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Regenerative endodontics aims to restore pulp tissues, thus preserving the vitality of the tooth. One promising approach involves the utilization of decellularized human dental pulp (DHDP) as a scaffold repopulated with Wharton's Jelly mesenchymal stem cells (WJMSCs). This study aimed to regenerate pulp tissues using DHDP and WJMSCs following pulpectomy in mature canine teeth of a feline animal model and to investigate the histological features of the regenerated pulp. A 12-month-old male domestic shorthaired felines were used as subjects. Teeth were categorized into untreated (Group 1), pulpectomy with mineral trioxide aggregate (MTA) (Group 2), and pulpectomy with DHDP-repopulated scaffold and MTA (Group 3). The animals were sacrificed six weeks post-intervention. H&E and immunohistochemistry using anti-collagen type 1 and laminin antibodies were used to stain the tissue sections. Histological examinations presented pulp-like tissues in Group 3, with tissue components similar to the structures found in Group 1. Immunohistochemical analysis demonstrated the presence of collagen type I and laminin within the regenerated tissues. The root canals of teeth in Group 2 were devoid of pulpal tissue. DHDP with WJMSCs can potentially be used for pulp regeneration, supporting the modality for developing new clinical protocols in stem cell therapy., Competing Interests: Declaration of Competing Interest The authors deny any conflicts of interest related to this study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aim: To evaluate the role of biomimetic pulp scaffolds derived from the extracellular matrix derived of stem cells from human exfoliated deciduous teeth (SHED-ECM-PS) in promoting pulp-dentine complex regeneration., Methodology: SHED-ECM-PS was prepared through cell aggregation and decellularization techniques. Histological and immunofluorescence analyses, scanning electron microscopy, and DNA quantification assays were used to characterize the SHED-ECM-PS. Additionally, a tooth slice implantation model was established to evaluate the effects of SHED-ECM-PS on regeneration of the pulp-dentine complex in vivo. Extraction medium for SHED-ECM-PS was prepared, and its effect on bone marrow mesenchymal stem cells (BMMSCs) was assessed in vitro. Cell counting kit-8 and Ki-67 staining assays were performed to determine cell proliferation. The rate of apoptosis was evaluated by flow cytometry. Wound healing and transwell assays were conducted to evaluate cell migration. Alizarin red S staining was performed to examine mineralized nodule formation. Western blotting was used to detect the expression of osteogenic and odontogenic markers. The results were analysed using an independent two-tailed Student's t-test. p < .05 was considered statistically significant., Results: SHED-ECM-PS was successfully constructed, exhibiting a striped dental pulp-like shape devoid of nuclear structures or DNA components, and rich in fibronectin, collagen I, DMP1 and DSPP. Notably, SHED-ECM-PS showed no impact on the proliferation or apoptosis of BMMSCs. Histological analysis revealed that dental pulp fibroblasts formed an interwoven mesh in the root canal, and angiogenesis was observed in the SHED-ECM-PS group. Moreover, a continuous, newly formed tubular dentine layer with polarized odontoblast-like cells was observed along the inner wall of the root canal. SHED-ECM-PS promoted the migration, polar alignment and mineralized nodule formation of BMMSCs and specifically elevated the expression levels of odontogenic markers, but not osteogenic markers, compared with the control group in vitro., Conclusion: SHED-ECM-PS exhibited no cytotoxicity and promoted pulp-dentine complex regeneration in vivo as well as cell migration and odontogenic differentiation of BMMSCs in vitro. These findings provide evidence that SHED-ECM-PS, as a novel biological scaffold, has the potential to improve the outcomes of REPs., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Successful dental pulp regeneration is closely associated with rapid revascularization and angiogenesis, processes driven by the Jagged1(JAG1)/Notch signaling pathway. However, soluble Notch ligands have proven ineffective in activating this pathway. To overcome this limitation, a Notch signaling hydrogel is developed by indirectly immobilizing JAG1, aimed at precisely directing the regeneration of vascularized pulp tissue. This hydrogel displays favorable mechanical properties and biocompatibility. Cultivating dental pulp stem cells (DPSCs) and endothelial cells (ECs) on this hydrogel significantly upregulate Notch target genes and key proangiogenic markers expression. Three-dimensional (3D) culture assays demonstrate Notch signaling hydrogels improve effectiveness by facilitating encapsulated cell differentiation, enhancing their paracrine functions, and promoting capillary lumen formation. Furthermore, it effectively communicates with the Wnt signaling pathway, creating an odontoinductive microenvironment for pulp-dentin complex formation. In vivo studies show that short-term transplantation of the Notch signaling hydrogel accelerates angiogenesis, stabilizes capillary-like structures, and improves cell survival. Long-term transplantation further confirms its capability to promote the formation of pulp-like tissues rich in blood vessels and peripheral nerve-like structures. In conclusion, this study introduces a feasible and effective hydrogel tailored to specifically regulate the JAG1/Notch signaling pathway, showing potential in advancing regenerative strategies for dental pulp tissue., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Objectives: This study aims to compare the application of three types of normal scaffolds-native chitosan, enzymatically modified chitosan, and blood clot (BC)-on pulp regeneration in the teeth of experimental dogs through histological examination, to determine the quantity and type of new tissues formed within the root canal., Materials and Methods: The research sample consisted of 32 root canals from 20 premolars of two male local experimental dogs. The sample was randomly divided into a control group, in which no intervention was performed on the teeth, and three experimental groups based on the type of scaffold used: the BC group, the native chitosan combined with BC (NCS + BC) group, and the enzymatically modified chitosan combined with BC (EMCS + BC) group. Mechanical and chemical cleaning of the canals was performed, followed by the application of the studied scaffolds within the root canals. After 3 months, the teeth were extracted and prepared for histological study, where two variables were studied: the percentage of total vital tissue (soft and hard; VT%) and the percentage of soft vital tissue only (ST%). A one-way ANOVA and Bonferroni tests were used to determine significant differences between the groups at a 95% confidence level., Results: The VT% values were significantly higher in the EMCS + BC group compared to both the NCS + BC and BC groups. The ST% values were also significantly higher in the EMCS + BC group compared to the BC group. However, no significant differences in ST% values were observed between the NCS + BC group and either the BC or EMCS + BC groups., Conclusions: Within the limitations of this study, we conclude that the application of enzymatically modified chitosan scaffolds combined with BC yields superior results in pulp regeneration, which contributes to the formation of pulp-like tissue and cells resembling odontoblasts, as well as apex closure with tissue resembling bone tissue., (© 2024 The Author(s). Clinical and Experimental Dental Research published by John Wiley & Sons Ltd.)

