## 3 Effects of Vitamin D\({}_{3}\) on Dental MSCs
### Effect of Vitamin D\({}_{3}\) on the Differentiation Potential of Dental MSCs In Vitro  
One of the most prominent biological effects of vitamin D\({}_{3}\) is its ability to stimulate osteogenic differentiation of different MSCs in vitro [37]. This process is usually achieved by culturing cells for 3-4 weeks in media containing specific supplements, such as dexamethasone, \(\beta\)-glycerophosphate, and L-ascorbic acid [51]. Besides, the osteogenic medium is supplemented with high amounts of fetal bovine serum (20%). Osteogenic differentiation is usually assessed by staining extracellular calcium deposits with Alizarin red or von Kossa [51]. Alternatively, it can be evaluated by measuring the expression of specific osteogenesis markers, including alkaline phosphatase (ALP), osteoclast (OCN), osteopontin (OPN), collagen 1 (Coll-1), bone sialoprotein (BSP), and runt-related transcription factor 2 (runx-2) [52]. Vitamin D\({}_{3}\) has been shown to stimulate osteogenic differentiation of MSCs from various dental tissues in vitro [44; 48; 53; 54; 55; 56; 57; 58; 59; 60; 61]. [PERSON] et al. reported that 1,25(OH)\({}_{2}\)D\({}_{3}\) and 25(OH)D\({}_{3}\) promote mineralization of hDPCs and hDPCs in the presence of osteogenic supplements, but fail to induce mineralization in their absence. However, the gene expression of OCN and OPN has been shown to be enhanced by both vitamin D\({}_{3}\) metabolites, which occurred independently of the presence of osteogenic media [48]. A later study of this group showed that 1,25(OH)\({}_{2}\)D\({}_{3}\) enhances the ALP expression, as well as the gene expression of osteoclast  
Figure 1: Systemic and local vitamin D\({}_{3}\) metabolism. and osteepontin in dental pulp cells cultured on a three-dimensional scaffold [55]. [PERSON] et al. reported that 1,25(OH)\({}_{2}\)D\({}_{3}\) promotes mineralization and stimulates alkaline phosphatase activity in human dental pulp stem cells (hDPSCs) [60]. The effects of 1,25(OH)\({}_{2}\)D\({}_{3}\) were accompanied by the activation of extracellular signal-regulated kinases (ERKs) and abolished by a specific ERKs' inhibitor [60]. In dental bud stem cells, 1,25(OH)\({}_{2}\)D\({}_{3}\) has been observed to stimulate mineralization, alkaline phosphatase activity, and the expression of Coll-1, runx-2, BSP, and OPN [58; 61]. [PERSON] et al. reported that 1,25(OH)\({}_{2}\)D\({}_{3}\) promotes the mineralization, as well as the gene expression of ALP, BSP, Coll-1, and OCN of MSCs derived from alveolar periosteum [59]. A recent report by [PERSON] et al. showed that 1,25(OH)\({}_{2}\)D\({}_{3}\) stimulates mineralization and alkaline phosphatase activity of hDPCs grown on calcium-aluminate-chitosan scaffolds [53]. In human periodontal ligament cells, [PERSON] et al. showed that 1,25(OH)\({}_{2}\)D\({}_{3}\) stimulates alkaline phosphatase activity and gene expression of OCN and OPNs [57]. [PERSON] et al. observed that 1,25(OH)\({}_{2}\)D\({}_{3}\) stimulates mineralization of hPDLCs assessed by Alizarin red and von Kossa staining as well as the expression of ALP, OCN, and BSP [44]. [PERSON] et al. showed that 1,25(OH)\({}_{2}\)D\({}_{3}\) stimulates the mineralization and alkaline phosphatase activity in human periodontal ligament stem cells (hPDLSCs) [54]. Besides, 1,25(OH)\({}_{2}\)D\({}_{3}\) has been reported to enhance the expression of transcriptional coactivator with PDZ-binding motif (TAZ) [54]. TAZ is the downstream effector of Hippo signaling, which regulates various cellular processes, including osteogenic differentiation. Therefore, there is a possibility that 1,25(OH)\({}_{2}\)D\({}_{3}\) might stimulate osteogenesis in hPDLSCs partially through TAZ [54]. Apart from osteogenesis, vitamin D\({}_{3}\) seems to enhance the differentiation of dental tissue-resident stem cells into other dental hard tissues, like dentin and cement. Particularly, 1,25(OH)\({}_{2}\)D\({}_{3}\) stimulated the production of dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1) by hDPCs through the activation of ERKs [60].