Investigating the vascularisation capacity of a decellularised dental pulp matrix seeded with human dental pulp stem cells: in vitro and preliminary in vivo evaluations

Aim

To investigate the vascularisation capacity of a decellularised dental pulp matrix (DDP) of bovine origin seeded with human dental pulp stem cells (hDPSCs) in vitro and to present preliminary in vivo findings.

Methodology

Bovine dental pulps were decellularised and then analysed using histological staining and DNA quantification. The resultant DDPs were characterised using immunohistochemical staining for the retention of vascular endothelial growth factor A (VEGF‐A) and fibroblast growth factor 2 (FGF‐2). Furthermore, DDPs were recellularised with hDPSCs and analysed histologically. The expression of markers involved in angiogenesis by hDPSCs colonising the DDPs was assessed in vitro. A preliminary in vivo study was then conducted in which hDPSCs‐seeded and unseeded DDPs were inserted in debrided human premolars root slices and implanted subcutaneously in immunodeficient mice. Samples were retrieved after 30 days and analysed using histological and immunohistochemical staining. The independent samples t‐test, analysis of variance and a Kruskal‐Wallis test were used to analyse the quantitative data statistically depending on the group numbers and normality of data distribution. The difference between the groups was considered significant when P‐value was less than 0.05.

Results

Acellular dental pulp matrices were generated following bovine dental pulp decellularisation. Evaluation of the developed DDPs revealed a significant DNA reduction (P < 0.0001) with preservation of the native histoarchitecture and vasculature and retention of VEGF‐A and FGF‐2. Upon recellularisation of the DDPs with hDPSCs, the in vitro analyses revealed cell engraftment with progressive repopulation of DDPs’ matrices and vasculature, and with enhanced expression of markers involved in angiogenesis. In vivo implantation of root slices with hDPSCs‐seeded DDPs revealed apparent vascularisation enhancement as compared to unseeded the DDPs group (P < 0.0001).

Conclusions

The developed decellularised dental pulp matrix has pro‐angiogenic properties and characterised by the retention of native vasculature and angiogenic growth factors. Seeding of hDPSCs into the DDP led to progressive repopulation of the vasculature, enhanced expression of markers involved in angiogenesis in hDPSCs and improved in vivo vascularisation capacity. The findings of this study suggest that a combination of DDP and hDPSCs might provide a promising vascularisation promoting strategy for dental pulp regeneration.