Native dental pulp extracellular matrix (DPEM) has proven to be an effective biomaterial for dental pulp regeneration. However, as a significant extracellular matrix glycoprotein, partial laminins were lost during the decellularization process, which were essential for odontoblast differentiation. Thereby, this study investigated the feasibility of LN supplementation to improve the surface of DPEM for odontoblast layer regeneration. The influences of laminin on cell adhesion and odontogenic differentiation were evaluated in vitro. Then, we fabricated laminin-modified DPEM based on the physical coating strategy and observed the location and persistency of laminin coating by immunofluorescent staining. Finally, laminin-modified DPEM combined with treated dentin matrix (TDM) was transplanted in orthotopic jaw bone of beagles (n = 3) to assess the effect of LNs on dental pulp tissue regeneration. The in vitro results showed that laminins could improve the adhesion of dental pulp stem cells (DPSCs) and promoted DPSCs toward odontogenic differentiation. Continuous odontoblastic layer-like structure was observed in laminin-modified DPEM group, expressing the markers for odontoblastogenesis, dentine matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP). Overall, these studies demonstrate that the supplementation of laminins to DPEM contributes to the odontogenic differentiation of cells and to the formation of odontoblast layer in dental pulp regeneration.

Direct pulp capping is a vital pulp therapy for a pin-point dental pulp exposure. Applying a pulp capping material leads to the formation of a dentin bridge and protects pulp vitality. The aim of this study was to compare the effects of four dental materials, DyCal®, ProRoot® MTA, Biodentine™, and TheraCal™ LC in vitro.

Human dental pulp stem cells (hDPSCs) are multipotent mesenchymal stem cells (MSCs) that are capable of self-renewal with multilineage differentiation potential. After being cryopreserved, hDPSCs were reported to maintain a high level of proliferation and multi-differentiation abilities. In order to optimize cryopreservation techniques, decrease storage requirements and lower contamination risks, the feasibility of new whole-tooth cryopreservation and its effects on hDPSCs were tested. The survival rates, morphology, proliferation rates, cell activity, surface antigens and differentiation abilities of hDPSCs isolated from fresh teeth were compared with those of one-month cryopreserved teeth in 5% and 10% DMSO. The data of the present study indicated that the new cryopreservation approach did not reduce the capabilities or stemness of hDPSCs, with the exception that it extended the first appearance time of hDPSCs in the teeth that were cryopreserved in 10% DMSO, and reduced their recovery rate. With the novel strategy of freezing, the hDPSCs still expressed the typical surface markers of MSCs and maintained excellent proliferation capacity. Three consecutive weeks of osteogenic and adipogenic induction also showed that the expression of the key genes in hDPSCs, including lipoprotein lipase (LPL), peroxisome proliferator-activated receptor-γ (PPAR-γ), alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), type I collagen (COL I) and osteocalcin (OSC) was not affected, indicating that their differentiation abilities remained intact, which are crucial parameters for hDPSCs as cell-therapy candidates. These results demonstrated that the new cryopreservation method is low-cost and effective for the good preservation of hDPSCs without compromising cell performance, and can provide ideas and evidence for the future application of stem-cell therapies and the establishment of dental banks.

Endodontic regeneration shows promise in treating dental pulp diseases; however, no suitable scaffolds exist for pulp regeneration. Acellular natural extracellular matrix (ECM) is a favorable scaffold for tissue regeneration since the anatomical structure and ECM of the natural tissues or organs are well-preserved. Xenogeneic ECM is superior to autologous or allogeneic ECM in tissue engineering for its unlimited resources. This study investigated the characteristics of decellularized dental pulp ECM from swine and evaluated whether it could mediate pulp regeneration. Dental pulps were acquired from the mandible anterior teeth of swine 12 months of age and decellularized with 10% sodium dodecyl sulfate (SDS) combined with Triton X-100. Pulp regeneration was conducted by seeding human dental pulp stem cells into decellularized pulp and transplanted subcutaneously into nude mice for 8 weeks. The decellularized pulp demonstrated preserved natural shape and structure without any cellular components. Histological analysis showed excellent ECM preservation and pulp-like tissue, and newly formed mineralized tissues were regenerated after being transplanted in vivo. In conclusion, decellularized swine dental pulp maintains ECM components favoring stem cell proliferation and differentiation, thus representing a suitable scaffold for improving clinical outcomes and functions of teeth with dental pulp diseases.

