In Vivo Evaluation of Decellularized Human Tooth Scaffold for Dental Tissue Regeneration

Abstract

Conventional root canal treatment may result in loss of tooth vitality, which can lead to unfavorable treatment outcomes. Notably, a ceased tooth development of immature permanent teeth with open apices, regeneration of periodontal ligaments (PDL), and pulp is highly expected healing process. For regeneration, the scaffold is one of the critical components that carry biological benefits. Therefore, this study evaluated a decellularized human tooth as a scaffold for the PDL and pulp tissue regeneration. A tooth scaffold was fabricated using an effective decellularization method as reported in previous studies. PDL stem cells (PDLSCs) and dental pulp stem cells (DPSCs) obtained from human permanent teeth were inoculated onto decellularized scaffolds, then cultured to transplant into immunosuppressed mouse. After 9 weeks, PDLSCs and DPSCs that were inoculated onto decellularized tooth scaffolds and cultured in an in vivo demonstrated successful differentiation. In PDLSCs, a regeneration of the cementum/PDL complex could be expected. In DPSCs, the expression of genes related to revascularization and the hard tissue regeneration showed the possibility of pulp regeneration. This study suggested that the potential possible application of decellularized human tooth could be a scaffold in regeneration PDL and pulp tissue along with PDLSCs and DPSCs, respectively, as a novel treatment method.

Keywords: decellularized tooth scaffold; dental pulp stem cell; periodontal ligament stem cell; tissue regeneration.

Figure 1.

Histological characteristics of PDLSC recellularization on dHPDL transplants. HE staining (A,D), MT staining (B,E), HN staining (C,F), HE, MT, and HN staining showed that dHPDL transplants were contributed to recellularization of cells, indicated by red arrows. Scale bars: 20 μm. Abbreviations: PDLSC, periodontal ligament stem cells; dHPDL, decellularized human periodontal ligament; HE, hematoxylin and eosin; MT, Masson’s trichrome; HN, human nuclei.

Figure 2.

Immunohistochemical staining of PDLSCs recellularization on dHPDL transplants. dHPDL transplants were immunostained with (A,D) anti-human Col XII, (B,E) anti-human CP23, and (C,F) anti-human OC antibodies. The red arrows indicate examples of positively immunostained cells. Scale bars: 20 μm. Abbreviations: PDLSCs, periodontal ligament stem cells; dHPDL, decellularized human periodontal ligament; Col XII, Collagen type XII; CP23, cementum-derived protein 23; OC, osteocalcin.

Figure 3.

Evaluation of gene expression level by relative fold changes for genes encoding CP23, Col XII, POSTN, OC, ALP, and Col I in dHPDL transplants. The expression levels of CP23, Col XII, OC, ALP, and Col I differed significantly between the two groups (* p < 0.05). The expression of POSTN did not differ significantly between the two groups (p > 0.05). Abbreviations: C, control group; P, PDLSC group, CP23, cementum-derived protein 23; Col XII, collagen type XII; POSTN, periostin; OC, osteocalcin; ALP, alkaline phosphatase; Col I, collagen type I; dHPDL, decellularized human periodontal ligament.

Figure 4.

Histological characteristics of recellularized dHDP transplants with DPSCs. (A,D) HE staining. (B,E) MT staining. (C,F) HN staining. HE, MT, and HN staining showed that dHDP transplants were recellularized. Recellularized cells are indicated by red arrows. Scale bars: 20 μm. Abbreviations: dHDP, decellularized human dental pulp; DPSCs, dental pulp stem cells; HE, hematoxylin and eosin; MT, Masson’s trichrome; HN, human nuclei.

Figure 5.

Immunohistochemical staining of dHDP transplants recellularized with DPSCs. dHDP transplants immunostained with (A,E), anti-human DSP, (B,F) anti-human OC, (C,G) anti-human VEGF, and (D,H) anti-human CD34 antibodies. Red arrows indicate positively immunostained cells. Scale bars: 20 μm. Abbreviations: dHDP, decellularized human dental pulp; DPSCs, dental pulp stem cells; DSP, dentin sialoprotein; OC, osteocalcin; VEGF, vascular endothelial growth factor; CD34, cluster of differentiation 34.

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. * idicates that there is a statistically significant difference. When p < 0.05, was used to show that there is a significant difference.