Investigation on In Vitro Osteogenic Properties of Multiple-doped Hydroxyapatite with Natural Bone Content

doi:10.15541/jim20160134

Abstract

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

CLC Number:

Q297

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.

Figures/Tables 8

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′

Fig. 1 MC3T3-E1 cell line

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

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

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)

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)

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)

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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