Fabrication of Dentin-Pulp-Like Organoids Using Dental-Pulp Stem Cells.
ABSTRACT: 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.
Project description:Dentin-pulp complex regeneration is a promising alternative treatment for the irreversible pulpitis caused by tooth trauma or dental caries. This process mainly relies on the recruitment of endogenous or the transplanted dental pulp stem cells (DPSCs) to guide dentin-pulp tissue formation. Platelet-derived growth factor (PDGF), a well-known potent mitogenic, angiogenic, and chemoattractive agent, has been widely used in tissue regeneration. However, the mechanisms underlying the therapeutic effects of PDGF on dentin-pulp complex regeneration are still unclear. In this study, we tested the effect of PDGF-BB on dentin-pulp tissue regeneration by establishing PDGF-BB gene-modified human dental pulp stem cells (hDPSCs) using a lentivirus. Our results showed that PDGF-BB can significantly enhance hDPSC proliferation and odontoblastic differentiation. Furthermore, PDGF-BB and vascular endothelial growth factor (VEGF) secreted by hDPSCs enhanced angiogenesis. The chemoattractive effect of PDGF-BB on hDPSCs was also confirmed using a Transwell chemotactic migration model. We further determined that PDGF-BB facilitates hDPSCs migration via the activation of the phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway. In vivo, CM-DiI-labeled hDPSCs were injected subcutaneously into mice, and our results showed that more labeled cells were recruited to the sites implanted with calcium phosphate cement scaffolds containing PDGF-BB gene-modified hDPSCs. Finally, the tissue-engineered complexes were implanted subcutaneously in mice for 12 weeks, the Lenti-PDGF group generated more dentin-like mineralized tissue which showed positive staining for the DSPP protein, similar to tooth dentin tissue, and was surrounded by highly vascularized dental pulp-like connective tissue. Taken together, our data demonstrated that the PDGF-BB possesses a powerful function in prompting stem cell-based dentin-pulp tissue regeneration. Stem Cells Translational Medicine 2017;6:2126-2134.
Project description:Dental pulp infection and necrosis are widespread diseases. Conventional endodontic treatments result in a devitalized and weakened tooth. In this work, we synthesized novel star-shaped polymer to self-assemble into unique nanofibrous spongy microspheres (NF-SMS), which were used to carry human dental pulp stem cells (hDPSCs) into the pulp cavity to regenerate living dental pulp tissues. It was found that NF-SMS significantly enhanced hDPSCs attachment, proliferation, odontogenic differentiation and angiogenesis, as compared to control cell carriers. Additionally, NF-SMS promoted vascular endothelial growth factor (VEGF) expression of hDPSCs in a 3D hypoxic culture. Hypoxia-primed hDPSCs/NF-SMS complexes were injected into the cleaned pulp cavities of rabbit molars for subcutaneous implantation in mice. After 4 weeks, the hypoxia group significantly enhanced angiogenesis inside the pulp chamber and promoted the formation of ondontoblast-like cells lining along the dentin-pulp interface, as compared to the control groups (hDPSCs alone group, NF-SMS alone group, and hDPSCs/NF-SMS group pre-cultured under normoxic conditions). Furthermore, in an in situ dental pulp repair model in rats, hypoxia-primed hDPSCs/NF-SMS were injected to fully fill the pulp cavity and regenerate pulp-like tissues with a rich vasculature and a histological structure similar to the native pulp.Vascularization is key to the regeneration of many vital tissues. However, it is challenging to create a suitable microenvironment for stem cells to regenerate vascularized tissue structure. This manuscript reports a novel star-shaped block copolymer that self-assembles into unique nanofibrous spongy microspheres, which as an injectable scaffold recapitulate the cell-cell and cell-matrix interactions in development. Using a clinically-relevant surgical procedure and a hypoxic treatment, the nanofibrous spongy microspheres were used to deliver stem cells and successfully regenerate dental pulp with a rich vasculature and a complex histologic structure similar to that of the native dental pulp. The novel microspheres can likely be used to regenerate many other vascularized tissues.
Project description:The presence of stem cells within the dental-pulp tissue as well as their differentiation into a new generation of functional odontoblast-like cells constitutes an important step of the dentin-pulp regeneration. Recent investigations demonstrated that the complement system activation participates in 2 critical steps of dentin-pulp regeneration: pulp progenitor's recruitment and pulp nerve sprouting. Surprisingly, its implication in odontoblastic differentiation has not been addressed yet. Since the complement receptor C5a receptor-like 2 (C5L2) is expressed by different stem cells, the aim of this study is to investigate if the dental pulp stem cells express C5L2 and if this receptor participates in odontoblastic differentiation. Immunohistochemistry performed on human third molar pulp sections showed a perivascular co-localization of the mesenchymal stem cell markers STRO1 and C5L2. In vitro immunofluorescent staining confirmed that hDPSCs express C5L2. Furthermore, we determined by real-time polymerase chain reaction that the expression of C5L2 is highly modulated in human dental pulp stem cells (hDPSCs) undergoing odontoblastic differentiation. Moreover, we showed that this odontogenesis-regulated expression of C5L2 is specifically potentiated by the proinflammatory cytokine TNF?. Using a C5L2-siRNA silencing strategy, we provide direct evidence that C5L2 constitutes a negative regulator of the dentinogenic marker DMP1 (dentin matrix protein 1) expression by hDPSCs. Our findings suggest a direct correlation between the odontoblastic differentiation and the level of C5L2 expression in hDPSCs and identify C5L2 as a negative regulator of DMP1 expression by hDPSCs during the odontoblastic differentiation and inflammation processes. This work is the first to demonstrate the involvement of C5L2 in the biological function of stem cells, provides an important knowledge in understanding odontoblastic differentiation of dental pulp stem cells, and may be useful in future dentin-pulp engineering strategies.