Aim: Angiogenesis is a key event in the successful healing of pulp injuries, and hypoxia is the main stimulator of pulpal angiogenesis. In this study, we investigated the effect of hypoxia on the proangiogenic potential of human dental pulp stem cells (hDPSCs) and the role of miR-143-5p in the process., Methodology: Human dental pulp stem cells were isolated, cultured and characterized in vitro. Cobalt chloride (CoCl 2 ) was used to induce hypoxia in hDPSCs. CCK-8 and Transwell assays were used to determine the effect of hypoxia on hDPSCs proliferation and migration. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting (WB) and ELISA were performed to assess the mRNA and protein levels of HIF-1α and angiogenic cytokines in hDPSCs. The effect of hypoxia on hDPSCs proangiogenic potential was measured in vitro using Matrigel tube formation and chick chorioallantoic membrane (CAM) assays. Recombinant lentiviral vectors were constructed to stably overexpress or inhibit miR-143-5p in hDPSCs, and the proangiogenic effects were assessed using qRT-PCR, WB, and tube formation assays. miR-143-5p target genes were identified and verified using bioinformatics prediction tools, dual-luciferase reporter assays and RNA pull-down experiments. Finally, a subcutaneous transplantation model in nude mice was used to determine the effects of hypoxia treatment and miR-143-5p overexpression/inhibition in hDPSCs in dental pulp regeneration., Results: Hypoxia promotes hDPSCs proliferation, migration and proangiogenic potential. The in vivo experiments showed that hypoxia treatment (50 and 100 μM CoCl 2 ) promoted pulp angiogenesis and dentine formation. In contrast to the levels of proangiogenic factors, miR-143-5p levels decreased with increasing CoCl 2 concentration. miR-143-5p inhibition significantly promoted proangiogenic potential of hDPSCs, whereas miR-143-5p overexpression inhibited angiogenesis in vitro. Dual-luciferase reporter assay identified retinoic acid receptor-related orphan receptor alpha (RORA) as an miR-143-5p target gene in hDPSCs. RNA pull-down experiments demonstrated that HIF-1α and RORA were pulled down by biotin-labelled miR-143-5p, and the levels of HIF-1α and RORA bound to miR-143-5p in the hypoxia group were lower than those in the normoxia group. Inhibition of miR-143-5p expression in hDPSCs promoted ectopic dental pulp tissue regeneration., Conclusions: CoCl 2 -induced hypoxia promotes hDPSCs-driven paracrine angiogenesis and pulp regeneration. The inhibition of miR-143-5p upregulates the proangiogenic potential of hDPSCs under hypoxic conditions by directly targeting HIF-1α and RORA., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Dental diseases, such as dental caries and periodontal disorders, constitute a major global health challenge, affecting millions worldwide and often resulting in tooth loss. Traditional dental treatments, though beneficial, typically cannot fully restore the natural functions and structures of teeth. This limitation has prompted growing interest in innovative strategies for tooth regeneration methods. Among these, the use of dental stem cells to generate functional tooth modules represents an emerging and promising approach in dental tissue engineering. These modules aim to closely replicate the intricate morphology and essential physiological functions of dental tissues. Recent advancements in regenerative research have not only enhanced the assembly techniques for these modules but also highlighted their therapeutic potential in addressing various dental diseases. In this review, we discuss the latest progress in the construction of functional tooth modules, especially on regenerating dental pulp, periodontal tissue, and tooth roots.

Objective: Pulp regeneration with bioactive dentin-pulp complex has been a research hotspot in recent years. Stem cell therapy provided an interest strategy to regenerate the dental-pulp complex. Hence, this study aimed to evaluate the effects of photosensitive gelatin methacrylate (GelMA) hydrogel encapsulating dental pulp stem cells (DPSCs) and silver nanoparticles (AgNPs) for dental pulp regeneration in vitro., Methods: First, the AgNPs@GelMA hydrogels were prepared by lithium phenyl-2,4,6-trimethyl-benzoyl phosphinate (LAP) initiation via blue-light emitting diode light. The physical and chemical properties of AgNPs@GelMA hydrogels were comprehensively analysed via scanning electron microscopy (SEM), and mechanical characterisation, such as swelling ability, degradation properties, and AgNP release profile. Then, AgNPs@GelMA hydrogels encapsulated DPSCs were used to establish an AgNPs@GelMA biomimetic complex, further analysing its biocompatibility, antibacterial properties, and angiogenic capacity in vitro., Results: The results indicated that GelMA hydrogels demontrated optimal characteristics with a monomer:LAP ratio of 16:1. The physico-chemical properties of AgNPs@GelMA hydrogels did not change significantly after loading with AgNPs. There was no significant difference in AgNP release rate amongst different concentrations of AgNPs@GelMA hydrogels. Fifty to 200 μg/mL AgNPs@GelMA hydrogels could disperse E faecalis biofilm and reduce its metabolic activity . Furthermore, cell proliferation was arrested in 100 and 200 μg/mL AgNPs@GelMA hydrogels. The inhibition of 50 μg/mL AgNPs@GelMA hydrogels on E faecalis biofilm was above 50%, and the cell viability of the hydrogels was higher than 90%. The angiogenesis assay indicated that AgNPs@GelMA hydrogels encapsulating DPSCs could induce the formation of capillary-like structures and express angiogenic markers CD31, vascular endothelial growth factor , and von willebrand factor (vWF) in vitro., Conclusions: Results of this study indicate that 50 μg/mL AgNPs@GelMA hydrogels encapsulating DPSCs had significant antibacterial properties and angiogenic capacity, which could provide a significant experimental basis for the regeneration of the dentin-pulp complex., Competing Interests: Conflict of interest None disclosed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Pulp and periapical diseases can lead to the cessation of tooth development, resulting in compromised tooth structure and functions. Despite numerous efforts to induce pulp regeneration, effective strategies are still lacking. Growth factors (GFs) hold considerable promise in pulp regeneration due to their diverse cellular regulatory properties. However, the limited half-lives and susceptibility to degradation of exogenous GFs necessitate the administration of supra-physiological doses, leading to undesirable side effects. In this research, a heparin-functionalized bioactive glass (CaO-P 2 O 5 -SiO 2 -Heparin, abbreviated as PSC-Heparin) with strong bioactivity and a stable neutral pH is developed as a promising candidate to addressing challenges in pulp regeneration. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis reveal the successful synthesis of PSC-Heparin. Scanning electron microscopy and X-ray diffraction show the hydroxyapatite formation can be observed on the surface of PSC-Heparin after soaking in simulated body fluid for 12 h. PSC-Heparin is capable of harvesting various endogenous GFs and sustainably releasing them over an extended duration by the enzyme-linked immunosorbent assay. Cytological experiments show that developed PSC-Heparin can facilitate the adhesion, migration, proliferation, and odontogenic differentiation of stem cells from apical papillae. Notably, the histological analysis of subcutaneous implantation in nude mice demonstrates PSC-Heparin is capable of promoting the odontoblast-like layers and pulp-dentin complex formation without the addition of exogenous GFs, which is vital for clinical applications. This work highlights an effective strategy of harvesting endogenous GFs and avoiding the involvement of exogenous GFs to achieve pulp-dentin complex regeneration, which may open a new horizon for regenerative endodontic therapy.

The process of neovascularization during cell-based pulp regeneration is difficult to study. Here we developed a tube model that simulates root canal space and allows direct visualization of the vascularization process in vitro. Endothelial-like cells (ECs) derived from guiding human dental pulp stem cells (DPSCs) into expressing endothelial cell markers CD144, vWF, VEGFR1, and VEGFR2 were used. Human microvascular endothelial cells (hMVECs) were used as a positive control. DPSC-ECs formed tubules on Matrigel similar to hMVECs. Cells were mixed in fibrinogen/thrombin or mouse blood and seeded into wells of 96-well plates or injected into a tapered plastic tube (14 mm in length and 1 or 2 mm diameter of the apex opening) with the larger end sealed with MTA to simulate root canal space. Cells/gels in wells or tubes were incubated for various times in vitro and observed under the microscope for morphological changes. Samples were then fixed and processed for histological analysis to determine vessel formation. Vessel-like networks were observed in culture from 1 to 3 d after cell seeding. Cells/gels in 96-well plates were maintained up to 25 d. Histologically, both hMVECs and DPSC-ECs in 96-well plates or tubes showed intracellular vacuole formation. Some cells showed merged large vacuoles indicating the lumenization. Tubular structures were also observed resembling blood vessels. Cells appeared healthy throughout the tube except some samples (1 mm apical diameter) in the coronal third. Histological analysis also showed pulp-like soft tissue throughout the tube samples with vascular-like structures. hMVECs formed larger vascular lumen size than DPSC-ECs while the latter tended to have more lumen and tubular structure counts. We conclude that DPSC-ECs can form vascular structures and sustained in the 3-dimensional fibrin gel system in vitro. The tube model appears to be a proper and simple system simulating the root canal space for vascular formation and pulp regeneration studies., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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.)

Stem cell-mediated tissue regeneration is a promising strategy for repairing tissue defects and functional reconstruction in periodontitis, a common disease that leads to the loss of alveolar bone and teeth. However, stem cell apoptosis, widely observed during tissue regeneration, impairs its efficiency. Therefore, the regulation of stem cell apoptosis is critical for improving regeneration efficiency. The LIM homeobox 8 gene LHX8, belongs to the LIM homeobox family, which was involved in tooth morphogenesis. Here, we found that LHX8 was significantly expressed in dental pulp. LHX8 knockdown significantly increased dental pulp mesenchymal stem cells (DPSCs) apoptosis, as confirmed by RT-PCR, western blotting, flow cytometry, and transmission electron microscopy. Additionally, LHX8 overexpression inhibited apoptosis and enhanced the osteo/odontogenic differentiation potential of hDPSCs in vitro. Furthermore, LHX8-overexpression could enhance the periodontal tissue regeneration efficiency of hDPSCs in mice with periodontitis. In conclusion, the present study indicates that LHX8 inhibits stem cell apoptosis and promotes functional tissue formation in stem cell-based tissue regeneration engineering, suggesting a new therapeutic target to increase the efficacy of periodontal tissue regeneration., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts or competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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., (© 2024. The Author(s).)

