钽涂层对hPDLSCs增殖及成骨分化的影响

doi:10.13591/j.cnki.kqyx.2023.05.005

摘要/Abstract

摘要：

目的探究钽涂层表面对人牙周膜干细胞(human periodontal ligament stem cells,hPDLSCs)增殖及成骨分化的影响。方法对hPDLSCs进行分离、培养和细胞鉴定。纯钛试件抛光清洗后,经喷砂、酸蚀处理,以等离子喷涂技术制备钽涂层。以抛光钛表面(P组)为对照组,喷砂酸蚀钛(SLA组)、钽涂层钛(Ta组)为实验组。通过扫描电镜、能谱仪分析各组钛表面的微形貌、元素组成;将hPDLSCs接种于各组试件表面,通过CCK-8法测定细胞的增殖情况,经成骨诱导培养后行碱性磷酸酶活性检测和茜素红染色,qPCR检测成骨基因表达。结果在钛试件上成功制备了钽涂层;成功分离培养并鉴定hPDLSCs;CCK-8实验结果显示,在培养3、5、7 d后,Ta组OD值明显高于SLA组及P组(P<0.05);ALP检测结果显示,在第7天时,三组间的差距无统计学意义;第14天时,Ta组ALP活性明显高于P组和SLA组(P<0.01)。茜素红染色显示,三组材料表面都有红色钙化结节形成,SLA组和Ta组的矿化结节明显多于P组。qPCR结果显示Ta组表面细胞ALP、RUNX2、OCN基因表达水平显著高于P组(P<0.01),RUNX2、OCN基因表达显著高于SLA组(P<0.01)。结论 SLA钛表面钽涂层对hPDLSCs的增殖、成骨分化具有促进作用。

关键词: 钽涂层, 人牙周膜干细胞, 成骨分化

Abstract:

Objective To investigate the effect of tantalum-coated surface on the proliferation and ossification of human periodontal ligament stem cells(hPDLSCs). Methods The hPDLSCs were separated and cultured, with the cell identified. After polishing and cleaning of pure titanium specimens, the tantalum coating was prepared by plasma spraying technique after sandblasting and acid etching treatment. The polished titanium specimens(P group)were used as control group, and sandblasted acid-etched titanium(SLA group)and tantalum-coated titanium(Ta group)were used as test groups. The micro-morphology and elemental composition of the titanium surface of each group were analyzed by scanning electron microscopy(SEM)and energy spectrometry(EDS). The hPDLSCs were inhibited on the surface of each group of specimens, and the proliferation activity of cells was measured by CCK-8 assay. After osteogenesis induction culture, alkaline phosphatase activity assay and alizarin red staining were performed. Osteogenic gene expression was detected by qPCR. Results Tantalum coating was successfully prepared on titanium specimens. The hPDLSCs were successfully isolated and identified. The results of CCK-8 experiment showed that after 3, 5 and 7 days of culture, the OD value of Ta group was significantly higher than that of SLA and P groups(P<0.05). The results of ALP assay showed that the difference between the three groups was not statistically significant at day 7;at day 14, the ALP activity in the Ta group was significantly higher than that in the P and SLA groups(P<0.01). Alizarin red staining showed that red calcified nodules were formed on the superficial side of the materials in all three groups, and the mineralized nodules in the SLA and Ta groups were significantly more than those in the P group. The qPCR data indicated that the expression levels of ALP, RUNX2, and OCN genes were significantly higher in the Ta group than in the P group(P<0.01), and the expression of RUNX2 and OCN genes were effectively higher than that in the SLA group(P<0.01). Conclusion Tantalum coating can promote the proliferation and osteogenic differentiation of periodontal stem cells.

Key words: tantalum coating, human periodontal ligament stem cells, osteogenic differentiation

中图分类号:

R783.1

葛潇,于淼,武伟,毕秀婷,吴小燕,于晨,李倜. 钽涂层对hPDLSCs增殖及成骨分化的影响[J]. 口腔医学, 2023, 43(5): 415-420.

GE Xiao,YU Miao,WU Wei,BI Xiuting,WU Xiaoyan,YU Chen,LI Ti. The effect of tantalum coating on the proliferation and osteogenic differentiation of human periodontal ligament stem cells[J]. Stomatology, 2023, 43(5): 415-420.

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基因名称	上游引物(5'—3')	下游引物(5'—3')
ALP	GGCGGTGAACGAGAGAATGT	GGACGTAGTTCTGCTCGTGG
OCN	TCACACTCCTCGCCCTATTG	CTCTTCACTACCTCGCTGCC
RUNX2	GGAGTGGACGAGGCAAGAGT	AGGCGGTCAGAGAACAAACT
GADPH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGGA

分组	1 d	3 d	5 d	7 d
P组	0.15±0.01	0.25±0.07	0.35±0.06	1.02±0.04
SLA组	0.15±0.00	0.27±0.06	0.40±0.03^#	1.28±0.08^#
Ta组	0.16±0.00	0.39±0.01^*#	0.64±0.05^*#	1.70±0.21^*#

分组	7 d	14 d
P组	7.80±2.71	51.58±0.72
SLA组	9.90±5.43	53.87±7.59
Ta组	15.08±6.19	73.58±5.20^**##

组别	ALP	RUNX2	OCN
P组	1.00±0.24	1.00±0.09	1.00±0.15
SLA组	1.47±0.17^*	1.30±0.10^*	1.49±0.10^**
Ta组	1.86±0.11^**	2.44±0.30^**##	1.88±0.16^**##