Decellularized Swine Dental Pulp as a Bioscaffold for Pulp Regeneration

Abstract

Endodontic regeneration shows promise in treating dental pulp diseases; however, no suitable scaffolds exist for pulp regeneration. Acellular natural extracellular matrix (ECM) is a favorable scaffold for tissue regeneration since the anatomical structure and ECM of the natural tissues or organs are well-preserved. Xenogeneic ECM is superior to autologous or allogeneic ECM in tissue engineering for its unlimited resources. This study investigated the characteristics of decellularized dental pulp ECM from swine and evaluated whether it could mediate pulp regeneration. Dental pulps were acquired from the mandible anterior teeth of swine 12 months of age and decellularized with 10% sodium dodecyl sulfate (SDS) combined with Triton X-100. Pulp regeneration was conducted by seeding human dental pulp stem cells into decellularized pulp and transplanted subcutaneously into nude mice for 8 weeks. The decellularized pulp demonstrated preserved natural shape and structure without any cellular components. Histological analysis showed excellent ECM preservation and pulp-like tissue, and newly formed mineralized tissues were regenerated after being transplanted in vivo. In conclusion, decellularized swine dental pulp maintains ECM components favoring stem cell proliferation and differentiation, thus representing a suitable scaffold for improving clinical outcomes and functions of teeth with dental pulp diseases.

Figure 1

Images of swine dental pulp undergoing decellularization. At 0 h (a) and 32 h (b), there was a visible change from a normal pink to a mostly white, translucent appearance. H&E staining showed the native structures of pulp were well-preserved (c, d), while no cellular components were observed after 32 h of decellularization (e–h). SEM analyses showed the decellularization pulp present porose structure in transverse section (i) and vertical section (j). n = 4, scale bars: 100 μm (c, d), 50 μm (e, f), and 100 μm (i, j).

Figure 2

Characterization of the ECM components of decellularized pulp. Immunofluorescent staining showed that ECM proteins, Col-IV, laminin, fibronectin, intergrin β1, and vimentin were retained in the decellularized pulp, which was similar to the native pulp, except vimentin, which was decreased in decellularized pulp. Sections were counterstained with DAPI (blue). n = 4, scale bars: 100 μm.

Figure 3

Cell reseeding and regeneration of dental pulp with decellularized pulp. Reseeded samples were transplanted subcutaneously in immunodeficient mice for 2 months, (a–c) H&E staining showed that pulp-like tissues were regenerated in recellularized matrix group, compared with natural pulp and blank control. (d–f) H&E staining showed the functional pulp was regenerated with a layer of mineral tissue regenerated (dashed line) between the dental pulp-like tissue and dentin interface, as well as a layer of polygonal cells (yellow arrow). (g) Quantitive analysis of pulp-like tissue regeneration area in control and recellularized matrix group. (h) Quantitive analysis of mineral tissue deposition area in natural pulp, blank control, and acellular pulp group. Values are mean ± SD, n = 10. One-way ANOVA was used to determine statistical significance, ^∗∗P ≤ 0.01. D: dentin; P: pulp; Od: odontoblasts; scale bars: 50 μm.

Figure 4

Immunohistochemistry analysis of regenerated pulp tissue. (a–c) Immunohistochemistry analysis showed polygonal cells layer highly expressing DSPP, which is a marker of odontoblast-like cells; no odontoblast, and mineralized tissue formation could be found in the control group. (d) Quantitive analysis of DSPP positive cells in natural pulp, control, and recellularized matrix group. Values are mean ± SD, n = 10. One-way ANOVA was used to determine statistical significance, ^∗∗P ≤ 0.01. Scale bars: 100 μm. D: dentin; P: pulp; Od: odontoblasts; scale bars: 50 μm.