Dental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry.

BACKGROUND Dental pulp cells (DPCs) play vital roles in the recovery of dental pulp tissue. Concentrated growth factor (CGF) can promote proliferation and mineralization of various cells. However, the functions of CGF on DPCs and dental pulp tissue are unclear. The object of our study was to identify the roles of CGF in DPCs proliferation and mineralization in vitro and to assess the effects of CGF on direct pulp capping in vivo. MATERIAL AND METHODS We performed CCK-8 and Transwell assay to detect proliferation and migration activity of DPCs. Alizarin Red staining was performed to examine mineralized nodules. Alkaline phosphatase activity test was used to measure the mineralization capacity of DPCs. We assessed the odontogenic differentiation gene expression level by Western blot and qPCR. The effect of CGF on direct pulp capping in vivo were evaluated by radiography and histopathology. RESULTS CGF increased the number of proliferative and migratory DPCs. CGF enhanced DPCs mineralized nodules and improved the gene expression levels of DSPP, DMP-1, BSP, and ALP. CGF upregulated the protein levels of ALP, BMP2, SMAD5, Runx2, and p-Smad, and the effect could be partially reversed by Noggin. CGF promoted pulp recovery and kept its vitality in directly pulp capping. CONCLUSIONS CGF promotes DPCs proliferation and mineralization. It regulates the mineralization of DPCs via the BMP2/SMAD5/Runx2 signaling pathway. CGF can be used as the effective graft for direct pulp capping.

We developed a novel dentin-pulp-like organoid. It has both stem-cell and odontoblast characteristics using a mesenchymal cell lineage of human dental-pulp stem cells (hDPSCs). The mixture of hDPSCs and Matrigel was transferred into the maintenance medium (MM) and divided into four different groups according to how long they were maintained in the odontogenic differentiation medium (ODM). All organoids were harvested at 21 days and analyzed to find the optimal differentiation condition. To assess the re-fabrication of dentin-pulp-like organoid, after dissociation of the organoids, it was successfully regenerated. Additionally, its biological activity was confirmed by analyzing changes of relevant gene expression and performing a histology analysis after adding Biodentine® into the ODM. The organoid was cultured for 11 days in the ODM (ODM 11) had the most features of both stem cells and differentiated cells (odontoblasts) as confirmed by relevant gene expression and histology analyses. Micro-computed tomography and an electron microscope also showed mineralization and odontoblastic differentiation. Finally, ODM 11 demonstrated a biologically active response to Biodentine® treatment. In conclusion, for the first time, we report the fabrication of a dentin-pulp-like organoid using mesenchymal stem cells. This organoid has potential as a future therapeutic strategy for tooth regeneration.

Senescence-related impairment of proliferation and differentiation limits the use of dental pulp cells for tissue regeneration. Deletion of sclerostin improves the dentinogenesis regeneration, while its role in dental pulp senescence is unclear. We investigated the role of sclerostin in subculture-induced senescence of human dental pulp cells (HDPCs) and in the senescence-related decline of proliferation and odontoblastic differentiation.

The present study aimed to determine whether dentine tissue and preparations of extracellular matrix (ECM) from pulp (pECM) and dentine (dECM), and breakdown products, influenced pulp cell migration. Chemotaxis transwell and agarose spot assays demonstrated that both dentine and pulp ECM molecules acted as chemoattractants for primary pulp cells. Chemoattractant activities of dECM and pECM were enhanced when subjected to acid and enzymatic breakdown, respectively. This enhanced activity following physiologically relevant breakdown may be pertinent to the disease environment. Pulp cell migration in response to dental ECMs was dependent on an active rho pathway. Recruited cells exhibited increased stem cell marker expression indicating that dental ECMs and their breakdown products selectively attract progenitor cells that contribute to repair processes. In conclusion, combined these results indicate that ECM molecules contribute to cell recruitment necessary for regeneration of the dentine-pulp complex after injury.

