The Effect Of RGD/biomimetic Calcium Phosphate Coatings Formed On Titanium On Adhesion,Proliferation And Differentiation Of MC3T3-E1 Cells

[Objective]To improve the osseointegration of Titanium implants by forming biomimetic calcium phosphate coatings containing RGD.[Materials and methods]A 2-step biomimetic coating approach was used to prepare the calcium phosphate coatings on the surface of Titanium(TA2,5mm in diameter,2mm in thickness),The RGD(Arg-Gly-Asp)were loaded within biomimetic calcium phosphate coatings through physical absorption and co-precipitation.Combine RGD into Titanium by Chemical coupling method,the characterization of the coatings was analyzed by scanning electron microscopy(SEM),energy dispersive X-ray spectroscopy analyzer(EDS),Fourier-transform infrared spectroscopy(FTIR)and X-ray diffractionometry(XRD).Detect the Release kinetics of RGD combined into coatings by physical absorption and co-precipitation.MC3T3-E1 cells were seeded on Ti,Ti/RGD,Ti/biomimetic calcium phosphate coatings,Ti/RGD/biomimetic calcium phosphate coatings(physical absorption),Ti/biomimetic calcium phosphate coatings(co-precipitation),respectively.The morphology of MC3T3-E1 cells cultured on samples for 4h and 24h was observed by SEM.The morphology of MC3T3-E1 cells after seeding on samples for 48h and 72h was observed by Laser scanning confocal microscope.The attachment of the MC3T3-E1 to various sample types was determined using the crystal violet staining assay.The proliferation of MC3T3-E1 cells was determined by MTT after cultured for 1,3,7days.The ALP activity of the MC3T3-E1 cells was measured after cells cultured on various sample of 3,7,14days.Real-time PCR was used to evaluate the expression of the osteogenesis-related genes after cells cultured on various sample of 7、14 days.[Results]A 2-step biomimetic coating approach could form the calcium phosphate coatings on the surface of Titanium.The results of SEM showed that coatings was composed of sharp crystal plates,the biomimetic calcium phosphate coatings loaded with RGD by physical absorption and co-precipitation could not affect the shape of the coatings;The result of EDS,XRD,FTIR indicated that the main structure of the coatings was Ca8(HPO4)2PO4,the loading of RGD by absorption and co-precipitation would not affect the structure of coatings.Release kinetics of coatings loaded within FITC-RGD by absorption and co-precipitation indicated FITC-RGD(absorption)was released completely by 48 hours,respectively,FITC-RGD(co-precipitation)was released through a long steady way with low amount.After the cells were seeded on different groups,The SEM images and Fluorescent microscopy images showed that RGD could promote the attachment and spreading of Cells on Ti,the biomimetic calcium phosphate coatings formed on Ti could promote the attachment and spreading of Cells.RGD/biomimetic calcium phosphate coatings(co-precipitation)grew tightly to each other and tended to form cell colonies compared with RGD/biomimetic calcium phosphate coatings(absorption).The crystal violet assay showed that the cell adhesion of RGD/biomimetic calcium phosphate coatings(co-precipitation)was greater than group Ti and TI/RGD(P<0.01)at 4h,there is no difference between two groups.At 8h,the cell adhesion of RGD/biomimetic calcium phosphate coatings(absorption)was greater than group Ti with coatings(P<0.01)Moreover,The results of MTT showed that,the OD value of RGD/biomimetic calcium phosphate coatings(co-precipitation)was greater than Ti and TI/RGD(P<0.01)at 1 day,the OD value of RGD/biomimetic calcium phosphate coatings(co-precipitation)was greater than RGD/biomimetic calcium phosphate coatings(absorption)(P<0.001)at 3 days,the OD value of RGD/biomimetic calcium phosphate coatings(co-precipitation)was greater than the other four groups(P<0.001)at 7days.In addition,the ALP activity showed that at 3,7,14days,the Ti with biomimetic calcium phosphate coatings and RGD/biomimetic calcium phosphate coatings(co-precipitation)got higher ALP activity than Ti and Ti/RGD(P<0.001).At 7,14day,Ti with coatings and RGD/biomimetic calcium phosphate coatings(co-precipitation)got a higher ALP activity than RGD/biomimetic calcium phosphate coatings(absorption)(P<0.001).The expression of the osteogenesis-related genes of the MC3T3-E1 cells cultured on various samples showed that the expression of ALP,OCN,OPN of cells cultured on RGD/biomimetic calcium phosphate coatings(co-precipitation)was higher than control groups RGD/biomimetic calcium phosphate coatings(absorption)(P<0.05)at 7days,the expression of OPN,ALP,COL-1,OCN of RGD/biomimetic calcium phosphate coatings(co-precipitation)was higher than group RGD/biomimetic calcium phosphate coatings(absorption)(P<0.05)at 14days.[Conclusion]A 2-step biomimetic coating approach could form the calcium phosphate coatings on the surface of Titanium,the SEM images and characterization examined by EDS,XRD and FTIR showed that the loading of RGD by absorption and co-precipitation would not affect the structure of coatings,Release kinetics of coatings loaded within FITC-RGD by absorption and co-precipitation indicated FITC-RGD(absorption)was released completely by 48 hours,respectively,FITC-RGD(co-precipitation)was released through a long steady way with low amount.After the cells were seeded on different groups,the results showed that RGD could promote adhesion and spreading of cells on samples,the biomimetic calcium phosphate coatings indicated better osteoconductivity and osteoinductivity than Ti,compared with RGD/biomimetic calcium phosphate coatings(absorption),the RGD/biomimetic calcium phosphate coatings(co-precipitation)had better osteoconductivity and osteoinductivity.