Inhibition of soluble epoxide hydrolase enhances the dentin-pulp complex regeneration mediated by crosstalk between vascular endothelial cells and dental pulp stem cells.

BACKGROUND: Revascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH. METHODS: In vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining. RESULTS: In vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-ß blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment. CONCLUSIONS: TPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-ß. TPPU boosts the angiogenic-odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration.

A carbon dot nanozyme hydrogel enhances pulp regeneration activity by regulating oxidative stress in dental pulpitis.

Preserving pulp viability and promoting pulp regeneration in pulpitis have attracted widespread attention. Restricted by the oxidative stress microenvironment of dental pulpitis, excessive reactive oxygen and nitrogen species (RONS) trigger uncontrolled inflammation and exacerbate pulp tissue destruction. However, modulating redox homeostasis in inflamed pulp tissue to promote pulp regeneration remains a great challenge. Herein, this work proposes an effective antioxidative system (C-NZ/GelMA) consisting of carbon dot nanozymes (C-NZ) with gelatin methacryloyl (GelMA) to modulate the pulpitis microenvironment for dental pulp regeneration by utilizing the antioxidant properties of C-NZ and the mechanical support of an injectable GelMA hydrogel. This system effectively scavenges RONS to normalize intracellular redox homeostasis, relieving oxidative stress damage. Impressively, it can dramatically enhance the polarization of regenerative M2 macrophages. This study revealed that the C-NZ/GelMA hydrogel promoted pulp regeneration and dentin repair through its outstanding antioxidant, antiapoptotic, and anti-inflammatory effects, suggesting that the C-NZ/GelMA hydrogel is highly valuable for pulpitis treatment.

Functional extracellular vesicles from SHEDs combined with gelatin methacryloyl promote the odontogenic differentiation of DPSCs for pulp regeneration.

BACKGROUND: Pulp regeneration is a novel approach for the treatment of immature permanent teeth with pulp necrosis. This technique includes the combination of stem cells, scaffolds, and growth factors. Recently, stem cell-derived extracellular vesicles (EVs) have emerged as a new methodology for pulp regeneration. Emerging evidence has proven that preconditioning is an effective scheme to modify EVs for better therapeutic potency. Meanwhile, proper scaffolding is of great significance to protect EVs from rapid clearance and destruction. This investigation aims to fabricate an injectable hydrogel loaded with EVs from pre-differentiated stem cells from human exfoliated deciduous teeth (SHEDs) and examine their effects on pulp regeneration. RESULTS: We successfully employed the odontogenic induction medium (OM) of SHEDs to generate functional EV (OM-EV). The OM-EV at a concentration of 20 µg/mL was demonstrated to promote the proliferation and migration of dental pulp stem cells (DPSCs). The results revealed that OM-EV has a better potential to promote odontogenic differentiation of DPSCs than common EVs (CM-EV) in vitro through Alizarin red phalloidin, alkaline phosphatase staining, and assessment of the expression of odontogenic-related markers. High-throughput sequencing suggests that the superior effects of OM-EV may be attributed to activation of the AMPK/mTOR pathway. Simultaneously, we prepared a photocrosslinkable gelatin methacryloyl (GelMA) to construct an OM-EV-encapsulated hydrogel. The hydrogel exhibited sustained release of OM-EV and good biocompatibility for DPSCs. The released OM-EV from the hydrogel could be internalized by DPSCs, thereby enhancing their survival and migration. In tooth root slices that were subcutaneously transplanted in nude mice, the OM-EV-encapsulated hydrogel was found to facilitate dentinogenesis. After 8 weeks, there was more formation of mineralized tissue, as well as higher levels of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1). CONCLUSIONS: The effects of EV can be substantially enhanced by preconditioning of SHEDs. The functional EVs from SHEDs combined with GelMA are capable of effectively promoting dentinogenesis through upregulating the odontogenic differentiation of DPSCs, which provides a promising therapeutic approach for pulp regeneration.

Chemically modified microRNA delivery via DNA tetrahedral frameworks for dental pulp regeneration.

