We generated periodontitis lesions in miniature swine and then transplanted hDPSC sheets or disassociated cells for tissue regeneration. The animals were sacrificed at 12 weeks post-transplantation. Intraoral photographs showed that, 12 weeks after transplantation, marked periodontal tissue healing was found in the hDPSC injection group (Fig. 2a) and the hDPSC sheet group (Fig. 2b). There were only limited reattached periodontal tissues in the control group (Fig. 2c). Three-dimensional CT images indicated marked bone regeneration in the hDPSC injection (Fig. 2d) and hDPSC sheet (Fig. 2e) groups after cell transplantation, while limited bone formation was seen in the control group (Fig. 2f). Three-dimensional models at 12 weeks post-transplantation and pre-transplantation were reconstructed using Mimics (Additional file 3: Figure S3). The regenerated bone volume was calculated (Fig. 2g). At 12 weeks post-transplantation, the AL was 3.1 ± 0.6 mm in the hDPSC sheet group, 3.5 ± 0.6 mm in the hDPSC injection group, and 5.7 ± 0.5 mm in the untreated control group (Fig. 3b). Statistical analysis indicated that both hDPSC sheet treatment and hDPSC injection significantly improved periodontal soft tissue healing in comparison with the control group (Fig. 3a and b). The heights of new bone regeneration were significantly higher in the hDPSC sheet group and hDPSC injection group than in the control group (Fig. 3c). The CT scan and three-dimensional CT imaging showed that the volumes of regenerative alveolar bone in the hDPSC sheet group and hDPSC injection group were 52.7 ± 4.1 mm3 and 32.4 ± 5.1 mm3, respectively, which were significantly larger than the volume in the control group (1.8 ± 2.3 mm3, Fig. 2g). At 12 weeks after cell implantation, experimental tissues were also sectioned in the buccal-lingual direction and stained with H&E to provide a view of the entire section. Image J semi-quantitative analysis showed the percentage of bone in the periodontium in the hDPSC injection group and hDPSC sheet group were 12.8 ± 4.4 % and 17.4 ± 5.3 %, respectively, which was significantly larger than the volume in the control group (7.2 ± 2.0 %) (Fig. 3d). New bone was regenerated in the hDPSC sheet group (Figs. 3d and 4d) and hDPSC injection group (Figs. 3d and 4a). A new cementum-like layer from the height of alveolar bone (HAB) to almost the CEJ was observed in the hDPSC injection group (Fig. 4a) and hDPSC sheet group (Fig. 4d). This structure is missing in the control group (Fig. 4g). There was new attachment of Sharpy’s fibers in the hDPSC sheet group (Fig. 4f) and hDPSC injection group (Fig. 4c), but attachment was irregular in the control group (Fig. 4i). Positive human β-globin expression was found in the tissues from the cell implantation group, while negative expression was found in the control group (Fig. 2h).

Fig. 2

Healing of periodontal defects mediated by hDPSCs. ac Intraoral photographs indicated that, 12 weeks after transplantation, marked periodontal soft tissue formation was found in the hDPSC injection group (the injection was performed without flap elevation), but could not restore soft tissues to healthy levels (a) (yellow dotted line). Periodontal soft tissue healing mediated by the hDPSC sheet (b) was close to the normal tissue level (yellow dotted line). Only limited periodontal soft tissues were recoverd in the control group (c) (yellow dotted line). df Three-dimensional CT images revealed marked bone formation in the hDPSC injection group (d), hDPSCs sheet group (e) after cell transplantation, and limited bone regeneration in the control group (f) (red dotted lines). g The bone regeneration volumes were larger in the hDPSC sheet group and hDPSC injection group compared with the control group (*P < 0.05). The bone regeneration volume was larger in the hDPSC sheet group than the hDPSC injection group (P < 0.05). h Genomic DNA was extracted from periodontal soft tissue and alveolar bone in the bone defect at 12 weeks after hDPSC implantation. Quantitative PCR was used to detect the human β-globin gene, and the results were normalized to the miniature pig receptor-associated protein at the synapse (RAPSYN) gene. Results are shown as means ± SD. *P < 0.05, versus control group. Statistical significance was evaluated by analysis of variance. All error bars represent SD (n = 5). hDPSC human dental pulp stem cell, M Mesial, W weeks

Fig. 3

Clinical and bone qualitative assessments of regenerated periodontal tissues mediated by hDPSC transplantation in miniature pigs. a, b Clinical assessments of the periodontal situation in the three groups. At week 0, there was no significant difference in PD (a) and AL (b) among the three groups. However, at 12 weeks post-transplantation, the PD (a), and AL (b) values were significantly improved in the hDPSC injection and hDPSC sheet groups compared with the control group. Data are expressed as the mean ± SD (mm). The differences in clinical indexes at each time point among the three groups were analyzed using one-way analysis of variance. The pairwise comparisons were analyzed using the Bonferroni method (*P <0.05, n = 5). c The bone regeneration length was highest in the hDPSC sheet group, while the height of bone regeneration in the hDPSC injection group was also higher than the control group, indicating there was more bone tissue regeneration in the hDPSC injection and hDPSC sheet groups than in the control group (*P < 0.05). d Semi-quantitative analysis shows the amount of bone formation in each group. The percentage of bone in the periodontium was larger in the hDPSC sheet group and hDPSC injection group compared with the control group (*P < 0.05); the bone area was larger in the hDPSC sheet group than that in the hDPSC injection group (P < 0.05). Statistical significance was evaluated by analysis of variance. All error bars represent SD (n = 5). hDPSC human dental pulp stem cell, W weeks

Fig. 4

Histopathological assessment of periodontal bone regeneration by H&E staining. New periodontal bone regeneration in the periodontal defects of the hDPSC injection group (a) and hDPSC sheet group (d). New bone was regenerated in the periodontal defect area in the hDPSC injection group (c) and hDPSC sheet group (f). The alveolar bone heights in the hDPSC sheet group and hDPSC injection group were much larger than those in the control group (i). B bone, C cementum, CEJ cemento-enameljunction, d dentin, HAB height of alveolar bone, hDPSC human dental pulp stem cell, PDL periodontal ligament, SE sulcular epithelium. However, deep periodontal pockets and shortages of new bone remainedin the control group (g). Much thicker sulcular epithelia and enlarged epithelial pegs were evident in the control group (h) compared with hDPSC injection (b) and hDPSC sheet group (e)