Cariogenic Streptococcus mutans (S. mutans) is implicated in the dental pulp necrosis but also in cardiovascular tissue infections. Herein, the purpose was to elucidate how human dental pulp derived stromal cells (DPSCs) react toward a direct interaction with S. mutans. DPSCs were challenged with S. mutans. Following 3 h of interaction, DPSCs were able to internalize S. mutans (rate < 1%), and F-actin fibers played a significant role in this process. S. mutans persisted in the DPSCs for 48 h without causing a cytotoxic effect. S. mutans was, however, able to get out of the DPSCs cytoplasm and to proliferate in the extracellular environment. Yet, we noticed several adaptive responses of bacteria to the extracellular environment such as a modification of the kinetic growth, the increase in biofilm formation on type I collagen and polyester fabrics, as well as a tolerance toward amoxicillin. In response to infection, DPSCs adopted a proinflammatory profile by increasing the secretion of IL-8, lL-1β, and TNF-α, strengthening the establishment of the dental pulp inflammation. Overall, these findings showed a direct impact of S. mutans on DPSCs, providing new insights into the potential role of S. mutans in infective diseases.

Dental pulp stem cells (DPSCs) are a source of postnatal stem cells essential for maintenance and regeneration of dentin and pulp tissues. Previous in vivo transplantation studies have shown that DPSCs are able to give rise to odontoblast-like cells, form dentin/pulp-like structures, and induce blood vessel formation. Importantly, dentin formation is closely associated to blood vessels. We have previously demonstrated that DPSC-induced angiogenesis is VEGFR-2-dependent. VEGFR-2 may play an important role in odontoblast differentiation of DPSCs, tooth formation and regeneration. Nevertheless, the role of VEGFR-2 signaling in odontoblast differentiation of DPSCs is still not well understood. Thus, in this study we aimed to determine the role of VEGFR-2 in odontoblast differentiation of DPSCs by knocking down the expression of VEGFR-2 in DPSCs and studying their odontoblast differentiation capacity in vitro and in vivo. Isolation and characterization of murine DPSCs was performed as previously described. DPSCs were induced by VEGFR-2 shRNA viral vectors transfection (MOI = 10:1) to silence the expression of VEGFR-2. The GFP+ expression in CopGFP DPSCs was used as a surrogate to measure the efficiency of transfection and verification that the viral vector does not affect the expression of VEGFR-2. The efficiency of viral transfection was shown by significant reduction in the levels of VEGFR-2 based on the Q-RT-PCR and immunofluorescence in VEGFR-2 knockdown DPSCs, compared to normal DPSCs. VEGFR-2 shRNA DPSCs expressed not only very low level of VEGFR-2, but also that of its ligand, VEGF-A, compared to CopGFP DPSCs in both transcriptional and translational levels. In vitro differentiation of DPSCs in osteo-odontogenic media supplemented with BMP-2 (100 ng/ml) for 21 days demonstrated that CopGFP DPSCs, but not VEGFR-2 shRNA DPSCs, were positive for alkaline phosphatase (ALP) staining and formed mineralized nodules demonstrated by positive Alizarin Red S staining. The expression levels of dentin matrix proteins, dentin matrix protein-1 (Dmp1), dentin sialoprotein (Dspp), and bone sialoprotein (Bsp), were also up-regulated in differentiated CopGFP DPSCs, compared to those in VEGFR-2 shRNA DPSCs, suggesting an impairment of odontoblast differentiation in VEGFR-2 shRNA DPSCs. In vivo subcutaneous transplantation of DPSCs with hydroxyapatite (HAp/TCP) for 5 weeks demonstrated that CopGFP DPSCs were able to differentiate into elongated and polarized odontoblast-like cells forming loose connective tissue resembling pulp-like structures with abundant blood vessels, as demonstrated by H&E, Alizarin Red S, and dentin matrix staining. On the other hand, in VEGFR-2 shRNA DPSC transplants, odontoblast-like cells were not observed. Collagen fibers were seen in replacement of dentin/pulp-like structures. These results indicate that VEGFR-2 may play an important role in dentin regeneration and highlight the potential of VEGFR-2 modulation to enhance dentin regeneration and tissue engineering as a promising clinical application.

