Investigation On In Vitro Osteogenic Properties Of Multiple-doped Hydroxyapatite With Natural Bone Content

Bone defect is a common difficult problem in clinical work,which greatly affects the patient’s appearance and mental health.Current clinical treatment of bone defect mainly adopt the method of implanted bone substitutes,namely in implanted with autogenous bone,allograft bone defect site,a sheep to repair bone defects.But as a result of these methods have some deficiencies,for example: autologous bone graft source has limited sources,intraoperative blood loss,postoperative pain,etc.Allograft transplantation immune rejection and the risk of disease transmission.With the constant improvement of the clinical requirements for bone replacement materials,developed has good biocompatibility,biodegradability,bone conductibility of synthetic bone defect repair material is gradually transformed into a hot spot in the field of treatment of bone defect.Hydroxyapatite（hydroxyapatite,HA）with inorganic structure similar to that of natural bone and has good biocompatibility,bone induction,make it become the most widely applied in clinical work of one of the synthetic bone defect repair material.However pure hydroxyapatite materials with refractory,bone replacement rate is low,poor mechanical strength weakness,within a certain range restriction on its clinical application.The study found that the natural bone inorganic composition of HA,is not completely in accordance with the standard of chemometrics to Ca₁₀（PO₄）₆（OH）₂,but be replaced by different anion and cation exchange.The study found that the natural bone inorganic composition contains a variety of different trace elements: magnesium,sodium,potassium,strontium,silicon,fluorine and carbon,the lack of these trace elements can increase the risk of fracture.At present on the basis of hydroxyapatite add one or more of the following elements to the method of modified hydroxyapatite get more extensive research,but for multivariate mixed simulation of the bionic bone composition both at home and abroad research of nanometer hydroxyapatite is still in the blank.Chinese Academy of Sciences,Shanghai Silicate Research the according to the content of trace elements（Na,K,Mg,Al,zinc,Sr,Si,F,C）in human bone,a variety of elements doped hydroxyapatite lattice internal,to manufacture by imitating human bone composition of multiple doping nano hydroxyapatite.Research purposes: This experiment was prepared in the same way the doping nano hydroxyapatite（HA₁）as reference,comparative study biomimetic multiple doping nanohydroxyapatite（HA₂）on osteogenesis precursor cells osteogenesis and the influence of the differentiation of osteogenesis.Methods: The first part of the experiment: The hydrothermal preparation of multivariate mixed hydroxyapatite powders and hydroxyapatite powders,electron microscope particle size form.Experiments in the second part: the preparation of different concentrations of multiple doping nano hydroxyapatite suspension and measure its impact on the distribution of precursor cells.Experiment in the third part: under the action of mineralized induced liquid compared to pure nanometer hydroxyapatite with multiple doping nano hydroxyapatite group of precursor cells for osteogenesis osteogenesis.Experiment the fourth part: after removal of mineralized induced liquid effect,study whether two different whether nanomaterials for osteogenesis precursor cells also have the function of the mineralized induced,and than the strength of the effect.Results: 1:Through the electron microscope scanning pure nanometer hydroxyapatite（HA₁）and multiple doping nano hydroxyapatite（HA₂）,found that HA₁ and HA₂ samples with hierarchical structures,doping and caused no significant HA morphology changes.2:（1）When multiple doping hydroxyapatite powders（HA₂）concentration in the 0-0.05mg/ml concentration range,HA₂ proliferation of MC3T3-E1 have a certain role in promoting,when HA₂ concentration higher than 0.05 mg/ml,HA₂ to MC3T3 E1 will produce certain inhibitory effect.（2）Through hochest33342 / PI double dyed further found that when the concentration of the medium inside HA₂ is 0.5 mg/ml,0.125 mg/ml will be to cause a certain degree of cell apoptosis,and apoptosis of the intensity of the staining was positively than with powder concentration and processing time.When the powder concentration to 0.05 mg/ml treatment group apoptosis phenomenon is not obvious.3,（1）Through the activity of alkaline phosphatase staining observation found that mineralization group,mineralized + HA₁ group,the mineralization + HA₂ group both can make the ALP activity of osteogenesis precursor cells,and the blank control group cannot make osteogenesis precursor cells mineralization.Mineralization + HA₁ group,mineralization +HA₂ than pure mineralized induced group is easier to promote cells produce ALP.Mineralization + HA₂ than mineralization + HA₁ easier to promote cells produce ALP.（2）The semi-quantitative PCR measurement,the author found out that the mineralization +HA₁ and mineralization + HA₂ group can promote osteogenesis related genes ALP,OCN and high expression of OPN.ALP levels of genes in the mineralized + HA₁ and mineralization + HA₂ group 3 days after the treatment had significant difference（p <0.005）,the mineralization + HA₂ group of ALP and promoting effect of the genetic level was obviously higher than that of mineralized + HA₁ group.The extension of incubation time as,increased difference between groups（p < 0.0001）.ALP levels peaked in mineralized induced by the 10 th day,fall in 14 days.OCN gene levels between groups there was no significant difference between the early stages of training,from handling the5 day begin to produce differences between groups（p < 0.05）,the mineralization + HA₂ group on the promoting effect of OCN gene level was obviously higher than that of mineralization + HA₁ group,and with the extension of incubation time,the increasing difference between the groups.OPN in dealing with the third day,mineralization + HA₁ and mineralization + HA₂ group no significant difference,but the two groups of OPN gene expression level was significantly higher mineralization group and the blank control group.Mineralization + HA₁ and mineralization + HA₂ group from 5 days difference between the groups began to reflect（p < 0.05）,the mineralization + HA₂ groups more prone to promote gene expression of OPN.（3）Dye alizarin red found: all treatment groups in 15 days you can see some mineralized small particles to form,continue to develop to 21 days is a typical mineralization nodules,red dye patches nodules on behalf of the effect of mineralized induced osteogenesis.Study found that does not make simple medium MC3T3E1 mineralization osteogenesis.But mineralized induced liquid group,the mineralization +HA₁ group,mineralization + HA₂ group can produce mineralized nodules.How much of the mineralized nodules and staining intensity relationship is: the mineralized + HA₂ group > mineralization + HA₁ group > mineralized induced group.Conclusion: 1 Silicate research institute of Chinese academy of sciences according to the ratio of the content of trace elements in human body skeleton was multivariate doping nano hydroxyapatite,observation found that doping elements did not cause the obvious change of the nano powder size and size,the preparation of material particle size and the same method preparation of pure nanometer hydroxyapatite is similar.2 The most suitable for the multivariate mixed hydroxyapatite powders for osteogenesis progenitor cell biology effect of solution concentration is 0.05 mg/ml.3 Simple medium can’t mineralization in the osteogenesis precursor cells,in the presence of mineralized induced liquid,HA₁ and HA₂ to mineralization osteogenesis effect in the osteogenesis precursor cells,and the mineralization + HA₂ than mineralization + HA₁ group promotes stronger for osteogenesis precursor cells.4 After removal of mineralized induced liquid effect,still can promote osteogenesis HA₁ and HA₂ precursor cells differentiation,two kinds of powders of werestronger than the mineralized induced liquid group,and pure HA₂ group precursor cells for osteogenesis of mineralized osteogenesis effect is stronger than HA₁ group.This study reveals the new bionic bone material has good biocompatibility and promote the performance of the osteogenetic differentiation,is a kind of promising hard tissue implant materials.