Dental pulp regeneration is a promising strategy for addressing tooth disorders. Incorporating this strategy involves the fundamental challenge of establishing functional vascular networks using dental pulp stem cells (DPSCs) to support tissue regeneration. Current therapeutic approaches lack efficient and stable methods for activating DPSCs. In the study, we used a chemically modified microRNA (miRNA)-loaded tetrahedral-framework nucleic acid nanostructure to promote DPSC-mediated angiogenesis and dental pulp regeneration. Incorporating chemically modified miR-126-3p into tetrahedral DNA nanostructures (miR@TDNs) represents a notable advancement in the stability and efficacy of miRNA delivery into DPSCs. These nanostructures enhanced DPSC proliferation, migration, and upregulated angiogenesis-related genes, enhancing their paracrine signaling effects on endothelial cells. This enhanced effect was substantiated by improvements in endothelial cell tube formation, migration, and gene expression. Moreover, in vivo investigations employing matrigel plug assays and ectopic dental pulp transplantation confirmed the potential of miR@TDNs in promoting angiogenesis and facilitating dental pulp regeneration. Our findings demonstrated the potential of chemically modified miRNA-loaded nucleic acid nanostructures in enhancing DPSC-mediated angiogenesis and supporting dental pulp regeneration. These results highlighted the promising role of chemically modified nucleic acid-based delivery systems as therapeutic agents in regenerative dentistry and tissue engineering.

Influence of extracellular matrix scaffolds on histological outcomes of regenerative endodontics in experimental animal models: a systematic review.

BACKGROUND: Decellularized extracellular matrix (dECM) from several tissue sources has been proposed as a promising alternative to conventional scaffolds used in regenerative endodontic procedures (REPs). This systematic review aimed to evaluate the histological outcomes of studies utilizing dECM-derived scaffolds for REPs and to analyse the contributing factors that might influence the nature of regenerated tissues. METHODS: The PRISMA 2020 guidelines were used. A search of articles published until April 2024 was conducted in Google Scholar, Scopus, PubMed and Web of Science databases. Additional records were manually searched in major endodontic journals. Original articles including histological results of dECM in REPs and in-vivo studies were included while reviews, in-vitro studies and clinical trials were excluded. The quality assessment of the included studies was analysed using the ARRIVE guidelines. Risk of Bias assessment was done using the (SYRCLE) risk of bias tool. RESULTS: Out of the 387 studies obtained, 17 studies were included for analysis. In most studies, when used as scaffolds with or without exogenous cells, dECM showed the potential to enhance angiogenesis, dentinogenesis and to regenerate pulp-like and dentin-like tissues. However, the included studies showed heterogeneity of decellularization methods, animal models, scaffold source, form and delivery, as well as high risk of bias and average quality of evidence. DISCUSSION: Decellularized ECM-derived scaffolds could offer a potential off-the-shelf scaffold for dentin-pulp regeneration in REPs. However, due to the methodological heterogeneity and the average quality of the studies included in this review, the overall effectiveness of decellularized ECM-derived scaffolds is still unclear. More standardized preclinical research is needed as well as well-constructed clinical trials to prove the efficacy of these scaffolds for clinical translation. OTHER: The protocol was registered in PROSPERO database #CRD42023433026. This review was funded by the Science, Technology and Innovation Funding Authority (STDF) under grant number (44426).

Does the use of different scaffolds have an impact on the therapeutic efficacy of regenerative endodontic procedures? A systematic evaluation and meta-analysis.

BACKGROUND: In the regenerative endodontic procedures, scaffolds could influence the prognosis of affected teeth. Currently, there is controversy regarding the postoperative evaluation of various scaffolds for pulp regeneration. The objective of this study was to access whether other scaffolds, used alone or in combination with blood clot (BC), are more effective than BC in regenerative endodontic procedures. METHODS: We systematically search the PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL), Embase, and Google Scholar databases. Randomized controlled trials examining the use of BC and other scaffold materials in the regenerative endodontic procedures were included. A random effects model was used for the meta-analysis. The GRADE method was used to determine the quality of the evidence. RESULTS: We screened 168 RCTs related to young permanent tooth pulp necrosis through electronic and manual retrieval. A total of 28 RCTs were related to regenerative endodontic procedures. Ultimately, 12 articles met the inclusion criteria and were included in the relevant meta-analysis. Only 2 studies were assessed to have a low risk of bias. High quality evidence indicated that there was no statistically significant difference in the success rate between the two groups (RR=0.99, 95% CI=0.96 to 1.03; 434 participants, 12 studies); low-quality evidence indicated that there was no statistically significant difference in the increase in root length or root canal wall thickness between the two groups. Medium quality evidence indicated that there was no statistically significant difference in pulp vitality testing between the two groups. CONCLUSIONS: For clinical regenerative endodontic procedures, the most commonly used scaffolds include BC, PRP, and PRF. All the different scaffolds had fairly high clinical success rates, and the difference was not significant. For regenerative endodontic procedures involving young permanent teeth with pulp necrosis, clinical practitioners could choose a reasonable scaffold considering the conditions of the equipment and patients.