Regenerating tissues similar to dental structure with normal function are putatively to be the aim in tooth regeneration filed. Currently, researchers preliminarily achieved tooth regeneration by applying dental pulp stem cells (DPSC) and stem cells from human exfoliated deciduous teeth (SHED). However, the regeneration efficiency remains unstable and needs further investigation. The development of single-cell RNA sequencing and organoid culture system provide potential of precise, targeted and controllable functional regeneration. This article reviews the current state of DPSC/SHED on tooth regeneration, and analyzes characteristics and hotspots of them, aiming to shed light on clinical translational application of stable and efficient tooth regeneration.

It remains an obstacle to induce the regeneration of hard dentin tissue in clinical settings. To overcome this, a P(VDF-TrFE) piezoelectric film with 2 wt% SrCl 2 addition is designed. The biofilm shows a high flexibility, a harmonious biocompatibility, and a large piezoelectric d 33 coefficient of 14 pC N -1 , all contributing to building an electric microenvironment that favor the recruitment of dental pulp stem cells (DPSCs) and their differentiation into odontoblasts during normal chewing, speaking, etc. On the other hand, the strontium ions can be gradually released from the film, thus promoting DPSC odonto-differentiation. In vivo experiments also demonstrate that the film induces the release of dentin minerals and regeneration of dentin tissue. In the large animal dentin defect models, this piezoelectric film induces in situ dentin tissue formation effectively over a period of three months. This study illustrates a therapeutic potential of the piezoelectric film to improve dentin tissue repair in clinical settings., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

Mesenchymal stem cells (MSCs) treatment has been widely explored as a therapy for myocardial infarction, peripheral ischemic vascular diseases, dilated cardiomyopathy, and pulmonary hypertension. Latest in vitro studies suggest that MSCs can differentiate into contractile cardiomyocytes. One of the best-characterized MSCs products are MSCs-derived extracellular vesicles (EVs). EVs are crucial paracrine effectors of MSCs. Based on previous works, paracrine effects of MSCs play a primary role in the regenerative ability. Hence, in the current paper, we focused our attention on an alternative approach, exploiting products derived from human dental pulp stem cells (hDPSCs) rather than MSCs themselves, which may denote a cost-effective and safer approach. The focus has been on EVs and the bioactive molecules they contain to evaluate their ability to influence the differentiation process toward cardiomyogenic lineage. The expression of GATA4, ACTC1, CX43, and Nkx2.5 was evaluated using Immunofluorescence, real time-PCR, and Western blotting analyses. Furthermore, the expression profiling analysis of the microRNA hsa-miR-200c-3p, targeting the GATA4 gene, was studied. The hsa-miR-200c-3p was found significantly down-regulated in both c-hDPSCs + EVs-hDPSCs and c-hDPSCs + EVs-HL-1 compared to untreated c-hDPSCs underlying a possible epigenetic mechanism behind the prevalent up-regulation of its targeted GATA4 gene. The aim of the present work was to develop an in vitro model of hDPSCs able to differentiate into cardiomyocytes in order to investigate the role of EVs derived from hDPSCs and derived from HL-1 cardiomyocyte cell line in modulating the differentiation process toward cardiomyogenic lineage., (© 2023 The Authors. BioFactors published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

The main goal of regenerative endodontics procedures (REPs) is to revitalize teeth by the regeneration of healthy dental pulp. In this study, we evaluated the potential of combining a natural and accessible biomaterial based on Platelet Poor Plasma (PPP) as a support for dental pulp stem cells (DPSC) and umbilical cord mesenchymal stem cells (UC-MSC). A comparison study between the two cell sources revealed compatibility with the PPP based scaffold with differences noted in the proliferation and angiogenic properties in vitro. Additionally, the release of growth factors including VEGF, HGF and DMP-1, was detected in the media of cultured PPP and was enhanced by the presence of the encapsulated MSCs. Dentin-Discs from human molars were filled with PPP alone or with MSCs and implanted subcutaneously for 4 weeks in mice. Histological analysis of the MSC-PPP implants revealed a newly formed dentin-like structure evidenced by the expression of Dentin sialophosphoprotein (DSPP). Finally, DPSC induced more vessel formation around the dental discs. This study provides evidence of a cost-effective, xenofree scaffold that is compatible with either autologous or allogenic strategy for dental pulp regeneration. This attempt if successfully implemented, could make REPs treatment widely accessible, contributing in improving global health conditions., (© 2022. The Author(s).)

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH) 2 , and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH 2 O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH) 2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH) 2 , CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.

Background: Dental trauma, restorative operative procedures and/or caries lesions can expose the dental pulp. Facing this clinical condition, where the maintenance of the dentin-pulp complex vitality is imperative, is challenging in Dentistry. Dental pulp stem cells conditioned medium contains trophic factors that could help in this task. This in vivo pilot study aimed to evaluate the effects of the human dental pulp stem cells conditioned medium on the dental pulp tissue response to vital pulp therapy., Material and Methods: Concentrated conditioned medium was obtained by incubating characterized human dental pulp stem cells with fresh culture medium. Pulp exposures performed at the first upper molars (n = 20) of Wistar rats were directly capped with: MTA or MTA + Conditioned Medium. Four and 8 weeks later, the samples were qualitatively analyzed in histological sections (H&E)., Results: When the conditioned medium was associated with MTA, there were a high percentage of samples presenting formation of dentin bridges and small percentage of pulp tissue with inflammatory signs in both experimental times. The conditioned medium improved the organization of the newly formed hard tissue., Conclusions: The association of dental pulp stem cell conditioned medium with MTA showed beneficial effects on dentin-pulp complex regeneration and has promising potential for studies in regenerative dentistry., (Copyright © 2021. Published by Elsevier Ltd.)

This research aimed to design innovative therapeutic bio-composites that enhance odontogenic and osteogenic differentiation of human dental pulp-derived mesenchymal stem cells (h-DPSCs) in-vitro regeneration. Herein, we report the fabrication of scaffolds containing chitosan, Ca-SAPO-34 monometallic and/or Fe-Ca-SAPO-34 bimetallic nanoparticles by freeze-drying technique. The scaffolds and nanoparticles were characterized using ICP-AES, FT-IR, XRD, TGA, TEM, BET, SEM, and EDS methods. The effects of SAPO-34 and nanoparticles were investigated by changes on the physicochemical properties of scaffolds including swelling ratio, density, porosity, bio-degradation, mechanical behavior, and biomineralization. Cell viability, cell adhesion and cytotoxicity of Ca-SAPO-34/CS and Fe-Ca-SAPO-34 scaffolds were investigated by MTT assay and SEM on h-DPSCs which revealed cell proliferation no toxicity on scaffolds. Cell tests demonstrated that Ca-SAPO-34/CS scaffold clearly displayed a positive effect on differentiation of hDPSCs into osteogenic/odontogenic cells and moderate effect on cell proliferation. Moreover, the incorporation of Fe 2 O 3 to Ca-SAPO-34/CS scaffold promoted the proliferation of hDPSCs and osteogenic differentiation. Alizarin red, Alkaline phosphatase and QRT-PCR results showed that Fe-Ca-loaded SAPO-34/CS can lead to osteoblast/odontoblast differentiation in DPSCs through the up-regulation of related genes, thus indicating that Fe-Ca-SAPO-34/CS has remarkable prospects as a biomaterial for hard tissue engineering., (Copyright © 2021. Published by Elsevier B.V.)

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.