Dental pulp stem cell (DPSC) transplantation has shown new prospects in dental pulp regeneration, and is of great significance in the treatment of pulpitis and pulp necrosis. The fate and regenerative potential of stem cells are dependent, to a great extent, on their microenvironment, which is composed of various tissue components, cell populations, and soluble factors. N-cadherin-mediated cell-cell interaction has been implicated as an important factor in controlling the cell-fate commitment of mesenchymal stem cells. In this study, the effect of N-cadherin on odontogenic differentiation of DPSCs and the potential underlying mechanisms, both in vitro and in vivo, was investigated using a cell culture model and a subcutaneous transplantation mouse model. It was found that the expression of N-cadherin was reversely related to the expression of odontogenic markers (dentin sialophosphoprotein, DSPP, and runt-related transcription factor 2, Runx2) during the differentiation process of DPSCs. Specific shRNA-mediated knockdown of N-cadherin expression in DPSCs significantly increased the expression of DSPP and Runx2, alkaline phosphatase (ALP) activity, and the formation of mineralized nodules. Notably, N-cadherin silencing promoted nucleus translocation and accumulation of β-catenin. Inhibition of β-catenin by a specific inhibitor XAV939, reversed the facilitating effects of N-cadherin downregulation on odontogenic differentiation of DPSCs. In addition, knockdown of N-cadherin promoted the formation of odontoblast-like cells and collagenous matrix in β-tricalcium phosphate/DPSCs composites transplanted into mice. In conclusion, N-cadherin acted as a negative regulator via regulating β-catenin activity during odontogenic differentiation of DPSCs. These data may help to guide DPSC behavior by tuning the N-cadherin-mediated cell-cell interactions, with implications for pulp regeneration.

Background and Objectives: Dental caries is one of the most common human pathological conditions resulting from the invasion of bacteria into the dentin. Current treatment options are limited. In many cases, endodontic therapy leads to permanent pulp tissue loss. Dentin-pulp complex regeneration involves dental pulp stem cells (DPSCs) that differentiate into odontoblast-like cells under an inflammatory context. However, limited information is available on how DPSC differentiation processes are affected under inflammatory environments. We identified the crucial role of complement C5a and its receptor C5aR in the inflammation-induced odontoblastic DPSC differentiation. Methodology: Here, we further investigated the role of a second and controversial C5a receptor, C5L2, in this process and explored the underlying mechanism. Human DPSCs were examined during 7-, 10-, and 14-day odontogenic differentiation treated with TNFα, C5L2 CRISPR, and tyrosine receptor kinase B (TrkB) antagonist [cyclotraxin-B (CTX-B)]. Results: Our data demonstrate that C5L2 CRISPR knockout (KO) enhances mineralization in TNFα-stimulated differentiating DPSCs. We further confirmed that C5L2 CRISPR KO significantly enhances dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP-1) expression after 14-day odontoblastic DPSC differentiation, and treatment with CTX-B abolished the TNFα/C5L2 CRISPR KO-induced DSPP and DMP-1 increase, suggesting TrkB's critical role in this process. Conclusion and Key applications: Our data suggest a regulatory role of C5L2 and TrkB in the TNFα-induced odontogenic DPSC differentiation. This study may provide a useful tool to understand the mechanisms of the role of inflammation in dentinogenesis that is required for successful DPSC engineering strategies.

