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Dental pulp is the only soft tissue in the tooth which plays a crucial role in maintaining intrinsic multi-functional behaviors of the dentin-pulp complex. Nevertheless, the restoration of fully functional pulps after pulpitis or pulp necrosis, termed endodontic regeneration, remained a major challenge for decades. Therefore, a bioactive and in-situ injectable biomaterial is highly desired for tissue-engineered pulp regeneration. Herein, a decellularized matrix hydrogel derived from porcine dental pulps (pDDPM-G) was prepared and characterized through systematic comparison against the porcine decellularized nerve matrix hydrogel (pDNM-G). The pDDPM-G not only exhibited superior capabilities in facilitating multi-directional differentiation of dental pulp stem cells (DPSCs) during 3D culture, but also promoted regeneration of pulp-like tissues after DPSCs encapsulation and transplantation. Further comparative proteomic and transcriptome analyses revealed the differential compositions and potential mechanisms that endow the pDDPM-G with highly tissue-specific properties. Finally, it was realized that the abundant tenascin C (TNC) in pDDPM served as key factor responsible for the activation of Notch signaling cascades and promoted DPSCs odontoblastic differentiation. Overall, it is believed that pDDPM-G is a sort of multi-functional and tissue-specific hydrogel-based material that holds great promise in endodontic regeneration and clinical translation. STATEMENT OF SIGNIFICANCE: Functional hydrogel-based biomaterials are highly desirable for endodontic regeneration treatments. Decellularized extracellular matrix (dECM) preserves most extracellular matrix components of its native tissue, exhibiting unique advantages in promoting tissue regeneration and functional restoration. In this study, we prepared a porcine dental pulp-derived dECM hydrogel (pDDPM-G), which exhibited superior performance in promoting odontogenesis, angiogenesis, and neurogenesis of the regenerating pulp-like tissue, further showed its tissue-specificity compared to the peripheral nerve-derived dECM hydrogel. In-depth proteomic and transcriptomic analyses revealed that the activation of tenascin C-Notch axis played an important role in facilitating odontogenic regeneration. This biomaterial-based study validated the great potential of the dental pulp-specific pDDPM-G for clinical applications, and provides a springboard for research strategies in ECM-related regenerative medicine., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Human dental pulp stem cells (hDPSCs) play a vital role in the regeneration of the pulp-dentin complex after pulp disease. While the regeneration efficiency relies on the odontoblastic differentiation capacity of hDPSCs, this is difficult to regulate within the pulp cavity. Although nicotinamide riboside (NR) has been found to promote tissue regeneration, its specific role in pulp-dentin complex regeneration is not fully understood. Here, we aimed to explore the role of NR in the odontoblastic differentiation of hDPSCs and its underlying molecular mechanism. It was found that NR enhanced the viability and retarded senescence in hDPSCs with higher NAD+/NADH levels. In contrast to the sustained action of NR, the multi-directional differentiation of hDPSCs was enhanced after NR pre-treatment. Moreover, in an ectopic pulp regeneration assay in nude mice, transplantation of hDPSCs pretreated with NR promoted the formation of a dentin-like structure surrounded by cells positively expressing DMP-1 and DSPP. RNA-Seq demonstrated inhibition of the HIF-1 signaling pathway in hDPSCs pretreated with NR. The number of HIF-1α-positive cells was significantly decreased in hDPSCs pretreated by NR in vivo. Similarly, NR significantly downregulated the expression of HIF-1α in vitro. The findings suggested that NR could potentially regulate hDPSC odontoblastic differentiation and promote the development of innovative strategies for dental pulp repair., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pulpal and periapical diseases account for a large proportion of dental visits, the current treatments for which are root canal therapy (RCT) and pulp revascularisation. Despite the clinical signs of full recovery and histological reconstruction, true regeneration of pulp tissues is still far from being achieved. The goal of regenerative endodontics is to promote normal pulp function recovery in inflamed or necrotic teeth that would result in true regeneration of the pulpodentinal complex. Recently, rapid progress has been made related to tissue engineering-mediated pulp regeneration, which combines stem cells, biomaterials, and growth factors. Since the successful isolation and characterisation of dental pulp stem cells (DPSCs) and other applicable dental mesenchymal stem cells, basic research and preclinical exploration of stem cell-mediated functional pulp regeneration via cell transplantation and cell homing have received considerably more attention. Some of this effort has translated into clinical therapeutic applications, bringing a ground-breaking revolution and a new perspective to the endodontic field. In this article, we retrospectively examined the current treatment status and clinical goals of pulpal and periapical diseases and scrutinized biological studies of functional pulp regeneration with a focus on DPSCs, biomaterials, and growth factors. Then, we reviewed preclinical experiments based on various animal models and research strategies. Finally, we summarised the current challenges encountered in preclinical or clinical regenerative applications and suggested promising solutions to address these challenges to guide tissue engineering-mediated clinical translation in the future.