Since the discovery of dental pulp stem cells, a lot of teams have expressed an interest in dental pulp regeneration. Many approaches, experimental models and biological explorations have been developed, each including the use of stem cells and scaffolds with the final goal being clinical application in humans. In this review, the authors' objective was to compare the experimental models and strategies used for the development of biomaterials for tissue engineering of dental pulp with stem cells. Electronic queries were conducted on PubMed using the following terms: pulp regeneration, scaffold, stem cells, tissue engineering and biomaterial. The extracted data included the following information: the strategy envisaged, the type of stem cells, the experimental models, the exploration or analysis methods, the cytotoxicity or viability or proliferation cellular tests, the tests of scaffold antibacterial properties and take into account the vascularization of the regenerated dental pulp. From the 71 selected articles, 59% focused on the "cell-transplantation" strategy, 82% used in vitro experimentation, 58% in vivo animal models and only one described an in vivo in situ human clinical study. 87% used dental pulp stem cells. A majority of the studies reported histology (75%) and immunohistochemistry explorations (66%). 73% mentioned the use of cytotoxicity, proliferation or viability tests. 48% took vascularization into account but only 6% studied the antibacterial properties of the scaffolds. This article gives an overview of the methods used to regenerate dental pulp from stem cells and should help researchers create the best development strategies for research in this field.

Background: The regeneration of dental pulp, especially in cases of pulp death of immature teeth, is the goal of the regenerative endodontic procedures (REPs) that are based on tissue engineering principles, consisting of stem cells, growth factors, and scaffolds. Photobiomodulation therapy (PBMT) showed to improve dental pulp regeneration through cell homing approaches in preclinical studies and has been proposed as the fourth element of tissue engineering. However, when a blood clot was used as a scaffold in one of these previous studies, only 30% of success was achieved. The authors pointed out the instability of the blood clot as the regeneration shortcoming. Then, to circumvent this problem, a new scaffold was developed to be applied with the blood clot. The hypothesis of the present study was that an experimental injectable chitosan hydrogel would facilitate the three-dimensional spatial organization of endogenous stem cells in dental pulp regeneration with no interference on the positive influence of PBMT., Methods: For the in vitro analysis, stem cells from the apical papilla (SCAPs) were characterized by flow cytometry and applied in the chitosan scaffold for evaluating adhesion, migration, and proliferation. For the in vivo analysis, the chitosan scaffold was applied in a rodent orthotopic dental pulp regeneration model under the influence of PBMT (660 nm; power output of 20 mW, beam area of 0.028 cm 2 , and energy density of 5 J/cm 2 )., Results: The scaffold tested in this study allowed significantly higher viability, proliferation, and migration of SCAPs in vitro when PBMT was applied, especially with the energy density of 5 J/cm 2 . These results were in consonance to those of the in vivo data, where pulp-like tissue formation was observed inside the root canal., Conclusion: Chitosan hydrogel when applied with a blood clot and PBMT could in the future improve previous results of dental pulp regeneration through cell homing approaches., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Maria Stella Moreira et al.)

Exiting developments in tissue engineering and new insights in stem cell biology have led to new possible strategies for the regeneration of damaged tissues in the oral cavity. The regeneration of the pulp-dentin complex regeneration in particular, has drawn the attention of many researchers because of the high clinical needs. While it is still important to perform in vitro research using a wide variety of cells, scaffolds and growth factors, it is also critical to have a reliable animal model for preclinical trials. In this chapter, we describe a mouse model in which a scaffold resembling a tooth containing dental pulp cells is implanted subcutaneously. We also describe which histological stainings could be used to examine blood vessel formation and the regeneration of the pulp-dentin complex.

Stem cells from Human Exfoliated Deciduous teeth (SHED) are considered one of the most attractive cell sources for tissue engineering due to their easy acquisition with no donor morbidity, ready availability, ability to self-renew with high proliferation, capacity for multilineage differentiation and immunomodulatory functions. To date, SHED are able to differentiate into odonto-/ osteoblasts, neuronal cells, endothelial cells, hepatocyte-like cells, chondrocytes, epidermal cells among many other cell types. Accordingly, SHED possess a promising potential to be used in the cell-based therapy for various diseases, including reversible pulpitis, orofacial bone defects, neurodevelopmental disease and ischemic injury. Despite this potential, it has been a concern that tissue specific stem cells do not differentiate with the same efficacy into all the different lineages as they may have an inherent tendency to differentiate toward the tissues from which they were originally derived. Furthermore, stem cell niche comprises of a complex microenvironment where various cells, soluble signals, extracellular matrix and physical cues interplay to maintain the stemness of SHED and modulate their differentiation. Therefore, it is of significant importance to identify the specific microenvironmental cues that regulate lineage specific differentiation of SHED, which could inspire to develop functional approaches in target tissue regeneration. In this review, we highlight the recent studies that demonstrated multilineage differentiation capacity of SHED, focusing on how the microenvironment could be modified using different cues in order to achieve tissue specific regeneration., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The effectiveness of stem cell-based periodontal tissue engineering need to be assessed by preclinical animal studies. Dog models are widely used animal models; however, there are not sufficient data on characterization of canine dental mesenchymal stem cells. Therefore, we aimed to compare the characteristics among canine and human periodontal ligament stem cells and canine and human dental pulp stem cells. Canine periodontal ligament stem cells and dental pulp stem cells showed significantly weaker clonogenic capability, and proliferation and migration capacity, and they displayed lower positive rates for CD90, CD73, CD105, and STRO-1. All of these canine and human cells showed multilineage differentiation potential. After osteogenic induction, the expression of alkaline phosphatase was obviously upregulated in human dental mesenchymal stem cells, but it was not upregulated in canine dental pulp stem cells. Other osteogenic genes, such as runt-related transcription factor 2 and bone morphogenetic protein 2, were upregulated in all induced canine and human cells, but their upregulation occurred later in canine cells. These results confirmed the stem cell properties of canine mesenchymal stem cells, but also suggested that more attention should be paid to the choice of appropriate research approaches, osteogenic gene markers, and time points for the utilization of canine dental mesenchymal stem cells due to their distinct characteristics., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Transforming growth factor-β1 (TGF-β1) regulates wound healing/regeneration and aging processes. Dental pulp stem cells from human exfoliated deciduous teeth (SHED) are cell sources for treatment of age-related disorders. We studied the effect of TGF-β1 on SHED and related signaling. SHED were treated with TGF-β1 with/without pretreatment/co-incubation by SB431542, U0126, 5Z-7-oxozeaenol or SB203580. Sircol collagen assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) assay, alkaline phosphatase (ALP) assay, RT-PCR, western blotting and PathScan phospho-ELISA were used to measure the effects. We found that SHED expressed ALK1, ALK3, ALK5, TGF-RII, betaglycan and endoglin mRNA. TGF-β1 stimulated p-Smad2, p-TAK1, p-ERK, p-p38 and cyclooxygenase-2 (COX-2) protein expression. It enhanced proliferation and collagen content of SHED that were attenuated by SB431542, 5Z-7-oxozeaenol and SB203580, but not U0126. TGF-β1 (0.5-1 ng/ml) stimulated ALP of SHED, whereas 5-10 ng/ml TGF-β1 suppressed ALP. SB431542 reversed the effects of TGF-β1. However, 5Z-7-oxozeaenol, SB203580 and U0126 only reversed the stimulatory effect of TGF-β1 on ALP. Four inhibitors attenuated TGF-β1-induced COX-2 expression. TGF-β1-stimulated TIMP-1 and N-cadherin was inhibited by SB431542 and 5Z-7-oxozeaenol. These results indicate that TGF-β1 affects SHED by differential regulation of ALK5/Smad2/3, TAK1, p38 and MEK/ERK. TGF-β1 and SHED could potentially be used for tissue engineering/regeneration and treatment of age-related diseases.