Research progress of dental pulp regeneration treatment. / å†ç”Ÿæ€§ç‰™é«“æ²»ç–—çš„ç ”ç©¶è¿›å±•.

The dental pulp is the only soft tissue structure within the tooth, serving functions such as sensation and nutrition. However, the dental pulp is highly susceptible to necrosis due to external factors. Currently, root canal therapy is the most commonly used treatment for pulp necrosis. Nevertheless, teeth treated with root canal therapy are prone to secondary infections and adverse outcomes like vertical root fractures. Regenerative endodontic therapy has emerged as a solution, aiming to replace damaged tooth structures, including dentin, root structure, and the pulp-dentin complex cells. This approach demonstrates significant advantages in addressing clinical symptoms and achieving regeneration of the root and even the pulp. Since the discovery of dental pulp stem cells, regenerative endodontic therapy has gained new momentum. Advances in cell transplantation and cell homing techniques have rapidly developed, showing promising potential for clinical applications.

Apoptotic vesicles activate autophagy in recipient cells to induce angiogenesis and dental pulp regeneration.

Extracellular vesicles (EVs) derived from living cells play important roles in donor cell-induced recipient tissue regeneration. Although numerous studies have found that cells undergo apoptosis after implantation in an ischemic-hypoxic environment, the roles played by the EVs released by apoptotic cells are largely unknown. In this study, we obtained apoptotic vesicles (apoVs) derived from human deciduous pulp stem cells and explored their effects on the dental pulp regeneration process. Our work showed that apoVs were ingested by endothelial cells (ECs) and elevated the expression of angiogenesis-related genes, leading to pulp revascularization and tissue regeneration. Furthermore, we found that, at the molecular level, apoV-carried mitochondrial Tu translation elongation factor was transported and regulated the angiogenic activation of ECs via the transcription factor EB-autophagy pathway. In a beagle model of dental pulp regeneration in situ, apoVs recruited endogenous ECs and facilitated the formation of dental-pulp-like tissue rich in blood vessels. These findings revealed the significance of apoptosis in tissue regeneration and demonstrated the potential of using apoVs to promote angiogenesis in clinical applications.

Research progress of odontogenic extracellular vesicles in regeneration of dental pulp.

It is well understood that maintaining viable pulp is critical for tooth retention. This review focused on cell-free therapy based on extracellular vesicles (EVs), a novel minimally invasive treatment strategy for endodontic restoration. This study was conducted by searching mainstream electronic databases such as Web of Science and PubMed for relevant studies on the therapeutic role of odontogenic EVs in pulp healing published in the last five years. We selected 89 relevant articles and discovered that dental stem cells (DSCs) derived EVs (DSC-EVs) have become a research hotspot in oral regenerative medicine, with significant advantages over cell transplantation in terms of low immunogenicity, ease of isolation, preservation, and management. Here, we introduce in detail the therapeutic effects of DSC-EVs for pulp restoration from three perspectives: excellent odontogenic properties, clinical applications, and possible molecular mechanisms. This article contributes a new viewpoint to the field of regenerative endodontics.

Injectable Xenogeneic Dental Pulp Decellularized Extracellular Matrix Hydrogel Promotes Functional Dental Pulp Regeneration.

The present challenge in dental pulp tissue engineering scaffold materials lies in the development of tissue-specific scaffolds that are conducive to an optimal regenerative microenvironment and capable of accommodating intricate root canal systems. This study utilized porcine dental pulp to derive the decellularized extracellular matrix (dECM) via appropriate decellularization protocols. The resultant dECM was dissolved in an acid pepsin solution to form dECM hydrogels. The analysis encompassed evaluating the microstructure and rheological properties of dECM hydrogels and evaluated their biological properties, including in vitro cell viability, proliferation, migration, tube formation, odontogenic, and neurogenic differentiation. Gelatin methacrylate (GelMA) hydrogel served as the control. Subsequently, hydrogels were injected into treated dentin matrix tubes and transplanted subcutaneously into nude mice to regenerate dental pulp tissue in vivo. The results showed that dECM hydrogels exhibited exceptional injectability and responsiveness to physiological temperature. It supported the survival, odontogenic, and neurogenic differentiation of dental pulp stem cells in a 3D culture setting. Moreover, it exhibited a superior ability to promote cell migration and angiogenesis compared to GelMA hydrogel in vitro. Additionally, the dECM hydrogel demonstrated the capability to regenerate pulp-like tissue with abundant blood vessels and a fully formed odontoblast-like cell layer in vivo. These findings highlight the potential of porcine dental pulp dECM hydrogel as a specialized scaffold material for dental pulp regeneration.

