骨髓间充质细胞在聚醚醚酮片和纯钛片表面的体外生物相容性比较

doi:10.12016/j.issn.2096-1456.2020.02.002

摘要/Abstract

摘要：

目的比较骨髓间充质细胞在聚醚醚酮（polyetheretherketone, PEEK）和纯钛（titanium, Ti）表面的体外生物相容性。方法制备厚度为1 mm,直径为10 mm的圆片状PEEK片和Ti片。将PEEK片和Ti片分别与SD乳鼠的骨髓间充质细胞在体外复合并定向成骨诱导培养,采用扫描电镜观察细胞黏附形态、Alamarblue检测细胞黏附与增殖能力、碱性磷酸酶（alkaline phosphatase,ALP）活性及茜素红染色的矿化结节半定量等方法检测成骨分化能力,对比骨髓间充质细胞在PEEK片和Ti片表面的细胞黏附、增殖及成骨分化的能力。结果 ①观察骨髓间充质细胞在PEEK片和Ti片表面培养1 h、4 h、24 h时的细胞形态无明显差异;② 骨髓间充质细胞接种于PEEK片和Ti片表面培养的1 h、3 h、1 d、3 d,PEEK组的活细胞数均高于Ti组（P < 0.05）;③在骨髓间充质细胞接种于PEEK片和Ti片表面并成骨诱导矿化培养后的7 d、14 d,PEEK组的细胞ALP活性均高于Ti组的细胞ALP活性（P < 0.05）;④成骨诱导的骨髓间充质细胞在PEEK片和Ti片表面接种培养后的7 d、14 d,经茜素红染色后半定量分析,PEEK组的矿化程度高于Ti组（P < 0.05）。结论 PEEK具有优于Ti的体外生物相容性,能更好地促进骨髓间充质细胞的黏附、增殖及成骨分化能力,有望成为牙种植体新材料。

关键词: 骨髓间充质细胞, 聚醚醚酮, 钛, 生物相容性, 牙种植体, 黏附, 增殖, 成骨分化, 碱性磷酸酶, 矿化结节

Abstract:

Objective To compare the in vitro biocompatibility of bone marrow mesenchymal cells on polyetheretherketone (PEEK) and titanium (Ti) surfaces. Methods PEEK and Ti foils with thicknesses of 1 mm and diameters of 10 mm were prepared. First, bone marrow mesenchymal cells were separated and purified by the whole bone marrow adherent culture method in vitro. Then, osteogenesis-induced bone marrow mesenchymal cells were cultivated on the surfaces of the PEEK and Ti foils. Scanning electron microscopy (SEM), the Alamar Blue test, an alkaline phosphatase (ALP) kit and Alizarin Red staining were used to analyze calcium nodules and compare the adhesion, proliferation and osteogenic differentiation ability of bone marrow mesenchymal cells on the surfaces of the PEEK and Ti foils. Results ① The morphology of the bone marrow mesenchymal cells cultured on the PEEK and Ti foils at 1 h, 4 h and 24 h showed no significant differences. ② In the 1 h, 3 h, 1 d and 3 d cultures of the bone marrow mesenchymal cells inoculated on the surfaces of the foils, the number of living cells in the PEEK group was greater than that in the Ti group (P < 0.05). ③ In the 7 d and 14 d osteogenesis-induced cultures of the inoculated bone marrow mesenchymal cells, the ALP activity of the PEEK group cells was significantly greater than that of the Ti group cells (P < 0.05). ④ Semiquantitative analysis after Alizarin Red staining showed that the mineralization degree of the bone marrow mesenchymal cells induced by osteoblasts was greater in the PEEK group than in the Ti group (P < 0.05). Conclusion PEEK has better in vitro biocompatibility than Ti and can better promote cell adhesion, proliferation and osteogenic differentiation compared with Ti, and so it is expected to become a new dental implant material.