In the crown pulp of brachydont teeth, a cell-free and a cell-rich zone are established beneath the odontoblastic layer, indicating a mature status. For the equine dental pulp, there are no descriptions which allow for a comparative analysis with regard to functional requirements in terms of lifelong secondary dentin production to compensate for occlusal wear. For histomorphological and immunohistological investigations, ten incisors and ten check teeth were used from seven adult horses and five foals. In the periphery of the equine dental pulp, a constant predentin and odontoblastic cell layer was present, followed by densely packed fibroblastic cells, capillary networks, and a high concentration of nerve fibers, suggesting a subodontoblastic supportive zone. Whilst the size of the equine dental pulp decreased with age, the numbers of blood vessels, nerve fibers, and fibroblastic cells increased with age. Histological analysis of the equine dental pulp did not show a cell-free and cell-rich zone as described in the brachydont crown pulp. The equine dental pulp remained in a juvenile status even in aged horses, with morphological features indicating a high capacity for dentine production.

Angiogenesis is considered a crucial event for dental pulp regeneration. The purpose of this study was to demonstrate neovascularization during coronal pulp regeneration in rat molars using rat dental pulp cells (rDPCs) and to examine whether rDPC-endothelial cell interactions promote proangiogenic capacity in vitro.

Dental pulp with special structure has become a good reference sample in paleomicrobiology-related blood-borne diseases, many pathogens were detected by different methods based on the diagnosis of nucleic acids and proteins.

Dental pulp plays an important role in the health of teeth. The aging of teeth is strongly related to the senescence of dental pulp cells. A novel adipokine, visfatin, is closely associated with cellular senescence. However, little is known about the effect of visfatin on the senescence of human dental pulp cells (hDPCs). Here, it was found that in vivo visfatin levels in human dental pulp tissues increase with age and are upregulated in vitro in hDPCs during premature senescence activated by H2O2, suggesting a correlation between visfatin and senescence. In addition, visfatin knockdown by small interfering RNA led to the reduction in hDPC senescence; however, treatment with exogenous visfatin protein induced the senescence of hDPCs along with increased NADPH consumption, which was reversed by FK866, a chemical inhibitor of visfatin. Furthermore, visfatin-induced senescence was associated with both the induction of telomere damage and the upregulation of senescence-associated secretory phenotype (SASP) factors as well as NF-κB activation, which were all inhibited by FK866. Taken together, these results demonstrate, for the first time, that visfatin plays a pivotal role in hDPC senescence in association with telomere dysfunction and the induction of SASP factors.

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.

Mesenchymal stromal/stem cell (MSC) therapies are currently being explored for dental pulp regeneration. As the therapeutic effects of MSCs in tissue repair are mediated mainly through the release of extracellular vesicles (EVs) including exosomes, we investigated here the cellular processes and molecular mechanisms modulated by MSC exosomes in dental pulp regeneration. Using dental pulp cell (DPC) cultures, we demonstrated that MSC exosomes could increase DPC migration, proliferation, and odontogenic differentiation. The enhancement of these cellular processes was mediated through exosomal CD73-mediated adenosine receptor activation of AKT and ERK signaling. Consistent with these observations, MSC exosomes increased the expression of dentin matrix proteins and promoted the formation of dentin-like tissue and bridge-like structures in a rat pulp defect model. These effects were comparable to that of mineral trioxide aggregate (MTA) treatment. MSC exosomes also yielded recellularized pulp-dentin tissues in the root canal of endodontically-treated human premolars, following subcutaneous implantation in the mouse dorsum. Together, our findings suggest that MSC exosomes could exert a multi-faceted effect on DPC functions including migration, proliferation and odontogenic differentiation to promote dental pulp regeneration. This study provides the basis for development of MSC exosomes as a cell-free MSC therapeutic alternative for pulp-dentin regeneration.

To evaluate the effect of SHED-CM on the proliferation, differentiation, migration ability, cell death, gene expression and production of VEGF of HUVEC in vitro and in a rodent orthotopic dental pulp regeneration.

Trauma or injury to the dental pulp causes inflammation. This study compared the proteome of healthy pulp with inflamed pulp and traumatic pulp to identify the differentially expressed proteins in the diseased state. Five participants were grouped based on the pulpal status of the teeth: healthy, inflamed, or traumatic pulp. Pulp was extirpated and stored immediately in liquid nitrogen. Pulp tissues were subjected to 2-dimensional gel electrophoresis, and spot selection was performed. The selected spots were analyzed using liquid chromatography-tandem mass spectrometry and identified by correlating mass spectra to the proteomic databases. Fifteen spots showed increased expression in the inflamed and traumatic pulp. Annexin V, type II keratin, and hemoglobin levels were increased two-fold in the inflamed and traumatic pulp group and annexin V, mutant beta-actin, and hemoglobin were increased by ten-fold in the inflamed or traumatic pulp group, compared to levels in the healthy pulp group. Annexin V constituted two out of fifteen protein spots, and seemed to play a critical role in inhibiting inflammation and promoting the immune reaction. Further studies on this protein concerning its role in pulp repair are necessary to elucidate the underlying mechanisms.