Transplantation of undifferentiated dental pulp stem cells (DPSCs) may suffer from tumorigenesis. Neuronal differentiated DPSCs (d-DPSCs) have emerged as an ideal source to treat central nervous system (CNS) disorders. Moreover, different components of culture medium functioned on the characteristics of d-DPSCs in vitro. In this study, d-DPSCs were cultured in three types of medium: Neurobasal®®-A medium supplemented with 2% B27 (the 2% B27 NM group), Neurobasal® -A medium supplemented with 2% B27 and 5% FBS (the 2% B27 + 5% FBS NM group), and α-MEM containing 10% FBS (the 10% FBS α-MEM group). We found that d-DPSCs in the 2% B27 + 5% FBS NM group had lower proliferation and reduced expression of transient receptor potential canonical 1 (TRPC1) and CD146, whereas up-regulated Nestin and microtubule-associated protein-2 (MAP-2). Notably, d-DPSCs in the 10% FBS α-MEM group possessed high proliferative capacity, decreased expression of neuron-like markers and partially restored stemness. It was demonstrated that d-DPSCs cultured in the 2% B27 + 5% FBS NM could maintain their neuron-like characteristics. Besides, d-DPSCs cultivated in the 10% FBS α-MEM could partially recover their stem cells properties, indicating that neural differentiation of DPSCs was reversible and could open novel avenues for exploring the pluripotency of DPSCs.

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation. Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A. Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs. Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3'UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

Extracellular matrix (ECM) represents an essential component of the cellular niche. In this conditioned microenvironment, the proliferation rates and differentiation states of stem cells are regulated by several factors. In contrast, in in vitro experimental models, cell growth, or induction procedures toward specific cell lines usually occur in contact with plastic, glass, or biogel supports. In this study, we evaluated the effect of a decellularized ECM, derived from bone marrow stem cells, on the neuronal differentiation of mesenchymal stem cells (MSCs) extracted from dental pulp (Dental Pulp Stem Cells - DPSCs). Since DPSCs derive from neuroectodermal embryonic precursors, they are thought to have a greater propensity toward neuronal differentiation than MSCs isolated from other sources. We hypothesized that the presence of a decellularized ECM scaffold could act positively on neuronal-DPSC differentiation through reproduction of an in vivo-like microenvironment. Results from scanning electron microscopy, immunofluorescence, and gene expression assays showed that ECM is able to positively influence the morphology of cells and their distribution and the expression of specific neuronal markers (i.e., NF-L, NF-M, NF-H, PAX6, MAP2).

Human dental pulp stem cells (hDPSCs) are characterized by high proliferation rate, the multi-differentiation ability and, notably, low immunogenicity and immunomodulatory properties exerted through different mechanisms including Fas/FasL pathway. Despite their multipotency, hDPSCs require particular conditions to achieve chondrogenic differentiation. This might be due to the perivascular localization and the expression of angiogenic marker under standard culture conditions. FasL stimulation was able to promote the early induction of chondrogenic commitment and to lead the differentiation at later times. Interestingly, the expression of angiogenic marker was reduced by FasL stimulation without activating the extrinsic apoptotic pathway in standard culture conditions. In conclusion, these findings highlight the peculiar embryological origin of hDPSCs and provide further insights on their biological properties. Therefore, Fas/FasL pathway not only is involved in determining the immunomodulatory properties, but also is implicated in supporting the chondrogenic commitment of hDPSCs.

A functional vascular network requires that blood vessels are invested by mural cells. We have shown that dental pulp stem cells (DPSC) can undergo vasculogenic differentiation, and that the resulting vessels anastomize with the host vasculature and become functional (blood carrying) vessels. However, the mechanisms underlying the maturation of DPSC-derived blood vessels remains unclear. Here, we performed a series of studies to understand the process of mural cell investment of blood vessels generated upon vasculogenic differentiation of dental pulp stem cells. Primary human DPSC were co-cultured with primary human umbilical artery smooth muscle cells (HUASMC) in 3D gels in presence of vasculogenic differentiation medium. We observed DPSC capillary sprout formation and SMC recruitment, alignment and remodeling that resulted in complex vascular networks. While HUASMC enhanced the number of capillary sprouts and stabilized the capillary network when co-cultured with DPSC, HUASMC by themselves were unable to form capillary sprouts. In vivo, GFP transduced human DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated functional human blood vessels invested with murine smooth muscle actin (SMA)-positive, GFP-negative cells. Inhibition of PDGFR-β signaling prevented the SMC investment of DPSC-derived capillary sprouts in vitro and of DPSC-derived blood vessels in vivo. In contrast, inhibition of Tie-2 signaling did not have a significant effect on the SMC recruitment in DPSC-derived vascular structures. Collectively, these results demonstrate that PDGF-BB signaling via PDGFR-β regulates the process of maturation (mural investment) of blood vessels generated upon vasculogenic differentiation of human dental pulp stem cells.