Key words: bone marrow mesenchymal cells, polyetheretherketone, titanium, biocompatibility, dental implants, adhesion, proliferation, osteogenesis differentiation, alkaline phosphatase, mineralized nodule

中图分类号:

R78
R318

连克乾,张昕,周捷宇,廖燕芬,司姗姗. 骨髓间充质细胞在聚醚醚酮片和纯钛片表面的体外生物相容性比较[J]. 口腔疾病防治, 2020, 28(2): 73-78.

LIAN Keqian,ZHANG Xin,ZHOU Jieyu,LIAO Yanfen,SI Shanshan. Biocompatibility of bone marrow mesenchymal cells on polyetheretherketone and titanium surfaces in vitro[J]. Journal of Prevention and Treatment for Stomatological Diseases, 2020, 28(2): 73-78.

图/表 7

图1 倒置显微镜下骨髓间充质细胞形态

Figure 1 Morphology of the bone marrow mesenchymal cells observed by inverted microscopy a: fourth day of primary culture （50 ×）; b: fourth day of the third generation （100 ×）

图2 茜素红矿化结节染色

Figure 2 Alizarin red staining mineralization nodules a: seventh day of osteoinductive cultivation （100 ×）; b: fourteenth day of osteoinductive cultivation （100 ×）

图3 PEEK片和Ti片实物图

Figure 3 PEEK and Ti images a: PEEK foils with thicknesses of 1 mm and diameters of 10 mm; b: Ti foils with thicknesses of 1 mm and diameters of 10 mm

图4 扫描电镜下PEEK与Ti试件表面骨髓间充质细胞接种1 h、4 h、24 h的形态

Figure 4 SEM showing the morphology of bone marrow mesenchymal cells cultured on PEEK and Ti substrates after 1, 4 and 24 hours a: morphology of cells inoculated for 1 hour in the PEEK group （4 000 ×）; b: morphology of cells inoculated for 4 hours in the PEEK group （2 000 ×）; c: morphology of cells inoculated for 24 hours in the PEEK group （2 000 ×）; d: morphology of cells inoculated for 1 hour in the Ti group （4 000 ×）; e: morphology of cells inoculated for 4 hours in the Ti group （2 000 ×）; f: morphology of cells inoculated for 24 hours in the Ti group （2 000 ×）

表1 骨髓间充质细胞在各组试件表面培养的黏附与增殖情况

Table 1 The adhesion and proliferation of the bone marrow mesenchymal cells on all substrates $\bar{x}$± s

	OD value
Group	1 h	3 h	1 d	3 d
Group of PEEK	0.321 ± 0.010	0.323 ± 0.008	0.233 ± 0.020	0.182 ± 0.007
Group of Ti	0.285 ± 0.008	0.296 ± 0.012	0.220 ± 0.007	0.127 ± 0.011
t	5.354	4.018	3.690	9.071
P	0.001	0.004	0.015	< 0.001
95% confidence interval of uPEEK-uTi	（0.020,0.051）	（0.001,0.041）	（0.003,0.022）	（0.040,0.069）

表2 成骨诱导的骨髓间充质细胞在各组试件表面培养7 d、14 d后的碱性磷酸酶活性

Table 2 ALP activity of the osteogenesis induced bone marrow mesenchymal cells cultured on all substrates for 7 and 14 days $\bar{x}$± s,gprot

	ALP
Group	7 d	14 d
Group of PEEK	10.849 ± 0.949	7.050 ± 1.001
Group of Ti	4.888 ± 0.784	4.938 ± 0.712
t	10.831	4.385
P	<0.001	0.001
95% confidence interval of uPEEK-uTi	（4.691,7.230）	（1.062,3.061）

表3 成骨诱导的骨髓间充质细胞在各组试件表面培养7 d、14 d的矿化程度

Table 3 The mineralization of the osteogenesis induced bone marrow mesenchymal cells cultured on all substrates for 7 and 14 days $\bar{x}$± s

	OD value
Group	7 d	14 d
Group of PEEK	0.552 ± 0.181	1.034 ± 0.233
Group of Ti	0.296 ± 0.055	0.583 ± 0.080
t	3.026	4.100
P	0.031	0.010
95% confidence interval of uPEEK-uTi	（0.035,0.477）	（0.167,0.735）

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