Optimizing Methods for Bovine Dental Pulp Decellularization

Introduction: This study aimed to characterize the decellularization effects of different treatment protocols on the bovine dental pulp extracellular matrix (ECM) for tissue regeneration. Methods: Seven different decellularization protocols consisting of trypsin/EDTA (for 1 hour, 24 hours, or 48 hours), sodium dodecyl sulfate (SDS, for 24 hours or 48 hours), Triton X-100 (for 1 hour), and deoxyribonuclease treatments were tested on bovine dental pulp tissue. The posttreatment samples were evaluated for remaining DNA and cellular contents, structural durability, immunofluorescence analysis, and in vivo immune responses. Results: A complete decellularization process in all of the experimental groups was observed. The protocol that included 1 hour of Triton X-100 treatment and 12 hours of trypsin/EDTA treatment with no SDS treatment (P7 [12E-0S-1T]) showed the highest retention of glycosaminoglycan and the absence of nuclei in 4,6-diamidino-2-phenylindole. All groups showed significantly lower DNA content compared with native pulp tissue (P < .05), whereas compared with other protocols, protocols 1 (1 hour of EDTA/trypsin, 24 hours of SDS, and 1 hour of Triton X-100) and 4 (1 hour of EDTA/Trypsin, 48 hours of SDS, and no Triton X-100) resulted in the highest DNA contents (P < .05). Based on these results, P7 was further evaluated by immunofluorescence and in vivo immunogenicity. P7 specimens preserved collagen type I, whereas mononuclear cell infiltration along with neovascularization was observed in vivo. Conclusions: All tested treatments displayed the potential ability to decellularize pulp tissue and are viable options for a xenogeneic dental pulp ECM scaffold. The P7 (12E-0S-1T) protocol resulted in decellularized ECM with minimal organic matrix/ultrastructural detriments and an acceptable host immune response.