Objective: To compare proteomics and biological function of human dentin matrix molecules (hDMMs) and bovine dentin matrix molecules (bDMMs)., Design: Dentin powder from human or bovine teeth (n = 4) was demineralized in 10% (v/v) ethylenediaminetetraacetic acid for 7 days. The extracts were dialyzed, lyophilized and proteins were characterized using liquid chromatography-tandem mass spectrometry and shotgun proteomic analysis. To study biological function, mouse-derived undifferentiated dental pulp cells (OD21) were treated with 0.01, 0.1 or 1 μg/mL of hDMMs or bDMMs and proliferation was measured after 24 hours and 48 hours using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell migration was assessed after 24 hours using a Boyden chamber. Alizarin Red S staining was used to evaluate mineral formation., Results: There were 307 proteins identified, of which 93 proteins were common to both species. Gene Ontology functional analysis demonstrated similar pattern of biological process in both species which consisted mainly of tissue development and biomineralization. hDMMs and bDMMs both enhanced cell proliferation. After 24 hours, all concentrations of bDMMs promoted cell proliferation (p ≤ 0.05), while hDMMs did not affect proliferation. After 48 hours, groups with 1μg/mL of bDMMs and 0.01μg/mL of hDMMs had increased cell proliferation compared to control (p ≤ 0.0001). All concentrations of hDMMs and bDMMs enhanced cell migration and mineralization (p ≤ 0.0001)., Conclusion: bDMMs has similar biological functions as hDMMs. Moreover, bDMMs stimulated cell proliferation, migration and differentiation similar to hDMMs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: Several scaffolds and cell sources are being investigated for cartilage regeneration. The aim of the study was to prepare nanocellulose-based thermosensitive injectable hydrogel scaffolds and assess their potential as 3D scaffolds allowing the chondrogenic differentiation of embedded human dental pulp stem and progenitor cells (hDPSCs)., Materials and Methods: The hydrogel-forming solutions were prepared by adding β-glycerophosphate (GP) to chitosan (CS) at different ratios. Nanocellulose (NC) suspension was produced from hemp hurd then added dropwise to the CS/GP mixture. In vitro characterization of the prepared hydrogels involved optimizing gelation and degradation time, mass-swelling ratio, and rheological properties. The hydrogel with optimal characteristics, NC-CS/GP-21, was selected for further investigation including assessment of biocompatibility. The chondrogenesis ability of hDPSCs embedded in NC-CS/GP-21 hydrogel was investigated in vitro and compared to that of bone marrow-derived mesenchymal stem cells (BMSCs), then was confirmed in vivo in 12 adult Sprague Dawley rats., Results: The selected hydrogel showed stability in culture media, had a gelation time of 2.8 minutes, showed a highly porous microstructure by scanning electron microscope, and was morphologically intact in vivo for 14 days after injection. Histological and immunohistochemical analyses and real-time PCR confirmed the chondrogenesis ability of hDPSCs embedded in NC-CS/GP-21 hydrogel., Conclusion: Our results suggest that nanocellulose-chitosan thermosensitive hydrogel is a biocompatible, injectable, mechanically stable and slowly degradable scaffold. hDPSCs embedded in NC-CS/GP-21 hydrogel is a promising, minimally invasive, stem cell-based strategy for cartilage regeneration., Competing Interests: The authors declare that they have no conflicts of interest for this work., (© 2020 Talaat et al.)

Hydrogel-based regenerative endodontic procedures (REPs) are considered to be very promising therapeutic strategies to reconstruct the dental pulp (DP) tissue in devitalized human teeth. However, the success of the regeneration process is limited by residual bacteria that may persist in the endodontic space after the disinfection step and contaminate the biomaterial. The aim of this work was to develop an innovative fibrin hydrogel incorporating clindamycin (CLIN)-loaded Poly (d,l) Lactic Acid (PLA) nanoparticles (NPs) to provide the hydrogel with antibacterial properties. CLIN-PLA-NPs were synthesized by a surfactant-free nanoprecipitation method and their microphysical properties were assessed by dynamic light scattering, electrophoretic mobility and scanning electron microscopy. Their antimicrobial efficacy was evaluated on Enteroccocus fæcalis by the determination of the minimal inhibitory concentration (MIC) and the minimal biofilm inhibition and eradication concentrations (MBIC and MBEC). Antibacterial properties of the nanocomposite hydrogel were verified by agar diffusion assays. NP distribution into the hydrogel and release from it were evaluated using fluorescent PLA-NPs. NP cytotoxicity was assessed on DP mesenchymal stem cells (DP-MSCs) incorporated into the hydrogel. Type I collagen synthesis was investigated after 7 days of culture by immunohistochemistry. We found that CLIN-PLA-NPs displayed a drug loading of 10 ± 2 μg per mg of PLA polymer and an entrapment efficiency of 43 ± 7%. Antibiotic loading did not affect NP size, polydispersity index and zeta potential. The MIC for Enterococcus fæcalis was 32 μg mL-1. MBIC50 and MBEC50 were 4 and 16 μg mL-1, respectively. CLIN-PLA-NPs appeared homogenously distributed throughout the hydrogel. CLIN-PLA-NP-loaded hydrogels clearly inhibited E. faecalis growth. DP-MSC viability and type I collagen synthesis within the fibrin hydrogel were not affected by CLIN-PLA-NPs. In conclusion, CLIN-PLA-NP incorporation into the fibrin hydrogel gave the latter antibacterial and antibiofilm properties without affecting cell viability and function. This formulation could help establish an aseptic environment supporting DP reconstruction and, accordingly, might be a valuable tool for REPs.

Tooth loss can cause a lot of physiological and psychological suffering. And tooth root engineering is a promising way for tooth loss treatment. Two kinds of seed cells are usually adopted for tooth root regeneration. In this study, a practical sandwich structure for tooth root regeneration was developed, which was constituted by only one kind of seed cell: human dental pulp stem cells (hDPSCs) and three kinds of graft materials: Vitamin C (VC) induced hDPSC sheet, human treated dentin matrix (hTDM), and Matrigel. It was found that VC could induce hDPSCs to form a cell sheet with two or three cell layers and promote their collagen type I ( COL1 ) mRNA expression obviously. hDPSCs could attach and grow on hTDM, and the mRNA expression of osteocalcin ( OCN ), dentin sialophosphoprotein ( DSPP ), vascular endothelial growth factor receptor 1 ( VEGFR1 ), and Nestin in hDPSCs was obviously upregulated by hTDM leaching solution. hDPSCs could stretch and proliferate in Matrigel. And when cultured in Matrigel condition medium, they positively expressed CD31, β3-Tubulin, and Nestin proteins, as well as increased the mRNA expression of OCN , ALP , and Nestin. Furthermore, periodontium, dentin, and pulp-like tissues were successfully regenerated after the sandwich structure of hDPSC sheet/TDM/Matrigel was transplanted in nude mice subcutaneously for 3 months. Periodontium-like dense connective tissue was regenerated around the hTDM, and a great mass of predentin was formed on the cavity side of hTDM. Odontoblast-like cells and blood vessel-like structures, even nerve-like fibers, were observed in the pulp cavity. In summary, the above results showed that hDPSCs could be used as seed cells for the whole tooth root regeneration, and the sandwich structure constituted by hDPSC sheet, TDM/hDPSCs, and Matrigel/hDPSCs could be utilized for tooth root regeneration.