A subset of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) developed a condition of hyper-inflammation, which can cause multi-organ damage and the more severe forms of coronavirus disease 2019 (COVID-19). Mesenchymal stem cells (MSCs) can promote tissue regeneration and modulate immune responses and, thus, have the rational requirements to be used to counteract SARS-CoV-2-induced pneumonia and hyper-inflammation. The aim of the present study was to gain insight into possible mechanisms of action of MSCs obtained from human dental pulp [dental pulp stem cells (DPSCs)] in COVID-19 patients. We investigated the concentrations of 18 cytokines in supernatants of peripheral blood mononuclear cells (PBMCs) obtained from COVID-19 patients cultured in vitro alone and in contact with DPSCs. The modulation of cytokines in PBMCs was confirmed by real-time PCR. IL-6 was the sole cytokine detected in supernatants of DPSCs. In resting conditions, co-culture increased IL-1β, IL-2, IL-5, IL-6, IL-10, IL-18, TNFα, and granulocyte macrophage colony-stimulating factor (GM-CSF) levels. When PBMCs were activated with anti-CD3/CD28 antibody-coated beads, co-culture increased IL-6 and GM-CSF, whereas it decreased IFNγ, TNFα, IL-2, IL-5, IL-9, IL-10, IL-12 (p70), IL-17A, IL-18, IL-21, IL-23, and IL-27 levels. Concentrations of IL-1β, IL-4, IL-13, and IL-22 were not affected. The comparison of cytokine concentrations in supernatants of PBMCs from COVID-19 patients vs. healthy subjects revealed lower concentrations of IL-10 and higher concentrations of IL-18 in supernatants of CD3/CD28-activated PBMCs from COVID-19 patients. Results are explorative but indicate that DPSCs can modulate the production of cytokines deregulated in COVID-19 patients, supporting their potential use in COVID-19.

Circular RNAs (circRNAs) have been found to be a crucial role in stem cell-associated bone regeneration. However, the functions and underlying mechanisms of circRNAs in the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain largely unclear. We found that overexpression of circRNA124534 unexpectedly promoted DPSCs osteogenesis in vitro and in vivo. Our results confirmed circRNA124534, acting as a miRNA sponge, directly interacts with miR-496 and consequently regulates β-catenin, which in turn exerts osteogenesis of DPSCs. Enforced expression of miR-496 reversed the osteogenesis of circRNA124534, and suppression of miR-496 enhanced the osteogenic differentiation of DPSCs by promoting β-catenin. In conclusion, our findings demonstrate functions of circRNA124534 in modulating osteogenic differentiation through the miR-496/β-catenin pathway; thus, providing a novel potential target for therapy.

Titanium modifications with different silver loading methods demonstrate excellent antibacterial properties. Yet pure silver nanoparticles with limited bioactive properties may delay regeneration of bone surrounding the dental implant. Therefore, loading silver with bioactive drugs on titanium surfaces seems to be a very promising strategy. Herein, we designed a silver (Ag) step-by-step cross-linking with the basic fibroblast growth factor (bFGF) by polydopamine (PDA) and heparin on titanium nanotube (TNT) as its cargo (TNT/PDA/Ag/bFGF) to improve the implant surface. Our results showed that TNT/PDA/Ag/bFGF significantly enhanced the osteogenic differentiation of dental pulp stem cells (DPSCs). It also showed an excellent effect in bacterial inhibition and a reduction of pro-inflammatory factors through inhibition of M1 macrophage activity. These results showed that bFGF cross-linked silver coating on TNTs presented good osteogenic differentiation and early anti-infiammatory and antibacterial properties. Together, this novel design on titanium provides a promising therapeutic for dental implants.