Poly(caprolactone)-aligned nanofibers associated with fibronectin-loaded collagen hydrogel as a potent bioactive scaffold for cell-free regenerative endodontics.

AIM: Guided tissue regeneration has been considered a promising strategy to replace conventional endodontic therapy of teeth with incomplete root formation. Therefore, the objective of this study was to develop a tubular scaffold (TB-SC) with poly (caprolactone)-aligned nanofibres associated with a fibronectin (FN)-loaded collagen hydrogel and assess the pulp regeneration potential mediated by human apical papilla cells (hAPCs) using an in vitro model of teeth with incomplete root formation. METHODOLOGY: Aligned nanofibre strips based on 10% poly(caprolactone) (PCL) were synthesized with the electrospinning technique to produce the TB-SCs. These were submitted to different treatments, according to the following groups: TB-SC (negative control): TB-SC without treatment; TB-SC + FN (positive control): TB-SC coated with 10 µg/ml of FN; TB-SC + H: TB-SC associated with collagen hydrogel; TB-SC + HFN: TB-SC associated with FN-loaded collagen hydrogel. Then, the biomaterials were inserted into cylindrical devices to mimic the regenerative therapy of teeth with incomplete root formation. The hAPCs were seeded on the upper surface of the TB-SCs associated or not with any treatment, and cell migration/proliferation and the gene expression of markers related to pulp regeneration (ITGA5, ITGAV, COL1A1 and COL1A3) were evaluated. The data were submitted to anova/Tukey's tests (α = 5%). RESULTS: Higher values of cell migration/proliferation and gene expression of all markers tested were observed in groups TB-SC + FN, TB-SC + H, and TB-SC + HFN compared with the TB-SC group (p < .05). The hAPCs in the TB-SC + HFN group showed the highest values of cell proliferation and gene expression of COL1A1 and COL3A1 (p < .05), as well as superior cell migration results to groups TB-SC and TB-SC + H (p < .05). CONCLUSION: Aligned nanofibre scaffolds associated with the FN-loaded collagen hydrogel enhanced the migration and proliferation of hAPCs and gene expression of pulp regeneration markers. Therefore, the use of these biomaterials may be considered an interesting strategy for regenerative pulp therapy of teeth with incomplete root formation.

A critical analysis of research methods and biological experimental models to study pulp regeneration.

With advances in knowledge and treatment options, pulp regeneration is now a clear objective in clinical dental practice. For this purpose, many methodologies have been developed in attempts to address the putative questions raised both in research and in clinical practice. In the first part of this review, laboratory-based methods will be presented, analysing the advantages, disadvantages, and benefits of cell culture methodologies and ectopic/semiorthotopic animal studies. This will also demonstrate the need for alignment between two-dimensional and three-dimensional laboratory techniques to accomplish the range of objectives in terms of cell responses and tissue differentiation. The second part will cover observations relating to orthotopic animal studies, describing the current models used for this purpose and how they contribute to the translation of regenerative techniques to the clinic.

Outcomes of root canal therapy or full pulpotomy using two endodontic biomaterials in mature permanent teeth: a randomized controlled trial.