Dental pulp is critical to maintain the vitality of a tooth. Regeneration of pulpo-dentinal complex is of great interest to treat pulpitis and pulp necrosis. In this study, through three-dimensional spheroid culture, a group of unique multipotent stem cells were identified from mouse dental papilla called multipotent dental pulp regenerative stem cells (MDPSCs). MDPSCs exhibited enhanced osteogenic/odontogenic differentiation capabilities and could form regenerative dentin and neurovascular-like structures that mimicked the native teeth in vivo. Further analysis revealed that CD24a was the bona fide marker for MDPSCs, and their expansion was highly dependent on the expression of a key transcriptional factor, Sp7 . Last, CD24a + cells could be detected in primary dental papilla in mice and human, suggesting that MDPSCs resided in their native niches. Together, our study has identified a previously unidentified group of multipotent pulp regenerative stem cells with defined molecular markers for the potential treatment of pulpitis and pulp necrosis., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Background: Symptomatic cartilage lesions and early osteoarthritis produce significant clinical and economic burdens. Cartilage repair can improve the symptoms and delay arthroplasty. The complete healing of damaged cartilage with the consistent reproduction of normal hyaline cartilage has not yet been achieved. The choice of harvesting site might influence the cells' abilities to modulate immunologic and inflammatory responses. Recently, dental pulp has been shown to contain a stem cell niche consisting of dental pulp stem cells (DPSCs) that maintain their self-renewal capacity due to the active environment in the dental pulp of deciduous teeth. Objective: The aim of this study was to critically review the current literature on the potential and limitations of the use of dental pulp-derived mesenchymal stem cells in cell-based therapies for cartilage regeneration. Methods: An electronic, customized search of scientific articles was conducted using the PubMed/MEDLINE and EMBASE databases from their inception to December 2018. The inclusion criteria were applied, and the articles that described the use of DPSC in cartilage treatment were selected for complete evaluation. The articles were classified according to the scaffold used, experimental model, chondrogenic differentiation features, defect location, cartilage evaluation, and results. After the application of the eligibility criteria, a total of nine studies were selected and fully analyzed. Results: A variety of animal models were used, including mice, rats, rabbits, and miniature pigs, to evaluate the quality and safety of human DPSCs in the repair of cartilage defects. Among the articles, two studies focused on preclinical models of cartilage tissue engineering. Five studies implanted DPSCs in other animal sites. Conclusion: The use of DPSCs is a potential new stem cell therapy for articular cartilage repair. The preclinical evidence discussed in this article provides a solid foundation for future clinical trials. Impact statement Osteoarthritis presents an ever-increasing clinical and socioeconomic burden. While cartilage repair has the potential to improve symptoms and delay joint replacement, complete regeneration of hyaline cartilage has been an elusive goal. Dental pulp has been shown to contain a niche that protects dental pulp stem cells (DPSCs) from the cumulative effects of genetic and environmental factors and maintains their self-renewal capacity due to the active environment. Transplantation and preclinical trials have demonstrated the strong potential of regenerative tissue-engineering protocols using DPSCs.

The continuous growth of rodent incisors is ensured by clusters of mesenchymal and epithelial stem cells that are located at the posterior part of these teeth. Genetic lineage tracing studies have shown that dental epithelial stem cells (DESCs) are able to generate all epithelial cell populations within incisors during homeostasis. However, it remains unclear whether these cells have the ability to adopt alternative fates in response to extrinsic factors. Here, we have studied the plasticity of DESCs in the context of mammary gland regeneration. Transplantation of DESCs together with mammary epithelial cells into the mammary stroma resulted in the formation of chimeric ductal epithelial structures in which DESCs adopted all the possible mammary fates including milk-producing alveolar cells. In addition, when transplanted without mammary epithelial cells, DESCs developed branching rudiments and cysts. These in vivo findings demonstrate that when outside their niche, DESCs redirect their fates according to their new microenvironment and thus can contribute to the regeneration of non-dental tissues., Competing Interests: The authors state that there are no conflicts of interest linked to the present manuscript.

The aim of this study was to prepare a promising biomaterial for dental pulp tissue repair and regeneration. The ultrasmall superparamagnetic iron oxide (USPIO)-labeled hydroxyapatite (HA)/silk fibroin (SF) scaffold loaded dental pulp stem cells (DPSCs) was designed. The odontogenic differentiation of DPSCs was investigated, and USPIO was used as magnetic resonance imaging (MRI) contrast agent. The results indicated that scaffolds freeze-dried from SF solution with 0.025 mg/mL USPIO and 5 mg/mL HA showed stable physical properties, accurate MRI images, and low cytotoxicity in vitro. The composite scaffolds were characterized and implanted to the subcutaneous space under the nude mice with tooth fragment. After 2 W of implantation, the relaxation rate (R2) values increased because of incorporation of USPIO, then decreased with the degradation of scaffolds. The tissue regeneration and scaffolds degradation behaviors were monitored by MRI. The expression of dentin sialophosphoprotein (DSPP) and dentin matrix acidic phosphoprotein 1 (DMP1) indicated that DPSCs differentiated into odontoblast-like cells. Revascularization and mineralization evaluated by HE-stained images and alizarin red staining images showed good formation. The USPIO/HA/SF composite scaffold loaded DPSCs promoted the repair and regeneration of dental pulp tissue and provided the ability of imaging noninvasively., (Copyright © 2019. Published by Elsevier B.V.)

Relying on their ease of isolation and remarkable tissue reparative/regenerative potential, dental pulp stem/progenitor cells (DPSCs) gained pronounced importance in the field of regenerative dentistry. Though inflammation is classically considered the reason for the damage of the dentin-pulpal complex, it continues to be an essential stage of any dentin-pulpal tissue repair or regeneration procedures. During their performance of a pulpal tissue repair or regeneration actions, DPSCs interact with their inflammatory microenvironment locally, possibly influencing their fate and the result of any DPSCs-mediated dentin-pulpal reparative/regenerative endeavor. Hence, this review aims at comprehensively elaborating on these complex interactions of DPSCs with their local pulpal inflammatory microenvironment, particularizing on the inflammatory aspects, affecting DPSCs' stemness, homing/migration, proliferation, differentiation as well as immunomodulation characteristics, and the potentially fundamental intracellular processes involved and their anticipated association with the noncanonical as well as canonical Wnt/β-Catenin intracellular signaling. Impact Statement This review particularizes on the current state of knowledge on the complex interrelation between dental pulp stem/progenitor cells and their pulpal inflammatory microenvironment; elaborates on inflammation aspects affecting their stemness, proliferation, migration/homing, differentiation and immunomodulation characteristics, and the fundamental intracellular processes involved and their anticipated association with the canonical and noncanonical Wnt pathways. All these aspects could significantly affect the dento-pulpal regenerative therapeutic approaches in vivo .

Impact Statement: This study is the first to investigate and confirm the effectiveness of single-cell Raman spectroscopy (SCRM), in its ability to discriminate between dental pulp stem cells (DPSCs) with contrasting proliferative and differentiation capabilities. The findings show that SCRM can rapidly and noninvasively distinguish and identify DPSC subpopulations in vitro with superior proliferative and multipotency properties, versus lesser quality DPSCs, thereby overcoming the significant heterogeneity issues surrounding DPSC ex vivo expansion and differentiation capabilities. Such findings support further SCRM assessment for the selective screening/isolation of superior quality DPSCs from whole dental pulp tissues, for more effective in vitro evaluation and therapy development.

Objectives: The current study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) overexpressing dental pulp stem cells (DPSCs) in vascularized dental pulp regeneration in vivo., Materials and Methods: Human DPSCs were transfected with VEGF or SDF-1α using premade lentiviral particles. Overexpression was verified by quantitative polymerase chain reaction (q-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot analysis. Effects of SDF-1α and VEGF overexpressing DPSCs on their proliferation (CCK-8 and MTT assays) and endothelial vascular-tube formation (Matrigel assay) were investigated in vitro. Human tooth roots sectioned into 6-mm segments were injected with gene-modified DPSCs encapsulated in PuraMatrix hydrogel and implanted in the dorsum of severe-combined-immunodeficient (SCID) mice. Implants were retrieved after 4 weeks and examined for regenerated pulp-like tissue and vascularization using histology and immunohistochemistry. p < 0.05 was considered statistically significant., Results: Gene-modified DPSCs expressed significantly high levels (p < 0.05) of SDF-1α and VEGF mRNA and proteins, respectively. Transfected DPSCs showed a significantly higher cell proliferation compared to that of wild-type DPSCs. Furthermore, they enhanced endothelial cell migration and vascular-tube formation on Matrigel in vitro. When injected into tooth root canals and implanted in vivo, DPSCs/SDF-1α + DPSCs/VEGF-mixed group resulted in significantly increased length of regenerated pulp-like tissue within the root canals compared to that of wild-type DPSCs/VEGF and DPSCs/SDF-1α groups. Vessel area density was significantly higher in DPSCs/SDF-1α and mixed DPSCs/SDF-1α + DPSCs/VEGF groups than in DPSCs-VEGF alone or wild-type DPSCs groups., Conclusion: A combination of VEGF-overexpressing and SDF-1α-overexpressing DPSCs could enhance the area of vascularized dental pulp regeneration in vivo., Clinical Relevance: Enhancing vascularization in pulp regeneration is crucial to overcome the clinical limitation of the limited blood supply to the root canals via a small apical foramen enclosed by hard dentin.

