To further elucidate the specific cell adhesive motif within Npnt, we investigated whether synthetic hexapeptide KPRGDV derived from mouse Npnt (Npnt-RGD) and its mutant counterpart KPRGEV (Npnt-RGE) in soluble form could block the adhesion of hDPSCs to surface-presenting Npnt (Figure 5).
The above results conclude that Npnt is a hDPSC adhesive and supports hDPSC growth in either serum-containing or serum-free conditions.
The results highlighted that Npnt triggered the upregulation of ITGA1 (1.31 [+ or -] 0.04-fold), ITGA4 (1.40 [+ or -] 0.05-fold), ITGA7 (1.33 [+ or -] 0.07-fold), and ITGB1 (1.87 [+ or -] 0.09-fold).
Further, the late-stage gene expression analysis in Figure 7 revealed that Npnt tended to direct the hDPSCs' differentiation toward osteoblastic lineage instead of odontoblastic lineage, as the DSPP, a well-established odontoblast differentiation marker , was moderately suppressed by Npnt, but BSP, a widely recognized osteoblast marker,  was markedly enhanced by Npnt compared to the other groups.
As expected, the induction of differentiation of cells in Npnt and FCOL1 resulted in the appearance of mineralized nodules revealed by alizarin red staining at day 30.
Based on the findings, it is suggested that Npnt possesses comparable capacity to FCOL1 in promoting proliferation and eliciting intensive mineralization in hDPSCs.
Taken together, the results demonstrated that Npnt facilitates hDPSC adhesion and spreading to hydrophobic nontissue culture-treated polystyrene.
Caption: FIGURE 1: Fluorescence visualization of coated Npnt. Npnt-coated polystyrene was positive for anti-Npnt (b).
Fluorescence microscopy photographs of hDPSCs that adhered and spread on polystyrene presenting Npnt. As positive controls, cells attached to substrate(s) having adsorbed layers of Fn and FCOL1.