The Related Research Of In Vitro Cytotoxicity Of Multi-doped Nano Hydroxyapatite

[Background] Hydroxyapatite has the similar chemical composition and crystal structure of human bone and teeth minerals,and is used as the hard tissue implant material.Hydroxyapatite has many advantages and good prospects,but also some shortcomings such as less biological active and resistant to degradation.Thus its clinical application is limited.It is founded that human bone apatite contains large amounts of trace elements besides calcium and phosphorus.Multi-element doped nano hydroxyapatite powders can be synthesized by hydrothermal method.But it is unclear whether the toxicity of nano hydroxyapatite powders will be affected by multi-Doping.And it will be launched by this study.[Objective] To provide certain theoretical basis for continue exploration of the clinical application and further study of preparation and biological effect of multi-doped nano hydroxyapatite coating of hard tissue implant following researches were carried out.The in vitro cytotoxicity and related mechanism of multi-doped nano hydroxyapatite and the extract of multi-doped nano hydroxyapatite were studied.Preliminarily study the effect of multi-doped nano hydroxyapatite on dental pulp cells.[Materials and methods] The first part: in vitro cytotoxicity and related mechanism of multi-doped nano hydroxyapatite.1.CCK-8 colorimetric method assay the impact on L929 cell proliferation of multi-doped nano hydroxyapatite;2.Flow cytometry assay apoptosis by Annexin V-PI double staining,fluorescent microscope assay apoptosis by Hochest33342-PI double staining;3.Fluorescent microscope and flow cytometry assay ROS by fluorescent probe technique;4.Flow cytometry assay the change of mitochondrial membrane surface potential by JC-1 fluorescent probe labeling;5.Western Blot analysis the related protein.The second part: in vitro cytotoxicity and related mechanism of the extract of multi-doped nano hydroxyapatite.1.Preparation of the extract of multi-doped nano hydroxyapatite by complete culture medium;2.CCK-8 colorimetric method assay the impact on L929 cell proliferation of the extract of multi-doped nano hydroxyapatite;3.Flow cytometry assay apoptosis by Annexin V-PI double staining;4.Western Blot analysis the related protein The third part: effect of multi-doped nano hydroxyapatite on dental pulp cells.1.Primary culture of the human dental pulp cells;2.CCK-8 colorimetric method assay the impact on human dental pulp cells of multi-doped nano hydroxyapatite.[Results] The first part: in vitro cytotoxicity and related mechanism of multi-doped nano hydroxyapatite.Both of nano hydroxyapatite and multi-doped nano hydroxyapatite can inhibit the proliferation of L929 cell,promote apoptosis,induce the production of ROS and changes in mitochondrial membrane potential,at the same time promote the expression of p53 protein and stimulate the activation of Caspase 3.The ratio of apoptosis is higher than nano hydroxyapatite,and also is the level of ROS and ratio of cells which the mitochondrial membrane potential changes.Although the effect of inhibition of proliferation is more obvious,the difference is not significant.The second part: in vitro cytotoxicity and related mechanism of the extract of multi-doped nano hydroxyapatite.Both of the extract of nano hydroxyapatite and multi-doped nano hydroxyapatite can inhibit the proliferation of L929 cell,promote apoptosis,induce expression of p53 protein and stimulate the activation of Caspase 3.But there is no obvious difference between them.The third part: effect of multi-doped nano hydroxyapatite on dental pulp cells.All of the nano hydroxyapatite,multi-doped nano hydroxyapatite and the direct pulp capping material calcium hydroxide can not inhibit the proliferation of human dental pulp cells significantly.The difference is not significant when compare with the control group,and there is no obvious difference between nano hydroxyapatite and multi-doped nano hydroxyapatite.[Conclusions] 1.Both of nano hydroxyapatite and multi-doped nano hydroxyapatite can induce expression of p53 protein by oxidative stress and promote cells apoptosis by a mitochondrial dependent pathway;Multi-doping do not obviously promote the cytotoxicity of nano hydroxyapatite.2.Both of the extract of nano hydroxyapatite and multi-doped nano hydroxyapatite have cytotoxicity.And multi-doping do not obviously promote the cytotoxicity of nano hydroxyapatite extract.3.Both of nano hydroxyapatite and multi-doped nano hydroxyapatite can not significantly affect the proliferation of human dental pulp cells.And multi-doping do not obviously promote the inhibition of nano hydroxyapatite on the proliferation of human dental pulp cells.