Effect Of Porous TC4 Metallic Scaffolds On Cytotoxicity And Osteogenic Effect

Objective:Effects of porous Ti-6Al-4V?TC4?metallic scaffolds adopt by SLM technology on cytotoxicity and osteogenic differentiation of bone marrowmesenchymal stem cells?BMSCs?.Methods:1.Primary BMSCs from SD rats are cultured by total bone marrow adherent method in vitro.2.The phenotype of BMSCs are identified by flow cytometry.The 3^rd generationBMSCs differentiated well in logarithm grown are selected to identify the phenotype by flow cytometry.3.The TC4 metallic scaffolds are fabricated,grouped and pretreated in the present work.SLM is used to fabricate square porous scaffolds with a length×width×height=5×5×2.5 mm.The holes on the scaffold are designed as round 9 holes,round 16 holes,round 64 holes,square 25 holes,square 64 holes and non-porous solid control group.4.The morphologies and adhesion of the 3^rd generation BMSCs inoculated onto TC4metallic scaffolds after 48 h are observed by scanning electron microscopy?SEM?.5.Cell Proliferation Activity Detection Kit?CCK-8?assay has been applied to examine the cytotoxicity of TC4 metallic scaffolds when the 3^rd generation BMSCs are inoculated at the 1^st day,3^rd day,5^th day and 7^th day,respectively.6.Alkaline phosphatase?ALP?has been detected when the 4^th generation BMSCs are inoculated onto TC4 porous metallic scaffolds and osteoblastic induced at the 7^th day and 14^thh day,respectively.7.Real-time fluorescence quantitative PCR?Q-PCR?have been applied to examine the expression of ON and CoL 1 of BMSCs when the 4^th generation BMSCs areinoculated onto TC4 porous metallic scaffolds and osteoblastic induced at the 7^th day and 14^th day,respectively.8.Alizarin red staining are performed when the 4^th generation BMSCs are inoculated onto TC4 porous metallic scaffolds and osteoblastic induced at the 21^st day.Results:1.The results of flow cytometry shows that the high expression of CD90?99.81%?and CD105?99.91%?on the surface markers of non-hematopoietic cell lines in cultured the 3^rdd generation cells is observed,and the hematopoietic stem cell surface marker CD45?3.38%?has a low expression,which consistented with the reported BMSCs phenotype.2.The results of the SEM shows that all the six groups of metallic scaffolds have adhered to the BMSCs around the surface.In the experimental group,as the pore size decreases,cell adhesion becomes better,the morphological extension,protrude pseudopodia and interconnect,distribute above the lamellar cell layer,which indicate that the cell morphology and adhesion are better on the smaller pore at the early stage of inoculation.3.The results of CCK-8 assay shows that when co-culture for 1^st day,scaffolds in each group have no significant effect on cell proliferation.From the 3^rd day,compared with the control group,the cell proliferation was significantly promoted by 9 holes round,16 holes round,25 holes square,64 holes round and 64 holes square.The results showed that the larger the pore size of the scaffold in the experimental group,the more conducive to cell proliferation.4.The results of ALP activity shows that comparing the 14th day with the 7th day,the ALP activity of scaffolds in 6 groups show an upward trend.Scaffolds can increase the expression of ALP activity.Compared with non-porous control group,ALP activity in porous stent group increase significantly.In the experimental group,small-aperture scaffolds show the highest ALP activity,but there is no significant difference between small-aperture round and square scaffolds.5.The results of Q-PCR shows that comparing the 14th day with the 7th day,the relative expression of ON and CoL1 in scaffold cells of 6 groups show an upward trend.The relative expression of mRNA is increased in the scaffold group,and the expression level is more obvious in the porous scaffold group than in the non-porous control group.In the experimental group,the expression level of small-aperture scaffolds is the highest,and the round 64-hole scaffolds is more significant than the square 64-hole scaffolds.6.The results of alizarin red staining shows that after osteogenesis induction,orange red complexes were produced in 6 scaffold cells in each group,which is positive for Alizarin Red staining,indicating that all the scaffolds have calcium deposits and all the cells of the six groups have osteogenesis.There is no significant difference between the non-porous control group and the round-9 pore scaffolds.As the pore size gradually decrease,the orange-red complex produce darker color and larger area.Obvious large orange-red complexes are observe in both round-64 pore and square-64pore scaffolds.Conclusion:The individualized TC4 porous metallic scaffolds adopt by SLM technology have no cytotoxicity to BMSCs and can promote cell proliferation and osteogenesis.In the experimental group,large-aperture scaffolds are beneficial to cell proliferation,and small-aperture scaffolds promote cell adhesion,aggregation,maturation,osteogenic differentiation and the relative expression of ON and CoL1.