仿骨成分多元掺杂羟基磷灰石体外成骨性能研究

doi:10.15541/jim20160134

摘要/Abstract

摘要：

羟基磷灰石因有良好的生物学活性被广泛应用于人工骨替代材料, 但纯羟基磷灰石存在的自身不足限制了其临床应用范围。人体硬组织中含有多种具有重要生化作用的微量元素, 本研究按照骨骼中微量元素含量对羟基磷灰石进行掺杂改性, 制备出一种新型仿骨成分多元掺杂纳米结构羟基磷灰石材料(HA₂)。通过碱性磷酸酶活性染色、半定量PCR测量和茜素红染色等不同方法对比了未掺杂纳米结构羟基磷灰石(HA₁)和多元掺杂纳米结构羟基磷灰石材料(HA₂)对小鼠成骨前体细胞(MC3T3-E1)增殖及分化的影响。碱性磷酸酶活性染色实验证明HA₂比HA₁具有更强的促进细胞产生ALP的作用; 半定量PCR测量证明HA₂比HA₁对成骨基因的表达有更为显著的促进作用; 茜素红对矿化结节的染色实验同样证明HA₂比HA₁更有利于成骨细胞发生矿化。相对于未掺杂纳米结构HA₁, 多元掺杂纳米结构HA₂对小鼠成骨前体细胞MC3T3-E1的分化及成骨有显著促进作用。多元掺杂改性使人工合成纳米结构羟基磷灰石更接近人体骨的无机成分, 具有优良的成骨性能, 是一种有前途的硬组织植入体材料。

关键词: 羟基磷灰石, 微量元素, 多元掺杂, 纳米结构, 成骨活性

Abstract:

Hydroxyapatite (HA) has been widely used as artificial bone substitutes because of its good biological activity, but the disadvantages of pure HA have limited its clinical applications. In human natural hard tissues, there are many trace elements doped in hydroxyapatite, which play beneficial roles in biological actions. In this study, multi-doped nanostructured hydroxyapatite (HA₂) was synthesized by doping trace elements in HA according to the contents in human bones. Alkaline phosphatase activity staining, semi-quantitative PCR measurement (ALP, OCN and OPN) and alizarin red staining were carried out to detect the effect of pure nanostructured hydroxyapatite (HA₁) and multi-doped nanostructured hydroxyapatite (HA₂) on proliferation and differentiation of MC3T3-E1cells. Alkaline phosphatase activity experiments prove that the HA₂ has a stronger role than HA₁ in promoting cell production. Semi quantitative PCR measurement confirms that HA₂ is more effective than HA₁ in promoting the expression of osteogenic genes. Alizarin red staining results show that HA₂ is better than HA₁ for MC3T3-E1 cells to produce mineralized nodules. Multiple-doped nanostructured hydroxyapatite (HA₂) has an obvious promoting effect on the differentiation and osteogenesis of MC3T3-E1 comparing with the undoped nanostructured HA₁. Multi-doped nanostructured hydroxyapatite (HA₂) is more beneficial to bone formation, which has the inorganic composition more close to human bone and is a promising biomaterial for human hard tissue implants.

Key words: hydroxyapatite, trace elements, multi-doping, nanostructure, osteogenic properties

中图分类号:

Q297

程瑶, 王铭, 王星星, 徐征丽, 汪大林, 祝迎春. 仿骨成分多元掺杂羟基磷灰石体外成骨性能研究[J]. 无机材料学报, 2016, 31(12): 1341-1346.

CHENG Yao, WANG Ming, WANG Xing-Xing, XU Zheng-Li, WANG Da-Lin, Zhu Ying-Chun. Investigation on In Vitro Osteogenic Properties of Multiple-doped Hydroxyapatite with Natural Bone Content[J]. Journal of Inorganic Materials, 2016, 31(12): 1341-1346.

图/表 8

表1 成骨基因相关引物

Table 1 Gene related primers

Gene	Sequence
ALP	F: 5′- AAGCTGGGAAGAACACTCCA-3′
ALP	R: 5′- CAAACAGGAGAGCCACTTCA-3′
OCN	F: 5′- AAGCAGGAGGGCAATAAGGT-3′
OCN	R: 5′- TTTGTAGGCGGTCTTCAAGC-3′
OPN	F: 5′- GCAGGCATTCTCGGAGGAAA-3′
OPN	R: 5′-TCTGAGCTGCCAGAATCAGTC-3′

图1 MC3T3-E1细胞株

Fig. 1 MC3T3-E1 cell line

图2 羟基磷灰石材料的SEM照片

Fig. 2 SEM images of hydroxyapatite materials^(a, b) HA1and (c,d) HA2. Inset in (c) is EDS of HA2, inset in (d) is the enlarge image of HA2

图3 碱性磷酸酶染色照片

Fig. 3 Light microscopic photoes of samples after alkaline phosphatase staining^(a) Control group; (b) Mineralization group; (c) HA1 group; (d) HA2 group

图4 ALP基因表达量培养时间变化图

Fig. 4 ALP genes expression with culture time^Ctr: control group; M, M+HA1, M+HA2: mineralizationgroups (*p<0.05; **p<0.005; ***p<0.0001)

图5 OCN基因表达量随培养时间变化图

Fig. 5 OCN genes expression with culture time^Ctr: control group; M, M+HA1, M+HA2: mineralizationgroups (*p<0.05; **p<0.005;***p<0.0001)

图6 OPN基因表达量随培养时间变化图

Fig. 6 OPN genes expression with culture time^Ctr: control group; M, M+HA1, M+HA2: mineralizationgroups. (*p<0.05; **p<0.005; ***p<0.0001)

图7 茜素红染色照片

Fig. 7 Light microscopic photoes of samples after Alizarin red staining^(a) Control group; (b) Mineralization group; (c) HA1 group; (d) HA2 group

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