In our previous study, we demonstrated that hyaluronan induces odontoblastic differentiation of dental pulp stem cells via interactions with CD44. However, it remains unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic differentiation. Therefore, we searched for a compound that induces odontoblastic differentiation of dental pulp stem cells, regardless of the chemical structure and function of hyaluronan, and examined whether CD44 is involved in the induction of odontoblastic differentiation by the compound. Because vitamin K analogues can promote bone formation and tissue calcification, we focused on derivatives of naphthoquinone, the skeleton of vitamin K; we verified whether those compounds could induce odontoblastic differentiation of dental pulp stem cells. We found that dentin sialophosphoprotein, a marker of odontoblasts, was expressed in dental pulp stem cells after treatment with shikonin. The shikonin-induced expression of dentin sialophosphoprotein was inhibited by PI3K, AKT, and mTOR inhibitors. In addition, in dental pulp stem cells transfected with siRNA against CD44, the shikonin-induced expression of dentin sialophosphoprotein was inhibited. Thus, shikonin can stimulate dental pulp stem cells to undergo odontoblastic differentiation through a mechanism involving the AKT-mTOR signaling pathway and CD44. Hyaluronan stimulated dental pulp stem cells to undergo CD44-mediated odontoblastic differentiation in our previous study; the present study indicated that CD44 is necessary for dental pulp stem cells to undergo odontoblastic differentiation. Although expression of CD44 is important for inducing odontoblastic differentiation of dental pulp stem cells, the relationship between the AKT-mTOR signaling pathway and CD44 expression, in the context of shikonin stimulation, has not yet been elucidated. This study suggested that shikonin may be useful for inducing odontoblastic differentiation of dental pulp stem cells, and that it may have clinical applications, including protection of dental pulp.

38In our previous study, we demonstrated that hyaluronan induces odontoblastic 39 differentiation of dental pulp stem cells via interactions with CD44. However, it remains 40 unclear whether CD44 expression by dental pulp stem cells is required for odontoblastic 41 differentiation. Therefore, we searched for a compound that induces odontoblastic 42 differentiation of dental pulp stem cells, regardless of the chemical structure and function 43 of hyaluronan, and examined whether CD44 is involved in the induction of odontoblastic 44 differentiation by the compound. Because vitamin K analogues can promote bone 45 formation and tissue calcification, we focused on derivatives of naphthoquinone, the 46 skeleton of vitamin K; we verified whether those compounds could induce odontoblastic 47 differentiation of dental pulp stem cells. We found that dentin sialophosphoprotein, a 48 marker of odontoblasts, was expressed in dental pulp stem cells after treatment with 49 shikonin. The shikonin-induced expression of dentin sialophosphoprotein was inhibited 50 by PI3K, AKT, and mTOR inhibitors. In addition, in dental pulp stem cells transfected 51 with siRNA against CD44, the shikonin-induced expression of dentin 52 sialophosphoprotein was inhibited. Thus, shikonin can stimulate dental pulp stem cells to 53 undergo odontoblastic differentiation through a mechanism involving the AKT-mTOR 54 signaling pathway and CD44. Hyaluronan stimulated dental pulp stem cells to undergo 55 CD44-mediated odontoblastic differentiation in our previous study; the present study 56 indicated that CD44 is necessary for dental pulp stem cells to undergo odontoblastic 57 differentiation. Although expression of CD44 is important for inducing odontoblastic 58 differentiation of dental pulp stem cells, the relationship between the AKT-mTOR 59 signaling pathway and CD44 expression, in the context of shikonin stimulation, has not 60 yet been elucidated. This study suggested that shikonin may be useful for inducing 61 odontoblastic differentiation of dental pulp stem cells, and that it may have clinical 62 applications, including protection of dental pulp. 63 64 67 CD44 [1]. However, the underlying cellular signaling mechanism has not yet been 68 elucidated, and it has been unclear whether small molecules can induce odontoblastic 69 differentiation of DPSCs. Therefore, we explored whether small molecules could induce 70 odontoblastic differentiation of DPSCs and attempted to identify the differentiation 71 induction mechanism. In addition, we assessed whether CD44 was essential for this 72 mechanism. 73In the field of DPSC research, several research groups have shown that collagenase-74 treated cells isolated from human dental pulp showed more rapid proliferation, compared 75 with hematopoietic stem cells, and a similar degree of multipotency [2,3]. Subsequently, 76 various groups have described possible DPSCs. There have been reports of the induction 77 of odontogenic differentiation from DPSCs in vitro [4-6]. Moreover, human DPSCs can 78 differentiate int...