Uncomplicated treatments for pulpitis and periodontitis continues to be challenging and regenerative approaches could meet this contingency. Dental pulp stem cells (DPSCs) represent a good candidate for oral recovering therapies. Here, we investigated changes in morphology, proliferation, and in vitro differentiation toward mesenchymal and neuronal phenotypes of human DPSCs harvested from differently aged donors. Aging is a physiologic phenomenon occurring with time that hamper body's capability to maintain homeostasis also affecting the functional reserve. Cytofluorimetric, immunohistochemical, quantitative reverse-transcription polymerase chain reaction (qRT-PCR), and western blot analyses were performed to gain insight for successful regenerative strategies in elderly. We observed a decline in DPSCs proliferation and differentiation potential with age. Interestingly, these cells behaved differently under osteogenic or odontogenic stimuli, showing different age-related mineralization capabilities. Similarly, neurogenic differentiation decreased with age. In conclusion, our observations represent a valid tool for the development of tailored regenerative strategies in an aging society., (© 2018 Wiley Periodicals, Inc.)

Impact Statement: Animal models are essential for tissue regeneration studies. This review summarizes and discusses the small and large animal models, including mouse, ferret, dog, and miniswine that have been utilized to experiment and to demonstrate stem cell-mediated dental pulp tissue regeneration. We describe the models based on the location where the tissue regeneration is tested-either ectopic, semiorthotopic, or orthotopic. Developing and utilizing optimal animal models for both mechanistic and translational studies of pulp regeneration are of critical importance to advance this field.

Mesenchymal stem cells (MSC) have a lot of potential in regenerative medicine, and MSC-based therapies are currently explored in numerous research fields. Among these cells, deciduous or permanent dental pulp-MSC represent a promising option in tissue engineering. This expectation is based on their capacity to self-renew, to repair various damaged tissues and organs due to their multipotency, as well as their ability to modulate immune system. They present other advantages such as the harvesting by a simple, painless, and noninvasive procedure and the absence of ethical considerations. The role played by these cells in the reparative process is mainly attributed to paracrine mechanisms mediated by their secreted factors, namely the secretome. The secreted factors can be found in the cell culture medium, called conditioned medium (CM). Moreover, CM presents many advantages compared with cells such as possible use in allogeneic therapies. This minireview aims at investigating the therapeutic use of dental pulp MSC-derived CM to develop cell-free therapies. The analysis of the available literature illustrates its massive panel of potential applications: mainly reduction of inflammation, promotion of angiogenesis and neurogenesis, reduction of stroke or ischemia, and organ regeneration. Furthermore, studies often highlight its superiority over the other sources of CM derived from other stem cells for the same applications. Dental pulp MSC-derived CM is an attractive, noninvasive, and acellular tool for therapeutic approaches in regenerative medicine. This promising novel approach should be further explored for clinical applications.

More than two thirds of the global population suffers from tooth decay, which results in cavities with various levels of lesion severity. Clinical interventions to treat tooth decay range from simple coronal fillings to invasive root canal treatment. Pulp capping is the only available clinical option to maintain the pulp vitality in deep lesions, but irreversible pulp inflammation and reinfection are frequent outcomes for this treatment. When affected pulp involvement is beyond repair, the dentist has to perform endodontic therapy leaving the tooth non-vital and brittle. On-going research strategies have failed to overcome the limitations of existing pulp capping materials so that healthy and progressive regeneration of the injured tissues is attained. Preserving pulp vitality is crucial for tooth homeostasis and durability, and thus, there is a critical need for clinical interventions that enable regeneration of the dentin-pulp complex to rescue millions of teeth annually. The identification and development of appropriate biomaterials for dentin-pulp scaffolds are necessary to optimize clinical approaches to regenerate these hybrid dental tissues. Likewise, a deep understanding of the interactions between the micro-environment, growth factors, and progenitor cells will provide design basis for the most fitting scaffolds for this purpose. In this review, we first introduce the long-lasting clinical dental problem of rescuing diseased tooth vitality, the limitations of current clinical therapies and interventions to restore the damaged tissues, and the need for new strategies to fully revitalize the tooth. Then, we comprehensively report on the characteristics of the main materials of naturally-derived and synthetically-engineered polymers, ceramics, and composite scaffolds as well as their use in dentin-pulp complex regeneration strategies. Finally, we present a series of innovative smart polymeric biomaterials with potential to overcome dentin-pulp complex regeneration challenges., (© 2018 John Wiley & Sons, Ltd.)

Background: Regenerative medicine is a growing branch of medicine aimed to treat damaged or lost tissues, to promote tissue formation and to restore both aesthetics and function. In the last years, several scientific articles have been focused on the regenerative procedures aimed to increase the survival rate of compromised teeth; the most effective approaches have been based on molecularbased and on cellular-based protocols; however, to date, both these techniques have not been carefully analysed and discussed, to know in details the advantages of each of them., Methods: A literature search was undertaken on three electronic scientific databases: Medline via PubMed, EMBASE and Google Scholar. Authors aimed to select such articles published in the time span from January 1961 until December 2017. The authors screened the titles and the abstracts including the following keywords combinations: "Pulp AND Therapy", "Regenerative AND Endodontic", and "Endodontics AND Tissue engineering". After the exclusion of any not related articles, the full text of such papers related to the topics was included in this review., Results: Following the removal of duplicate articles and of other types of publications (such as erratum and corrigendum), 621 articles were selected to be included and analysed in our topical review. The articles were analysed into the following sections: cellular-based approaches for dental regeneration, molecular-based and combined cellular/molecular-based approaches for dental regeneration, and translational applications of regenerative dentistry., Conclusion: This topical review has been focused on the main, the most promising and the most innovative strategies for achieving the regeneration of dental pulp or dental tissues. The main and surprising "take-home message" is related to the great interest towards the dental-derived stem cells, characterized by a high angiogenic and neurogenic commitment. Future challenges will be focused on the development of biological-friendly regenerative strategies: the new approaches should overcome the current biological limitations, to promote the combined cellular and molecular-based treatments, able to ensure predictable clinical evidence, with the achievement of the regeneration/repairing of the compromised dental pulp and of the entire tooth structure., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Following the basis of tissue engineering (Cells-Scaffold-Bioactive molecules), regenerative endodontic has emerged as a new concept of dental treatment. Clinical procedures have been proposed by endodontic practitioners willing to promote regenerative therapy. Preserving pulp vitality was a first approach. Later procedures aimed to regenerate a vascularized pulp in necrotic root canals. However, there is still no protocol allowing an effective regeneration of necrotic pulp tissue either in immature or mature teeth. This review explores in vitro and preclinical concepts developed during the last decade, especially the potential use of stem cells, bioactive molecules, and scaffolds, and makes a comparison with the goals achieved so far in clinical practice. Regeneration of pulp-like tissue has been shown in various experimental conditions. However, the appropriate techniques are currently in a developmental stage. The ideal combination of scaffolds and growth factors to obtain a complete regeneration of the pulp-dentin complex is still unknown. The use of stem cells, especially from pulp origin, sounds promising for pulp regeneration therapy, but it has not been applied so far for clinical endodontics, in case of necrotic teeth. The gap observed between the hope raised from in vitro experiments and the reality of endodontic treatments suggests that clinical success may be achieved without external stem cell application. Therefore, procedures using the concept of cell homing, through evoked bleeding that permit to recreate a living tissue that mimics the original pulp has been proposed. Perspectives for pulp tissue engineering in the near future include a better control of clinical parameters and pragmatic approach of the experimental results (autologous stem cells from cell homing, controlled release of growth factors). In the coming years, this therapeutic strategy will probably become a clinical reality, even for mature necrotic teeth.

