## 4 Discussion  
Recently, clinical research of stem cell therapies has been increasing. Although the mechanisms underlying the therapeutic benefit of stem cells remain unclear, many reports suggest the efficacy of related cell therapies [24; 25]. In this study, two mechanisms of enhanced healing of the tooth extraction site can be assumed. First, administered MSCs differentiate into gingiva cells and facilitate wound closure. Second, MSCs secrete cytokines and growth factors that activate epithelial cells to migrate and proliferate to achieve primary closure of the damaged tissue [26]. We previously reported that intravenous administration of MSCs in rats contributed to the healing of damaged tissue [27]. However, delivering cells subcutaneously without a scaffold did not result in a positive effect on tissue healing [28]. As such, we focused on the scaffold as a foundation for cells. First, we searched for a suitable scaffold material in which MSCs could retain their characteristics for long periods of time. Although we verified the competence of other materials (data not shown), we selected collagen as a scaffold because it is fluid at low temperatures, which makes it easy to deliver by injection and less damaging to the cells. In addition, collagen forms a gel at body temperature, which facilitates retention at the application site [29]. Generally, if artificial materials exist in the body for a long time, problems such as infection or degradation may arise; thus, the characteristics of type I collagen, which can be absorbed by the body, might be suitable. In addition, collagen seems to provide an effective scaffold with little toxicity, as suggested by other studies [30; 31]. Consistent with previous reports [32; 33], MSCs could remain alive in the scaffold without major changes in cell number for 2 weeks. Meanwhile, as shown in Figure 1C, MSCs maintained expression of MSC markers (CD29, CD90, and CD105), and MSCs extracted at each stage exhibited differentiation  
Figure 5: Effect of MSCs from collagen gel for tooth extraction. (**A**) Experimental protocol for in vivo experiments. (**B**) At 7 days after MSC administration, numbers of accumulated MSCs around the tooth extraction socket. MSCs injected via the tail vein and MSCs from collagen gel were observed as many GFP/CD90 double-positive cells around the socket after extraction by immunofluorescence staining. Injected MSCs accumulated similarly to scaffold MSCs. (**C**) Extraction socket healing. The scaffold MSC group and the injected MSC group showed good mucosal healing at 7 days. Scale bars, 100 \(\upmu\)m. * \(p<0.05\). (**D**) localization of Ln-332. Laminin-332 was observed as a line on connective tissue in both the systemic injection and collagen scaffold groups (white arrowheads). and self-renewal potential. Therefore, MSCs could maintain their \"stemness\", which is an important characteristic for at least 14 days. Second, we checked the competence of collagen as a scaffold. As shown in Figure 2A, the injected 2 mL of collagen retained its form at 2 weeks after insertion into back subcutaneous tissue. Our results were consistent with previous reports suggesting that a 2-week period was required to absorb approximately 5-mm diameter (corresponding to 2 mL) of collagen containing MSCs after insertion into the body as a scaffold for exogenous cells [34]. Safety of the collagen scaffold was determined by the survival rate of MSCs in the scaffold in vivo experiments. As a result, MSCs in collagen gels implanted in back subcutaneous tissue remained alive for at least 2 weeks, similar to the in vitro data shown. Thus, the injected collagen scaffold had the potential to act as a reservoir for exogenous MSCs in the body. Third, we investigated whether MSCs in the collagen gel could maintain their chemotactic properties. A previous report indicated that systemically delivered MSCs accumulated at sites of inflammation [27; 35]. As shown in Figure 3, MSCs could be guided from the collagen scaffold to the wound area after tooth extraction.