Previous studies have found that circular RNA (circRNA) hsa_circ_0026827 plays a role during osteoblast differentiation, but the mechanism is unclear. The aim of this study was to illuminate the role of hsa_circ_0026827 in human dental pulp stem cells (DPSCs) during osteoblast differentiation. The results show that hsa_circ_0026827 expression significantly increased during osteoblast differentiation, while knockdown of hsa_circ_0026827 suppressed DPSC-derived osteoblast differentiation. microRNA (miRNA) expression profile analysis showed that downregulation of hsa_circ_0026827 promoted miR-188-3p expression. miR-188-3p downregulation restored osteogenic differentiation of DPSCs after hsa_circ_0026827 was silenced. Luciferase reporter assays verified that miR-188-3p was the target of hsa_circ_0026827 and also demonstrated that Beclin1 and RUNX1 were miR-188-3p downstream targets. miR-188-3p overexpression suppressed DPSC osteogenic differentiation by targeting Beclin-1-mediated autophagy and runt-related transcription factor 1 (RUNX1). In vivo studies using a heterotopic bone model also found that hsa_circ_0026827 overexpression plays an important role in promoting heterotopic bone formation. In conclusion, our research indicates that hsa_circ_0026827 promotes osteoblast differentiation of DPSCs via Beclin1 and the RUNX1 signaling pathways by sponging miR-188-3p, which suggests novel therapeutics for osteoporosis treatment.

Dental stem cells (DSCs) holds the ability to differentiate into numerous cell types. This property makes these cells particularly appropriate for therapeutic use in regenerative medicine. We report evidence that when DSCs undergo osteogenic differentiation, the osteoblast-like cells can be reverted back to a stem-like state and then further differentiated toward the osteogenic phenotype again, without gene manipulation. We have investigated two different MSCs types, both from dental tissues: dental follicle progenitor stem cells (DFPCs) and dental pulp stem cells (DPSCs). After osteogenic differentiation, both DFPCs and DPSCs can be reverted to a naïve stem cell-like status; importantly, dedifferentiated DSCs showed a greater potential to further differentiate toward the osteogenic phenotype. Our report aims to demonstrate for the first time that it is possible, under physiological conditions, to control the dedifferentiation of DSCs and that the rerouting of cell fate could potentially be used to enhance their osteogenic therapeutic potential. Significantly, this study first validates the use of dedifferentiated DSCs as an alternative source for bone tissue engineering.

Fibroblast activation protein-α (FAPα) is a membrane protein with dipeptidyl-peptidase and type I collagenase activity and is expressed during fetal growth. At the age of adolescence, FAPα expression is greatly reduced, only emerging in pathologies associated with extracellular matrix remodeling. We determined whether FAPα is expressed in human dental tissue involved in root maturation i.e., dental follicle and apical papilla and in dental pulp tissue. The dental follicle revealed a high concentration of FAPα and vimentin-positive cells within the stromal tissue. A similar observation was made in cell culture and FACS analysis confirmed these as dental follicle stem cells. Within the remnants of the Hertwigs' epithelial root sheath, we observed FAPα staining in the E-cadherin positive and vimentin-negative epithelial islands. FAPα- and vimentin-positive cells were encountered at the periphery of the islands suggesting an epithelial mesenchymal transition process. Analysis of the apical papilla revealed two novel histological regions; the periphery with dense and parallel aligned collagen type I defined as cortex fibrosa and the inner stromal tissue composed of less compacted collagen defined as medulla. FAPα expression was highly present within the medulla suggesting a role in extracellular matrix remodeling. Dental pulp tissue uncovered a heterogeneous FAPα staining but strong staining was noted within odontoblasts. In vitro studies confirmed the presence of FAPα expression in stem cells of the apical papilla and dental pulp. This study identified the expression of FAPα expression in dental stem cells which could open new perspectives in understanding dental root maturation and odontoblast function.