Extracellular vesicles (EVs) are lipid bound particles derived from their original cells, which play critical roles in intercellular communication through their cargoes, including protein, lipids, and nucleic acids. According to their biogenesis and release pathway, EVs can be divided into three categories: apoptotic vesicles (ApoVs), microvesicles (MVs), and small EVs (sEVs). Recently, the role of EVs in oral disease has received close attention. In this review, the main characteristics of EVs are described, including their classification, biogenesis, biomarkers, and components. Moreover, the therapeutic mechanism of EVs in tissue regeneration is discussed. We further summarize the current status of EVs in pulp/periodontal tissue regeneration and discuss the potential mechanisms. The therapeutic potential of EVs in pulp and periodontal regeneration might involve the promotion of tissue regeneration and immunomodulatory capabilities. Furthermore, we highlight the current challenges in the translational use of EVs. This review would provide valuable insights into the potential therapeutic strategies of EVs in dental pulp and periodontal regeneration.

Dental Caries affects approximately 90% of the world's population. At present, the clinical treatment for dental caries is root canal therapy. This treatment results in loss of tooth sensitivity and vitality. Tissue engineering can potentially solve this problem by enabling regeneration of a functional pulp tissue. Dental pulp stem cells (DPSCs) have been shown to be an excellent source for pulp regeneration. However, limited availability of these cells hinders its potential for clinical translation. We have investigated the possibility of using somatic mesenchymal stem cells (MSCs) from other sources for dental pulp tissue regeneration using a biomimetic dental pulp extracellular matrix (ECM) incorporated scaffold. Human periodontal ligament stem cells (PDLSCs) and human bone marrow stromal cells (HMSCs) were investigated for their ability to differentiate toward an odontogenic lineage. In vitro real-time PCR results coupled with histological and immunohistochemical examination of the explanted tissues confirmed the ability of PDLSCs and HMSCs to form a vascularized pulp-like tissue. These findings indicate that the dental pulp stem derived ECM scaffold stimulated odontogenic differentiation of PDLSCs and HMSCs without the need for exogenous addition of growth and differentiation factors. This study represents a translational perspective toward possible therapeutic application of using a combination of somatic stem cells and extracellular matrix for pulp regeneration.

CD146 and STRO-1 are endothelial biomarkers that are co-expressed on the cellular membranes of blood vessels within human dental pulp tissue. This study characterized the percentage of dentin-like structures produced by CD146-positive (CD146+) human dental pulp stem cells (DPSCs), compared with their CD146-negative (CD146-) counterparts. DPSC populations were enriched using magnetic-activated cell sorting (MACS), yielding CD146+ and CD146- cells, as well as mixtures composed of 25% CD146+ cells and 75% CD146- cells (CD146+/-). Cell growth assays indicated that CD146+ cells exhibit an approximate 3-4 h difference in doubling time, compared with CD146- cells. Cell cycle distributions were determined by flow cytometry analysis. The low percentage of CD146+ cells' DNA content in G0/G1 phase were compared with CD146- and non-separated cells. In contrast to CD146- and non-separated cells, prompt mineralization was observed in CD146+ cells. Subsequently, qRT-PCR revealed high mRNA expression of CD146 and Alkaline phosphatase in mineralization-induced CD146+ cells. CD146+ cells were also observed high adipogenic ability by Oil red O staining. Histological examinations revealed an increased area of dentin/pulp-like structures in transplanted CD146+ cells, compared with CD146- and CD146+/- cells. Immunohistochemical studies detected dentin matrix protein-1 (DMP1) and dentin sialophosphoprotein (DSPP), as well as human mitochondria, in transplanted DPSCs. Co-expression of CD146 and GFP indicated that CD146 was expressed in transplanted CD146+ cells. CD146+ cells may promote mineralization and generate dentin/pulp-like structures, suggesting a role in self-renewal of stem cells and dental pulp regenerative therapy.