Effect of the porous Ti-6Al-4V bio-metallic implants on cytoactive and osteo-genic properties of bone marrow mesenchymal stem cells
Objective The porous(Ti-6Al-4V,TC4)titanium alloy scaffold was employed to investigate the cell viability,proliferation and osteogenic differentiation behavior of SD rat bone marrow mesenchymal stem cells(rBMSCs).Methods Primary rBMSCs were cultured using the whole bone marrow adherent method.Flow cy-tometry was employed to identify the phenotype of rBMSCs.TC4 titanium scaffolds were 9,16 and 64 round-like wells and 25 and 64 square-like wells,fabricated by selective laser melting technology(SLM).rBMSCs were seeded into TC4 wells as the experimental groups and TC4 titanium scaffolds without wells was set as the control group.Cell morphology and adherence of rBMSCs cultured for 48 h were observed by scanning electron microsco-py(SEM).Cell viability was measured by CCK-8 kit.Alkaline phosphatase(ALP)activity in 7 d and 14 d was measured by enzyme-linked immunosorbent assay.The expression of osteogenic genes including bone connexin(Osteonectin)and collagen type Ⅰ(Col1a1)were examined by quantitative real-time fluorescence PCR(qRT-PCR).The rBMSCs osteogenic ability in 21 d was assessed using alizarin red staining.Results rBMSCs in TC4 scaffolds of 64 round-like wells and 64 square-like wells showed the excellent cell adhesion behavior.CCK-8 as-say demonstrated that the scaffold with 9 round-like wells promoted rBMSCs proliferation.ALP activity assay showed that all experimental groups exhibited higher ALP activity compared with the control group,scaffolds with both 64 round-like wells and 64 square-like wells showed the highest ALP activity among experimental groups.qRT-PCR revealed that the expression levels of both Osteonectin and Col1a1 gradually increased with increasing wells,and 64 round-like and 64 square-like wells showed the highest expression levels of Osteonectin and Col1a1.rBMSCs osteogenic differentiation in TC4 scaffolds was significantly higher compared with the control group.Alizarin red staining illustrated that TC4 scaffolds with both 64 round-like wells and 64 square-like wells produced orange-red complexes with darker color and larger area compared with the control group.Conclusion The porous TC4 titanium scaffolds fabricated by SLM technology showed excellent rBMSCs adherence,cell via-bility,proliferation and osteogenic differentiation,with 9-wells promoting proliferation and 64-wells promoting adherence and differentiation.
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