Pulpitis is a frequent bacterially driven inflammation featured with the local accumulation of inflammatory products in human dental pulps. A GEO dataset GSE16134 comprising data of inflamed dental pulp tissues was used for bioinformatics analyses. A protein-protein interaction (PPI) analysis suggested that chemokine receptor 4 (CXCR4) owned a high correlation with platelet endothelial cell adhesion molecule-1 (PECAM1). A rat model with pulpitis was established, and lipopolysaccharide (LPS)-induced human dental pulp fibroblasts (HDPFs) were used for in vitro experiments. Then, high expression of PECAM1 and CXCR4 was validated in the inflamed dental pulp tissues in rats and in LPS-induced HDPFs. Either downregulation of PECAM1 or CXCR4 suppressed inflammatory cell infiltration in inflamed tissues as well as the inflammation and apoptosis of HDPFs. A transcription factor myocyte-enhancer factor 2 (MEF2C) was predicted and validated as a positive regulator of either PECAM1 or CXCR4, which activated the NF-κB signaling pathway and promoted pulpitis progression. To sum up, this study suggested that MEF2C transcriptionally activates PECAM1 and CXCR4 to activate the B-cell and NF-κB signaling pathways, leading to inflammatory cell infiltration and pulpitis progression.

Matrix-metalloproteinase-13 (MMP13) is important for bone formation and remodeling; however, its role in tooth development remains unknown. To investigate this, MMP13-knockout (Mmp13 -/- ) mice were used to analyze phenotypic changes in the dentin-pulp complex, mineralization-associated marker-expression, and mechanistic interactions. Immunohistochemistry demonstrated high MMP13-expression in pulp-tissue, ameloblasts, odontoblasts, and dentin in developing WT-molars, which reduced in adults, with human-DPC cultures demonstrating a >2000-fold increase in Mmp13-expression during mineralization. Morphologically, Mmp13 -/- molars displayed critical alterations in the dentin-phenotype, affecting dentin-tubule regularity, the odontoblast-palisade and predentin-definition with significantly reduced dentin volume (∼30% incisor; 13% molar), and enamel and dentin mineral-density. Reactionary-tertiary-dentin in response to injury was reduced at Mmp13 -/- molar cusp-tips but with significantly more dystrophic pulpal mineralization in MMP13-null samples. Odontoblast differentiation-markers, nestin and DSP, reduced in expression after MMP13-loss in vivo, with reduced calcium deposition in MMP13-null DPC cultures. RNA-sequencing analysis of WT and Mmp13 -/- pulp highlighted 5,020 transcripts to have significantly >2.0-fold change, with pathway-analysis indicating downregulation of the Wnt-signaling pathway, supported by reduced in vivo expression of the Wnt-responsive gene Axin2. Mmp13 interaction with Axin2 could be partly responsible for the loss of odontoblastic activity and alteration to the tooth phenotype and volume which is evident in this study. Overall, our novel findings indicate MMP13 as critical for tooth development and mineralization processes, highlighting mechanistic interaction with the Wnt-signaling pathway.

Dentin, which composes most of the tooth structure, is formed by odontoblasts, long-lived post-mitotic cells maintained throughout the entire life of the tooth. In mature odontoblasts, however, cellular activity is significantly weakened. Therefore, it is important to augment the cellular activity of mature odontoblasts to regenerate physiological dentin; however, no molecule regulating the cellular activity of mature odontoblasts has yet been identified. Here, we suggest that copine-7 (CPNE7) can reactivate the lost functions of mature odontoblasts by inducing autophagy. CPNE7 was observed to elevate the expression of microtubule-associated protein light chain 3-II (LC3-II), an autophagy marker, and autophagosome formation in the pre-odontoblast and mature odontoblast stages of human dental pulp cells. CPNE7-induced autophagy upregulated DSP and DMP-1, odontoblast differentiation and mineralization markers, and augmented dentin formation in mature odontoblasts. Furthermore, CPNE7 also upregulated NESTIN and TAU, which are expressed in the physiological odontoblast process, and stimulated the elongation of the odontoblast process by inducing autophagy. Moreover, lipofuscin, which progressively accumulates in long-lived post-mitotic cells and hinders their proper functions, was observed to be removed in recombinant CPNE7-treated mature odontoblasts. Thus, CPNE7-induced autophagy reactivated the function of mature odontoblasts and promoted the formation of physiological dentin in vivo. On the other hand, the well-known autophagy inducer, rapamycin, promoted odontoblast differentiation in pre-odontoblasts but did not properly reactivate the function of mature odontoblasts. These findings provide evidence that CPNE7 functionally reactivates mature odontoblasts and introduce its potential for dentinal loss-targeted clinical applications.

