| Periodontitis is a biofilm-induced chronic inflammatory disease that destroys periodontal supporting tissue.It has attracted profound attention in the arena of periodontal regeneration of severe periodontitis by guided tissue regeneration（GTR）,especially for guided bone regeneration（GBR）of the bone defect after tooth extraction.With the progress of engineering technology,custom-made titanium alloy mesh fabricated by selective laser melting（SLM）,one of 3D printing techniques,has the potential to be applied to GBR and GTR therapy to achieve ideal tissue regeneration.ObjectiveCopper-bearing titanium alloy was fabricated by SLM and co-cultured with macrophage cell line RAW264.7,osteoblast cell line MC3T3-E1,osteoporotic macrophages and osteoblasts to explore its influences on biological behaviors of cells above.Methods1.Copper-bearing titanium alloy Ti6Al4V-Cu was fabricated by SLM,and its material properties were evaluated by SEM,XRD,EDS,ICP and XPS.2.To co-culture Ti6Al4V-Cu with macrophage cell line RAW264.7 and assess its effects of cell proliferation,attachment,cell polarization and expression of inflammatory cytokines.Meanwhile,RANKL was adding to induce osteoclastogenesis of RAW264.7 cells.Morphology of osteoclasts was observed by fluorescence microscopy.Osteoclastogenic gene expression was evaluated by real-time PCR.3.MC3T3-E1 cells were co-culture with Ti6Al4V-Cu and its proliferation,attachment,ALP activity and osteoblastic gene expression were evaluated.4.The OP model of SD rats were established by bilateral ovariectomy and fed for 12 weeks.Osteoporotic macrophages and osteoblasts were separated and cultured from the rats and co-cultured with Ti6Al4V-Cu.5.Attachment and cell polarization of oteoporotic macrophages on Ti6Al4V-Cu were observed by fluorescence microscopy.Inflammatory cytokines and polarization markers were evaluated by real-time PCR,ELISA,western blot and immunofluorescent staining.6.Osteoporotic macrophages on Ti6Al4V-Cu were induced to osteoclastogenesis by addition of RANKL.The expression of osteoclastogenic genes and proteins was assess by real-time PCR,western blot and immunofluorescent staining.7.Attachment and proliferation of osteoporotic osteoblasts on Ti6Al4V-Cu was observed.Osteogenic gene expression was assessed.Osteoblastic extracellular matrix genes and proteins（Col-1α1and LOX）were evaluated by real-time PCR,western blot and immunofluorescent staining.Results1.Copper ions released from SLMed Ti6Al4V-Cu.The diffraction peaks of the Ti2Cu intermetallic phase were observed in the Ti6Al4V-Cu.Copper on the surface of Ti6Al4V-Cu may exist predominantly in Cu2O or TiCuOx.2.Ti6Al4V-Cu inhibited the activation,proliferation,and pro-inflammatory cytokine secretion of macrophages,promoted anti-inflammatory cytokine expression,down-regulated M1 macrophage marker expression and up-regulated M2 macrophage marker expression.3.Ti6Al4V-Cu inhibited the formation of osteoclasts and osteoclastogenic gene expression.4.Ti6Al4V-Cu did not alter attachment,proliferation and osteogenesis of MC3T3-E1 cells.5.Ti6Al4V-Cu inhibited proliferation and attachment of osteoporotic macrophages,down-regulated pro-inflammatory cytokines and M1 macrophage marker expression,and up-regulated anti-inflammatory cytokines and M2 macrophage marker expression.6.Ti6Al4V-Cu inhibited osteoporotic macrophages osteotogenic differentiation.7.There was not significant difference of osteoporotic osteoblasts on Ti6Al4V-Cu and Ti6Al4V,but the expression of Col-1α1 and LOX was up-regulated.Conclusions1.Copper-bearing titanium alloy Ti6Al4V-Cu was successfully fabricated by SLM.2.Ti6Al4V-Cu inhibited inflammatory response and osteoclastogenesis of macrophages,and did not alter osteogenic differentiation of osteoblasts,but promoted extracellular matrix formation.3.Ti6Al4V-Cu has the potential to apply to fabricate custom-made titanium alloy mesh and GTR/GBR treatment of periodontits and periodontits patients with osteoporosis. |