OBJECTIVE: The concept of minimally invasive endodontics recommends less-invasive vital pulp therapy (VPT) modalities over more aggressive traditional endodontic approaches in mature permanent teeth with carious pulp exposure, including irreversible pulpitis (IP) cases. Consequently, VPT needs to be compared with root canal therapy (RCT) in terms of treatment outcomes. This randomized clinical trial compares the results of full pulpotomy using two calcium-silicate cements, i.e., mineral trioxide aggregate (MTA) and calcium-enriched mixture (CEM) cement, with RCT in mature permanent teeth. MATERIALS AND METHODS: A total of 157 carious pulp exposure cases in two academic centers with/without established IP were selected/included/randomly appointed to three study arms; (i) RCT (n = 51) as the reference treatment, (ii) pulpotomy with ProRoot MTA (PMTA; n = 55), and (iii) pulpotomy with CEM cement (PCEM; n = 51) as two alternative VPT treatments. Two-year clinical/radiographic results were the outcomes of interest. Data were statistically analyzed through the analysis of variance, chi-square, Fisher exact test, and Kruskal-Wallis. RESULTS: At 2-year recall, 147 teeth were examined (6.4% dropout). All molars, except for one, were clinically functional/symptom-free, and there was no statistical difference between the three study arms (p = 0.653). The radiographic success rates in RCT, PMTA, and PCEM arms were 98%, 100%, and 97.9%, respectively, without statistically significant differences (p = 0.544). CONCLUSION: In the management of mature permanent teeth with/without established IP, all experimental groups exhibited equivalent/comparable results. CLINICAL RELEVANCE: Simple VPT using MTA/CEM can be suggested/recommended as a viable advantageous alternative to RCT for the management of carious pulp exposures with/without sign/symptoms of IP.

Evaluation of a hyaluronic acid hydrogel (Restylane Lyft) as a scaffold for dental pulp regeneration in a regenerative endodontic organotype model.

Scaffolds are crucial elements for dental pulp regeneration. Most of the currently used scaffolds in regenerative endodontic procedures (REPs) are unsuitable for chairside clinical use. This study aimed to evaluate the effect of an injectable synthetic scaffold (Restylane Lyft) on human bone marrow mesenchymal stem cell (hBMSC) viability, proliferation, and osteo/dentinogenic differentiation in a regenerative endodontic organotype model (REM). hBMSC were loaded in an REM either alone (hBMSC group) or mixed with the Restylane Lyft scaffold (Restylane/hBMSC group) and cultured in basal culture medium (n = 9/group). hMSC on culture plates served as controls. Cell viability and proliferation were measured using AlamarBlue assay. The loaded REM was cultured in an osteogenic differentiation medium to measure alkaline phosphatase activity (ALP) and examine the expression of the osteo/dentinogenic markers using real-time reverse transcriptase polymerase chain reaction. Cell viability in all groups increased significantly over 5 days. The Restylane/hBMSC group showed significantly higher ALP activity and dentin sialophosphoprotein, osteocalcin, and bone sialoprotein genes expression than the hBMSC and the control groups. Restylane Lyft, a hyaluronic acid (HA) injectable, FDA-approved hydrogel, maintained cell viability and proliferation and promoted osteo/dentinogenic differentiation of hBMSC when cultured in an REM. Henceforth, it could be a promising chairside scaffold material for REPs.

Exosome-Based Cell Homing and Angiogenic Differentiation for Dental Pulp Regeneration.

Exosomes have attracted attention due to their ability to promote intercellular communication leading to enhanced cell recruitment, lineage-specific differentiation, and tissue regeneration. The object of this study was to determine the effect of exosomes on cell homing and angiogenic differentiation for pulp regeneration. Exosomes (DPSC-Exos) were isolated from rabbit dental pulp stem cells cultured under a growth (Exo-G) or angiogenic differentiation (Exo-A) condition. The characterization of exosomes was confirmed by nanoparticle tracking analysis and an antibody array. DPSC-Exos significantly promoted cell proliferation and migration when treated with 5 × 108/mL exosomes. In gene expression analysis, DPSC-Exos enhanced the expression of angiogenic markers including vascular endothelial growth factor A (VEGFA), Fms-related tyrosine kinase 1 (FLT1), and platelet and endothelial cell adhesion molecule 1 (PECAM1). Moreover, we identified key exosomal microRNAs in Exo-A for cell homing and angiogenesis. In conclusion, the exosome-based cell homing and angiogenic differentiation strategy has significant therapeutic potential for pulp regeneration.

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

AIM: To investigate the vascularization capacity of a decellularized 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 decellularized and then analysed using histological staining and DNA quantification. The resultant DDPs were characterized using immunohistochemical staining for the retention of vascular endothelial growth factor A (VEGF-A) and fibroblast growth factor 2 (FGF-2). Furthermore, DDPs were recellularized with hDPSCs and analysed histologically. The expression of markers involved in angiogenesis by hDPSCs colonizing 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 the P-value was less than 0.05. RESULTS: Acellular dental pulp matrices were generated following bovine dental pulp decellularization. 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 recellularization 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 vascularization enhancement as compared to the unseeded DDP group (P < 0.0001). CONCLUSIONS: The developed decellularized dental pulp matrix had pro-angiogenic properties characterized 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 vascularization capacity. The se suggest that a combination of DDP and hDPSCs have the potential to provide a promising vascularization promoting strategy for dental pulp regeneration.