Introduction: Dental pulp is a major composition in the pulp-dentin complex, which serves as protective system against dental trauma/infection. Functional dental pulp regeneration is highly desirable after pulpitis or pulp necrosis. However, endodontic regeneration has remained challenging for decades because of the deconstructive microenvironment and the lack of functional cells within the root canal system. The present study developed a decellularized matrix hydrogel derived from human dental pulp (hDDPM-G), which might serve as a growth-permissive microenvironment for dental pulp regeneration., Methods: Human dental pulps extracted from healthy wisdom teeth were decellularized and digested and then underwent sol-gel transition to form hDDPM-G. The protein compositions were identified by proteomic analysis. Human dental pulp stem cells (hDPSCs) were seeded on hDDPM-G-coated surfaces and evaluated by immunofluorescence staining, transwell migration, and Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) assays. Induced hDPSC differentiation was examined in vitro and characterized by immunostaining, Western blotting, and reverse transcription polymerase chain reaction., Results: Complete decellularization was implemented. Protein contents found in the human decellularized dental pulp matrix were identified to contribute in promoting cell proliferation, migration, and regulation of stem cell differentiation. The hDDPM-G-coated surfaces promoted hDPSC adhesion, migration, and proliferation. Furthermore, hDDPM-G coatings facilitated odontoblastlike, neural-like, and angiogenic differentiation of the seeded hDPSCs after being cultured in induction media for 14 days., Conclusions: This study showed that hDDPM-G effectively contributed in promoting hDPSC proliferation and migration and induced multidirectional differentiation. Considering the injectability and gelation at body temperature, hDDPM-G may hold translational potential for endodontic regeneration., (Copyright © 2020 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The AIM2 inflammasome pathway has been determined to play an important role in cellular immune defense against bacterial and viral infections; however, its function and regulatory mechanism in human dental pulp cells (HDPCs) during pulpitis remains poorly understood. In this study, we explored whether the AIM2 inflammasome pathway was activated in HDPCs in response to dsDNA and defined its role in regulating IL-1β secretion. We demonstrated that stimulation with IFN-γ and cytoplasmic DNA significantly activated the AIM2 inflammasome and increased IL-1β secretion in HDPCs. Moreover, AIM2 overexpression significantly up-regulated both cleaved Caspase-1 expression and IL-1β release in HDPCs, while suppression of ASC and Caspase-1 resulted in down-regulation of cleaved Caspase-1 and IL-1β secretion. These results suggest that Caspase-1-dependent IL-1β processing and secretion require the AIM2 inflammasome pathway in HDPCs and that the AIM2 inflammasome pathway is critical for regulation of the dental pulp immune response.

This present study investigated the potential of adeno-associated virus serotype 2 (AAV2) mediated BMP-7 (AAV2-BMP-7) to induce odontoblastic differentiation of human dental pulp cells (DPCs) in vitro. AAV2-BMP-7 was constructed to overexpress BMP-7, and the biologic effects of BMP-7 on DPCs were investigated by the evaluation of the activity of alkaline phosphatase (ALPase), the detection of the expression of dentin sialophosphoprotein (DSPP) and osteocalcin (OCN) expression and the analysis of the proliferative ability of the cells. DPCs that were infected with AAV2-BMP-7 displayed significantly upregulated ALP activity and formed mineralized nodules. Moreover, AAV2-BMP-7 promoted the expression of mineralization-related genes, which included DSPP and OCN. In addition, there was no significant difference between the proliferative ability of AAV2-BMP-7 and the control group. In conclusion, AAV2-BMP-7 promoted the odontoblastic differentiation in DPCs, a clear indication of the therapeutic potential of AAV2-BMP-7 in dental tissue regeneration.

Enhanced odontoblast differentiation of human dental pulp cells (hDPCs) is considered a keystone in dentin-pulp complex formation. We have revealed lncRNA DANCR was implicated in this differentiation program, however, its mechanism in odontoblast differentiation of hDPCs remains further explored. In this study, by employing loss-of-function approach, we identified downregulation of DANCR drived odontoblast differentiaion of hDPCs. Bioinformatics analysis was utilized to show that DANCR contained binding site for miR-216a and an inverse correlation between DANCR and miR-216a was obtained. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) were applied to further confirm that DANCR conferred its functions by directly binding to miR-216a. Notably, miR-216a was able to bind to the 3′-UTR of c-Cbl and repressed its expression. In addition, the protein level of c-CBL was significantly downregulated during hDPCs differentiation, while c-Cbl overexpression inhibited odontoblast differentiation of hDPCs. Moreover, downregulation of miR-216a efficiently reversed the suppression of c-Cbl level and odontoblast differentiation induced by knockdown of DANCR. Taken together, these analyses indicated that DANCR positively regulated the expression of c-Cbl, through sponging miR-216a, and inhibited odontoblast differentiation of hDPCs. Our results will extend the field of clinical application for cell-based therapy in regenerative medicine. • DANCR exerts important roles in controlling odontoblast differentiation. • Downregulation of DANCR augments odontoblast differentiation. • DANCR acts as a molecular sponge for miR-216a. • MiR-216a directly targets c-Cbl 3′-UTR. • DANCR inhibits odontoblast differentiation by controlling the miR-216a target, c-Cbl. [ABSTRACT FROM AUTHOR]