| ObjectiveTo explore the effects of human tooth bone graft materials prepared from autologous human fresh teeth on the proliferation,differentiation and inflammatory response of macrophages,and to investigate the potential biocompatibility and cytotoxicity of human tooth bone graft materials in vitro.MethodsFresh human teeth were collected and treated with reagents to prepare human tooth bone graft materials,while untreated dental particles were obtained without reagents and artificial bone implants,a commonly used bone material in clinics,was also prepared.The surface morphology of the three different materials was observed by scanning electron microscopy.The materials were co-cultured with cells for 24 hours.The materials were stained with Hoechst 33342 and Rhodamine B cells under single photon confocal microscopy in order to observe the morphological and quantitative changes of cells and determine cellular cytotoxicity.The extracts of these three materials were prepared according to ISO 10993-12 standard and divided into four groups: Group A consisted of 10% serum culture solution(blank control group),group B consisted of reagent-treated tooth powder granules(human tooth bone graft materials group);group C consisted of clinically used bone materials(artificial bone implants),and group D consisted of untreated tooth powder granules(untreated tooth powder group)which acts as a positive control group.MTT method was used to detect the OD values of cells in the four groups with 1,3,5,and 7days;The cell viability of each group was measured by Trypanblue staining,and the effect of extract of each material on cell growth curve was compared in 1-6 days.Flow cytometry was used to detect the effects of four extracts on apoptosis of RAW264.7 monocytes and macrophages in mice.Western blot and ELISA have been used to detect the expression of inflammatory related genes TNF-ɑand IL-6 protein quantitatively from each group of materials on the 1st,3rd and 5th day respectively.ResultsIt has been shown by scanning electron microscopy that the surface of human tooth bone graft materials had uniform porous morphology,with an effective protein carrier and scaffold structure,while the structure of collagen fibers in the group of untreated tooth powder and the demineralized dentin layer had collapsed without specific structure.The results showed that mouse mononuclear macrophages have normal cell morphology and have no difference quantity on human tooth bone graft materials and artificial bone implants(P>0.05);.The cell viability of MTT assay showed that the light absorption value(A492nm)of group B and C was higher than that of group D(P<0.05),but they were same as group A(P>0.05).Trypan blue staining showed that the number of cells in group A,B and C was higher than that in group D at each point(P<0.05).Flow cytometry showed that the apoptotic rates of groups A,B and C were lower than those of group D(P<0.05).The results of western blot and ELISA showed that the TNF-α and IL-6 released by macrophages in groups A,B and C were significantly lower than those in group D(untreated tooth powder group)(P<0.05).ConclusionsHuman tooth bone graft materials prepared from fresh human teeth in vitro can grow together with RAW264.7 cells of mouse monocyte macrophages.There was no significant difference in the extracts of artificial bone implants on the proliferation and differentiation of RAW264.7 cells.They have no effect on cell apoptosis,no obvious cytotoxicity,and could not increase the release of TNF-ɑand IL-6 on monocyte macrophages.Human tooth bone graft materials are identical to artificial bone implants in terms of cytotoxicity.It has good biocompatibility in vitro and is expected to be used for clinical bone defect repair. |
PDF Full Text Request