Large three-dimensional cell constructs for tissue engineering.

Much research has been conducted on fabricating biomimetic biomaterials in vitro. Tissue engineering approaches are often conducted by combining cells, scaffolds, and growth factors. However, the degradation rate of scaffolds is difficult to control and the degradation byproducts occasionally limit tissue regeneration. To overcome these issues, we have developed a novel system using a thermo-responsive hydrogel that forms scaffold-free, three-dimensional (3D) cell constructs with arbitrary size and morphology. 3D cell constructs prepared using bone marrow-derived stromal stem cells (BMSCs) exhibited self-organizing ability and formed bone-like tissue with endochondral ossification. Endothelial cells were then introduced into the BMSC construct and a vessel-like structure was formed within the constructs. Additionally, the bone formation ability was promoted by endothelial cells and cell constructs could be freeze-dried to improve their clinical application. A pre-treatment with specific protein protectant allowed for the fabrication of novel bone substitutes composed only of cells. This 3D cell construct technology using thermo-responsive hydrogels was then applied to other cell species. Cell constructs composed of dental pulp stem cells were fabricated, and the resulting construct regenerated pulp-like tissue within a human pulpless tooth. In this review, we demonstrate the approaches for the in vitro fabrication of bone and dental pulp-like tissue using thermo-responsive hydrogels and their potential applications.

A Cell-Based Approach to Dental Pulp Regeneration Using Mesenchymal Stem Cells: A Scoping Review.

Despite the recent explosion of investigations on dental pulp regeneration using various tissue engineering strategies, the translation of the findings from such studies into therapeutic applications has not been properly achieved. The purpose of this scoping review was to systematically review the efficacy of mesenchymal stem cell transplantation for dental pulp regeneration. A literature search was conducted using five electronic databases from their inception to January 2021 and supplemented by hand searches. A total of 17 studies, including two clinical trials and 15 animal studies using orthotopic pulp regeneration models, were included for the review. The risk of bias for the individual studies was assessed. This scoping review demonstrated that the regeneration of vascularized pulp-like tissue was achieved using the stem cell transplantation strategy in animal models. Autologous cell transplantation in two clinical studies also successfully regenerated vascularized vital tissue. Dental pulp stem cell subpopulations, such as mobilized dental pulp stem cells, injectable scaffolds such as atelocollagen, and a granulocyte-colony forming factor, were the most commonly used for pulp regeneration. The overall risk of bias was unclear for animal studies and was moderate or judged to raise some concerns for clinical studies. More high-quality clinical studies are needed to further determine the safety and efficacy of the stem cell transplantation strategy for dental pulp regeneration.

Functional Dental Pulp Regeneration: Basic Research and Clinical Translation.

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.

Polymeric Scaffolds for Dental, Oral, and Craniofacial Regenerative Medicine.

Dental, oral, and craniofacial (DOC) regenerative medicine aims to repair or regenerate DOC tissues including teeth, dental pulp, periodontal tissues, salivary gland, temporomandibular joint (TMJ), hard (bone, cartilage), and soft (muscle, nerve, skin) tissues of the craniofacial complex. Polymeric materials have a broad range of applications in biomedical engineering and regenerative medicine functioning as tissue engineering scaffolds, carriers for cell-based therapies, and biomedical devices for delivery of drugs and biologics. The focus of this review is to discuss the properties and clinical indications of polymeric scaffold materials and extracellular matrix technologies for DOC regenerative medicine. More specifically, this review outlines the key properties, advantages and drawbacks of natural polymers including alginate, cellulose, chitosan, silk, collagen, gelatin, fibrin, laminin, decellularized extracellular matrix, and hyaluronic acid, as well as synthetic polymers including polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), poly (ethylene glycol) (PEG), and Zwitterionic polymers. This review highlights key clinical applications of polymeric scaffolding materials to repair and/or regenerate various DOC tissues. Particularly, polymeric materials used in clinical procedures are discussed including alveolar ridge preservation, vertical and horizontal ridge augmentation, maxillary sinus augmentation, TMJ reconstruction, periodontal regeneration, periodontal/peri-implant plastic surgery, regenerative endodontics. In addition, polymeric scaffolds application in whole tooth and salivary gland regeneration are discussed.