Physiological root resorption of deciduous teeth is a normal phenomenon. How the angiogenesis process is regulated to provide adequate levels of oxygen and nutrients in hypoxic conditions when the dental pulp tissue is reduced at the stage of root resorption is not fully understood. In this study, we designed hypoxic preconditioning (2%) to mimic the physiological conditions. We isolated exosomes from hypoxic-preconditioned SHED (Hypo-exos) cells and from normally cultured SHED cells (Norm-exos). We found that treatment with Hypo-exos significantly enhanced the growth, migration and tube formation of endothelial cells in vitro compared with Norm-exos. We also performed matrigel plug assays in vivo and higher expression of VEGF and higher number of lumenal structures that stained positive for CD31 were found in the Hypo-exos treated group. To understand the potential molecular mechanism responsible for the positive effects of Hypo-exos, we performed exosomal miRNA sequencing and validated that Hypo-exos transferred both let-7f-5p and miR-210-3p to promote the tube formation of endothelial cells. Further study revealed that those two miRNAs regulate angiogenesis via the let-7f-5p/AGO1/VEGF and/or miR-210-3p/ephrinA3 signal pathways. Finally, we found that the increased release of exosomes regulated by hypoxia treatment may be related to Rab27a. Taking these data together, the present study demonstrates that exosomes derived from hypoxic-preconditioned SHED cells promote angiogenesis by transferring let-7f-5p and miR-210-3p, which suggests that they can potentially be developed as a novel therapeutic approach for pro-angiogenic therapy in tissue regeneration engineering.

Adult erythropoiesis is a highly controlled sequential differentiation of hematopoietic stem cells (HSCs) to mature red blood cells in the bone marrow (BM). The bones which contain BM are diverse in their structure, embryonic origin, and mode of ossification. This has created substantial heterogeneity in HSCs function in BM of different bones, however, it is not known if this heterogeneity influences erythropoiesis in different bones and different regions of the same bone. In this study, we examined steady state BM erythroid progenitors and precursors from different bones - the femur, tibia, pelvis, sternum, vertebrae, radius, humerus, frontal, parietal bone, and compared all to the femur. Trabecular and cortical regions of the femur were also compared for differences in erythropoiesis. In addition, mouse spleen was studied to determine at which age erythropoietic support by the spleen was lost postnatally. We report that total erythroid cells, and erythroid precursors in the femur are comparable to tibia, pelvis, humerus and sternum, but are significantly reduced in the vertebrae, radius, frontal, and parietal bones. Erythroid progenitors and multipotential progenitor numbers are comparable in all the bones except for reduced number in the parietal bone. In the femur, the epiphysis and metaphysis have significantly reduced number of erythroid precursors and progenitors, multipotential progenitors and myeloid progenitors compared to the diaphysis region. These results show that analysis of erythroid precursors from diaphysis region of the femur is representative of tibia, pelvis, humerus and sternum and have significant implications on the interpretation of the steady-state erythropoiesis finding from adult BM. Postnatal spleen supports erythroid precursors until 6 weeks of age which coincides with reduced number of red pulp macrophages. The residual erythroid progenitor support reaches the adult level by 3 months of age. In conclusion, our findings provide insights to the differences in erythropoiesis between different bones, between trabecular and cortical regions of the femur, and developmental changes in postnatal spleen erythropoiesis.