Background: Recent studies have demonstrated mesenchymal stem cells (MSCs) as effective mitochondrial donors with therapeutic success in multiple experimental models of human disease. MSCs obtained from different tissue sources such as bone marrow (BM), adipose (AD), dental pulp (DP), and Wharton's jelly (WJ) are routinely used in clinical trials with no known study of their mitochondrial donor capacity. Here, we show for the first time that MSCs derived from different tissue sources have different mitochondrial donor properties and that this is correlated with their intrinsic respiratory states., Methods: MitoTracker ® -labeled MSCs were co-cultured with Cell Trace-labeled U87-MG cells or rat cardiomyocytes. Mitochondrial transfer abilities of MSCs were assessed by using flow cytometry analysis and fluorescence imaging. Mitochondrial reactive oxygen species (mtROS) levels were analyzed by using MitoSOX red-based staining, and mitochondrial respiration parameters were analyzed by using a Seahorse XF Analyzer., Results: AD-MSCs and BM-MSCs displayed higher mitochondrial transfer than DP-MSCs and WJ-MSCs. Counterintuitively, DP-MSCs and WJ-MSCs were more effective in suppressing mtROS levels in stressed recipient cells than AD-MSCs or BM-MSCs. Interestingly, the oxygen consumption rates and intrinsic mitochondrial respiration parameters like ATP levels, basal and maximal respiration, and mitochondrial DNA copy number in donor MSCs showed a highly significant inverse correlation with their mitochondrial donation., Conclusions: We find that there are intrinsic differences in the mitochondrial respiration, donation capacity, and therapeutic efficacy among MSCs of different tissue origin. MSCs with high mitochondrial respiration capacities are associated with lower mitochondrial transfer but more effective suppression of mtROS in stressed recipient cells. This is most compatible with a model where recipient cells optimally regulate mitochondrial transfer such that they take more mitochondria from MSCs with lower mitochondrial function. Furthermore, it appears to be advantageous to use MSCs such as DP-MSCs or WJ-MSCs with higher mitochondrial respiratory abilities that achieved better therapeutic effect with lower mitochondrial transfer in our study. This opens up a new direction in stem cell therapeutics.

Introduction: On stimulation by lipoteichoic acid or by a physical injury, fibroblasts have been shown to play a major role in the initiation of the pulp inflammatory reaction and healing through secretion of complement proteins and growth factors. The application of direct pulp-capping materials on these cells may interfere with the inflammatory and the healing processes within the pulp's inextensible environment. This work was designed to study in vitro the effects of silicate-based materials on pulp fibroblast modulation of the initial steps of pulp inflammation and healing., Methods: The effects of Biodentine, TheraCal, and Xeno III eluates were studied on lipoteichoic acid-stimulated and physically injured fibroblasts. Cytokine secretion (interleukin 6, vascular endothelial growth factor, fibroblast growth factor-2, and transforming growth factor-β1) was quantified by enzyme-linked immunosorbent assay. Inflammatory THP-1 adhesion to endothelial cells and their migration and activation were studied in vitro. Human pulp fibroblast proliferation was investigated with the MTT test, and their migration to the injury site was studied with the scratch healing assay., Results: Interleukin 6 and vascular endothelial growth factor secretion increased with all materials but to a lesser extent with Biodentine. Fibroblast growth factor-2 and transforming growth factor-β1 secretion was significantly higher with Biodentine than with all other materials. THP-1 cell adhesion to endothelial cells and their activation were reduced by Biodentine and TheraCal. However, their migration decreased only with Biodentine. Fibroblast proliferation significantly increased with Biodentine but significantly decreased with Xeno III after day 6. Finally, only Biodentine induced fibroblast migration to the injury site in the scratch assay., Conclusions: These results confirm that pulp-capping materials affect the early steps of pulp inflammation and healing. They show that Biodentine had the highest pulp healing and anti-inflammatory potential when compared with the resin-containing materials. This highlights the interest of the material choice for direct pulp-capping., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from human and animal sources such as rats. Recently, an in vivo protocol for pulp tissue engineering using implantation of bone marrow MSCs into rat pulpotomized molars was established by our research group. This coronal pulp regeneration model showed almost complete regeneration/healing with dentin bridge formation when the cavity was sealed with mineral trioxide aggregate (MTA) to create a biocompatible seal of the pulp. This method is a powerful tool for elucidating the processes of dental pulp tissue regeneration following implantation of MSCs. In the present review, we discuss the literature in the field of dental pulp tissue engineering using MSCs including dental pulp stem cells and stem cells from exfoliated deciduous teeth. In addition, we present a brief step-by-step protocol of the coronal pulp regeneration model focusing on the implantation of rat bone marrow MSCs, biodegradable scaffolds, and hydrogels in pulpotomized rat molars. The protocol may lay the foundation for studies aiming at defining further histological and molecular mechanism of the rat pulp tissue engineering.

FGF8 specifies early tooth development by directing the migration of the early tooth founder cells to the site of tooth emergence. To date the effect of the FGF8 in adult dental pulp has not been studied. We have assessed the regenerative potential of FGF8 by evaluating changes in the proteome landscape of dental pulp following short- and long-term exposure to recombinant FGF8 protein. In addition, we carried out qRT PCR analysis to determine extracellular/adhesion gene marker expression and assessed cell proliferation and mineralization in response to FGF8 treatment. 2D and mass spectrometry data showed differential expression of proteins implicated in cytoskeleton/ECM remodeling and migration, cell proliferation and odontogenic differentiation as evidenced by the upregulation of gelsolin, moesin, LMNA, WDR1, PLOD2, COPS5 and downregulation of P4HB. qRT PCR showed downregulation of proteins involved in cell-matrix adhesion such as ADAMTS8, LAMB3 and ANOS1 and increased expression of the angiogenesis marker PECAM1. We have observed that, FGF8 treatment was able to boost dental pulp cell proliferation and to enhance dental pulp mineralization. Collectively, our data suggest that, FGF8 treatment could promote endogenous healing of the dental pulp via recruitment of dental pulp progenitors as well as by promoting their angiogenic and odontogenic differentiation., Significance: Dental pulp cells (DP) have been studied extensively for the purposes of mineralized tissue repair, particularly for the reconstruction of hard and soft tissue maxillofacial defects. Canonical FGF signaling has been implicated throughout multiple stages of tooth development by regulating cell proliferation, differentiation, survival as well as cellular migration. FGF8 expression is indispensible for normal tooth development and particularly for the migration of early tooth progenitors to the sites of tooth emergence. The present study provides proteome and qRT PCR data with regard to the future application and biological relevance of FGF8 in dental regenerative medicine., Authors With Orcid: Rozaliya Tsikandelova - 0000-0003-0178-3767 Zornitsa Mihaylova - 0000-0003-1748-4489 Sébastien Planchon - 0000-0002-0455-0574 Nikolay Ishkitiev - 0000-0002-4351-5579., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Current dental restorations have short longevity, and consequently, there is a need for novel tissue engineering strategies that aim to regenerate the dentin-pulp complex. Dentin matrix contains a myriad of bioactive growth factors and extracellular matrix proteins associated with the recruitment, proliferation, and differentiation of dental pulp progenitor cells. In this study, we show that demineralized dentin matrix (DDM), from noncarious dentine, can be encapsulated into liposomes for delivery to dental tissue to promote regeneration. Liposomes were formulated to encapsulate 0-100 μg/mL DDM, lysed with Triton X, and used in vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) enzyme-linked immunosorbent assays to quantify release. The encapsulation efficiencies were calculated to be 25.9% and 28.8% (VEGF/TGF-β1) for 50 μg/mL DDM liposomes and 39% and 146.7% (VEGF/TGF-β1) for 100 μg/mL DDM liposomes. All liposome formulations had no cytotoxic effects on a dental pulp stem cell (DPSC) clone, as shown by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide), Caspase 3/7 assays, and cell counts. The ability of the liposomes to stimulate DPSC chemotactic recruitment was tested by Boyden chamber chemotaxis assays. Unloaded liposomes alone stimulated significant progenitor cell recruitment, while DDM-loaded liposomes further promoted chemotactic recruitment in a dose-dependent manner. DDM liposomes promoted the upregulation of "osteodentin" markers osteocalcin and RUNX2 (Runt-related transcription factor 2) in DPSCs after 9 days of treatment, determined by real-time quantitative PCR. Furthermore, Alizarin Red S staining showed that unloaded liposomes alone induced biomineralization of DPSCs, and DDM liposomes further increased the amount of mineralization observed. DDM liposomes were more effective than free DDM (10 μg/mL) at activating recruitment and osteogenic differentiation of DPSC, which are key events in the endogenous repair of the dentin-pulp complex. The study has highlighted the therapeutic potential of bioactive DDM liposomes in activating dental tissue repair in vitro, suggesting that liposomal delivery from biomaterials could be a valuable tool for reparative dentistry and hard-tissue engineering applications.