Proliferation And Odontogenic Differentiation Of Umbilical Cord Mesenchymal Stem Cells Cocultured In RADA16-I

Objective:We assessed the compatibility of self-assembling peptide hydrogel RADA16-I with human dental pulp cells?hDPCs?and human umbilical cord mesenchymal stem cells?hUCMSCs?growth and proliferation,to explore the optimal hydrogel concentration from different concentrations?1%,0.75%,0.5%,0.25%,0.125%?.This study evaluated the role of hDPCs in triggering the differentiation of hUCMSCs into odontoblast-like cells in the three-dimensional coculture system which was established with the help of bone morphogenetic protein 2?BMP-2?and such a scaffold.Moreover,it prepared experimental basis for the subsequent related research.Methods:To evaluate the effect of three-dimensional culture on the biological properties of hDPCs and hUCMSCs,cells of passages 4⁶ were mixed with hydrogel,cell morphology was observed in each group using inverted phase contrast microscope.Cell viability was measured by the MTT assay after 3,6,9,and 12 days of incubation respectively,and cell growth curves were made according to the optical density?OD490?.Analysis of these data,the optimal hydrogel concentration for the subsequent experiments was determined.hUCMSCs,hDPCs induced by BMP-2,or coculture of both cell types were encapsulated in the hydrogel.MTT assay and cell growth curves were used to measure the viability of cells after 1,3,6,9,and 12 days.When the cultures were performed for two or three weeks,ALP activity in each group was assessed,and the differentiation into odontoblast-like cells were measured by the mRNA expression of DSPP,DMP-1,ALP and OCN.Alizarin red staining was performed for the formation of mineralized nodules after 21 days.Results:Compared with two-dimensional spindle shape which was a typical form of fibroblasts cells,cells encapsulated in the hydrogel were of the conglobating growing style.The MTT assay revealed that:?1?2D culture cells entered logarithmic growth phase on the third day,and reached the platform on the 6th day.?2?The hydrogel scaffolds supported cell survival.hUCMSCs entered logarithmic growth phase on the 6^th day after several days of adaption,while the logarithmic growth period of hDPCs came on the third day,and the proliferation slowed down after 6 days.?3?The three-dimensional hydrogel scaffold supported cell growth and prolification.The growth rate of cells in lower concentration hydrogels was higher than that in high concentration hydrogels?P<0.05?.We observed that 0.25%hydrogel scaffold showed to be better than the other groups in terms of cellular compatibility and structural stability,which was suitable for the follow-up experiments.Notably,the coculture groups exhibited more proliferative potential,ALP activity and mineralization nodule generation ability than either hUCMSCs-monocultures or hDPCs-monocultures?P<0.05?.The coculture groups also expressed DSPP,DMP-1,ALPand OCN mRNA,putative markers of odotoblastic differentiation,which was close to,or even more than that of the hDPCs-monocultures under some circumstances.The mRNA expression of those genes in coculture groups were higher than that of hUCMSCs-monocultures.Conclusion:RADA16-I is a kind of hydrogel scaffold with favorable biocompatibility,and we choose 0.25%hydrogel for future research through comprehensive consideration.At the ratio of 1:1,both hDPCs and hUCMSCs were encapsulated in RADA16-I with BMP-2 added to these cultures,and cells maintain the proliferation and differentiation abilities in this cell-gel complex.The coculture groups even have stronger abilities of odotoblastic differentiation and mineralization than hDPCs-monoculture groups,suggesting that coculture conditions can regulate the proliferation and differentiation of cocultured cells in a certain range.