和7 d细胞数量显著多于对照组（P<0.05）;实验2组4、7和12 d ALP活性显著大于对照组及实验1组（P<0.05）;与对照组相比,实验1组有少量红色视野,钙化沉淀量较少且分布不均匀,实验2组大部分视野呈红色,钙化沉淀量较多且分布均匀;实验1组与实验2组第4天Runt相关转录因子2（runt related transcription factor 2,Runx2）mRNA表达水平显著高于对照组（P<0.05）;实验2组第7天成骨相关转录因子（osterix,OSX）mRNA表达水平显著高于对照组（P<0.05）;实验2组第4天骨涎蛋白（bone sialoprotein,BSP）mRNA表达水平显著高于对照组和实验1组（P<0.05）。结论：明胶-HA-TCP支架的微观表征与天然骨结构相似,具有良好的抗压性,不易变形,对hDPSCs增殖和分化具有促进作用,并具有显著的骨诱导性,可作为骨组织工程新型复合材料。

关键词: 溶剂浇铸/粒子沥滤法, 明胶支架, 明胶-羟基磷灰石-磷酸三钙支架, 人牙髓干细胞, 成骨分化

Abstract: PURPOSE: To investigate the osteogenic stimulation effect of gelatin-hydroxyapatite-tricalcium phosphate (gelatin-HA-TCP) scaffold on human dental pulp stem cells(hDPSCs). METHODS: Gelatin scaffold and gelatin-HA-TCP scaffold were developed via solution casting/particle washing method, and the physical and mechanical properties of the scaffolds were examined using scanning electron microscopy and a universal testing machine, respectively. The stress intensity(SS) and elastic modulus(E) of the scaffold were measured by vertical electronic universal testing machine. The osteogenic medium was divided into control group (osteogenic medium), experimental group 1 (gelatin scaffold extract + osteogenic medium) and experimental group 2(gelatin-HA-TCP scaffold + osteogenic medium) according to different mediums. The effects of scaffolds on proliferation and alkaline phosphatase(ALP) activity of hDPSCs were evaluated by CCK8 and ALP staining. The expression of osteogenic factors in hDPSCs was detected by alizarin red S staining and reverse transcription-polymerase chain reaction(RT-PCR). Data were processed by SPSS 20.0 software package. RESULTS: Compared with gelatin scaffold, gelatin-HA-TCP scaffold had higher SS and lower E(P<0.05); CD146 and CD105 were positively expressed in hDPSCs, and CD34 and CD45 were negatively expressed in hDPSCs. The number of cells on the 3rd, 5th and 7th day of experimental group 1 was significantly higher than that of the control group (P<0.05); The number of cells on the 1st, 3rd, 5th and 7th day of experimental group 2 was significantly higher than that of the control group (P<0.05); ALP activities on the 4th, 7th and 12th day of experimental group 2 were significantly higher than those of the control group and experimental group 1(P<0.05). Compared with the control group, visual fields were red in both experimental groups, and the unevenly distributed calcified precipitation was also found in both experimental groups. The mRNA expression level of Runt-related transcription factor 2(Runx2) on the 4th day of experimental group 1 and experimental group 2 was significantly higher than that of the control group(P<0.05). The mRNA expression level of osterix (OSX) on 7th day of experimental group 2 was significantly higher than that of the control group(P<0.05). The mRNA expression level of bone sialoprotein (BSP) on the 4th day of experimental group 2 was significantly higher than that of the control group and experimental group 1 (P<0.05). CONCLUSIONS: The microscopic characterization of gelatin-HA-TCP scaffold is similar to that of natural bone structure with significant osteoinductive effect on the proliferation and differentiation of hDPSCs, and may be a promising biomaterial for bone tissue engineering.

Key words: Solvent casting and particulate leaching, Gelatin scaffold, Gelatin-hydroxyapatite-tricalcium phosphate scaffold, Human dental pulp stem cells, Osteogenic differentiation

中图分类号:

R783.1

常思佳, 杨柳, 李冉. 明胶-羟基磷灰石-磷酸三钙支架对hDPSCs的成骨刺激作用[J]. 上海口腔医学, 2020, 29(5): 492-498.

CHANG Si-jia, YANG Liu, LI Ran. Osteogenic stimulation of human dental pulp stem cells with gelatin-hydroxyapatite-tricalcium phosphate scaffold[J]. Shanghai Journal of Stomatology, 2020, 29(5): 492-498.

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