# In Vivo Evaluation of Decellularized Human Tooth Scaffold for Dental Tissue Regeneration
## 4 Discussion  
The current study examined the cell differentiation of the stem cells inoculated in decellularized human dental tooth scaffolds. Scaffolds are an important component contributing to tissue regeneration. They provide structural support and play an important role in the cell differentiation required for new tissue to form [41; 42]. Many decellularized tissues, such as skin, blood vessels, bone, and cartilage, are used as graft materials in treating patients [43; 44]. Appropriately decellularized tissues preserve ECM integrity, bioactivity, spatial structures, and vascular, lymphatic, and nervous networks [45]. However, all cellular components must be removed from decellularized scaffolds as they can cause immune rejection [46]. The results of scaffold decellularization and recellularization are affected by various factors, such as the type, composition, thickness, density, and cellularity of the tissue being regenerated [47]. Previous studies have developed an optimal decellularization method and an effective method for removing residual DNA and cellular \(\beta\)-actin from human teeth [23; 24]. The recellularization of decellularized PDL and pulp scaffolds was evaluated by qPCR and IHC staining. Stem cells repopulated on samples with pulp and PDL scaffolds but not on the control group that was not inoculated with cells. Similar to the results of in vitro experiments [24], in this study, PDLSCs were observed to be well engrafted around the cement and they retained their unique characteristics through the high expression of the  
Figure 6: Relative expression levels of the genes encoding _DSPP_, _DMP1_, _NES_, _OC_, _Col I_, _ALP_, _CD31_, and _CD34_ in dHDP transplants. Data are mean and standard deviation. The expression levels of _Col I_, _DMP1_, _NES_, _Col I_, _ALP_, _CD31_, and _CD34_ differed significantly between the DPSC recellularization and control groups (_p_ < 0.05). The expression of _OC_ did not differ significantly between the two groups (_p_ > 0.05). Abbreviations: C, control group; D, DPSC recellularization group, _DSPP_, dentin sialophosphoprotein; _DMP1_, dentin matrix acidic phosphoprotein 1; _NES_, nestin; _CD31_, cluster of differentiation 31; _CD34_, cluster of differentiation 34; dHDP, decellularized human dental pulp; DPSC, dental pulp stem cells. * indicates that there is a statistically significant difference. When \(p\) < 0.05, was used to show that there is a significant difference. PD LSC-specific markers CP23 and POSTN. CP23 is a cementoblast marker and a regulator of the bio-mineralization of cementum [48; 49]. POSTN is a PDL marker and is mainly found in cells with a mesenchymal lineage, such as osteoblasts, periodontal ligaments, and periostea [50; 51]. The PDLSCs migrated deeply to the part of the semi-cylindrical dHPDLs close to the cementum by passing through the dHPDL's collagen network (Figure 1D-F and D-F). The expression of Col XII, a marker of mature PDL, increased [52], indicating that PDLSCs differentiated into more mature PDLs. It was difficult to confirm the newly formed hard tissue through HE staining, but OC and ALP, which are involved in the mineralization of cementum and bone, were expressed [53; 54]. During recellularization, PDLSCs moved close to the cementum of dHPDL, which affected the generation of mature PDL, and caused the differentiation of hard tissues in vivo. These results indicate that transplanting stem cells onto scaffolds can help repair or regenerate damaged periodontal tissue. Recellularization on DPSCs had different characteristics than recellularization on PD LSCs. In the DPSC recellularization group, the formation of newly formed hard tissue was shown through HE staining. The newly created hard tissue did not have a dentin-like structure, but the hard tissue-related markers DSPP, DMP1, ALP, OC, and Col I were expressed [54; 55; 56; 57; 58]. In addition, nestin, which is related to nerves, and CD31 and CD34, which are related to angiogenesis, were expressed, indicating that hard tissue, nerves, and blood vessels may have been generated.