Project description:Aim:The purpose of the current study was to investigate the effects of nephronectin (Npnt) in human dental pulp stem cells (hDPSCs). Methodology:Npnt was coated to nontissue culture-treated polystyrene (non-PS) plates. The presence of immobilized protein on the surface was detected by polyclonal rabbit primary anti-Npnt antibody. Then the cell number was counted and compared with PBS-, bovine serum albumin- (BSA-), fish scale type I collagen- (FCOL1-), and human fibronectin- (Fn-) coated wells. Cell proliferation was assessed using CCK-8 assay. Cell morphology was observed under light microscopy and fluorescence microscopy. Lastly, the mRNA expression profiles of integrins, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), and mineralization capacity of hDPSCs were investigated by real time RT-PCR and alizarin red staining, respectively. Results:Npnt mediates hDPSC adhesion and spreading partially via the Arg-Gly-Asp (RGD) motif. Npnt enhanced the mRNA expression of ITGA1, ITGA4, ITGA7, and ITGB1 on day five. Npnt downregulated DSPP but significantly upregulated BSP mRNA expression at day 28. Further, Npnt and FCOL1 accelerated the matrix mineralization in hDPSCs. Conclusions:The current findings implicate that Npnt would be favorable to recruit hDPSCs and conducive to mineralization in hDPSCs. The combination of Npnt with hDPSCs may offer a promising approach for hard tissue regeneration.
Project description:Human dental pulp stem/progenitor cells (hDPSC) can differentiate into odontoblast-like cells and express dentin sialophosphoprotein (DSPP) and osteocalcin (OCN); thus, they may be used to regenerate dentin. However, residual bacterial components in the root canal may suppress this activity.This study investigated the effect of a Porphyromonas gingivalis component on the expression of DSPP and OCN by stimulated hDPSCs and the influence of blockade of TLR2-mediated P. gingivalis host recognition.Stimulated hDPSCs were exposed to varying concentrations of P. gingivalis lipopolysaccharide (LPS), and the expression of DSPP and OCN was measured. Similar groups of stimulated hDPSCs were exposed to TLR2 blocking agents before exposure to LPS.hDPSCs exposed to 5, 10, and 20 ?g/mL LPS exhibited a dose-dependent reduction in the expression of DSPP (3.19 ± 0.18, 2.60 ± 0.49, and 1.15 ± 0.29, respectively) and OCN (3.51 ± 1.18, 2.60 ± 0.67 and 1.66 ± 0.89, respectively). The expression of DSPP and OCN after exposure to 20 ?g/mL of LPS was significantly lower than measured for unexposed stimulated cells (analysis of variance and post hoc Tukey test, P < .05). The blockade of TLR2 using an extra- and intracellular agent affected DSPP (4.67 ± 0.97 and 5.29 ± 1.66, respectively) and OCN (5.25 ± 1.69 and 5.82 ± 2.38, respectively) expression at levels comparable to stimulated cells unexposed to 20 ?g/mL LPS (6.32 ± 2.47 and 4.70 ± 1.60 for DSPP and OCN, respectively).The suppressing effect of P. gingivalis on mineralized matrix formation by hDPSCs is confirmed, and this suppression can be moderated by TLR2 blockade.
Project description:A computer-designed, solvent-free scaffold offer several potential advantages such as ease of customized manufacture and in vivo safety. In this work, we firstly used a computer-designed, solvent-free scaffold and human dental pulp stem cells (hDPSCs) to regenerate neo-bone within cranial bone defects. The hDPSCs expressed mesenchymal stem cell markers and served as an abundant source of stem cells with a high proliferation rate. In addition, hDPSCs showed a phenotype of differentiated osteoblasts in the presence of osteogenic factors (OF). We used solid freeform fabrication (SFF) with biodegradable polyesters (MPEG-(PLLA-co-PGA-co-PCL) (PLGC)) to fabricate a computer-designed scaffold. The SFF technology gave quick and reproducible results. To assess bone tissue engineering in vivo, the computer-designed, circular PLGC scaffold was implanted into a full-thickness cranial bone defect and monitored by micro-computed tomography (CT) and histology of the in vivo tissue-engineered bone. Neo-bone formation of more than 50% in both micro-CT and histology tests was observed at only PLGC scaffold with hDPSCs/OF. Furthermore, the PLGC scaffold gradually degraded, as evidenced by the fluorescent-labeled PLGC scaffold, which provides information to tract biodegradation of implanted PLGC scaffold. In conclusion, we confirmed neo-bone formation within a cranial bone defect using hDPSCs and a computer-designed PLGC scaffold.
Project description:Calcium silicate-based cements are biomaterials with calcium oxide and carbonate filler additives. Their properties are close to those of dentin, making them useful in restorative dentistry and endodontics. The aim of this study was to evaluate the in vitro biological effects of two such calcium silicate cements, Biodentine (BD) and Bioroot (BR), on dental stem cells in both direct and indirect contact models. The two models used aimed to mimic reparative dentin formation (direct contact) and reactionary dentin formation (indirect contact). An original aspect of this study is the use of an interposed thin agarose gel layer to assess the effects of diffusible components from the materials.The two biomaterials were compared and did not modify dental pulp stem cell (DPSC) proliferation. BD and BR showed no significant cytotoxicity, although some cell death occurred in direct contact. No apoptosis or inflammation induction was detected. A striking increase of mineralization induction was observed in the presence of BD and BR, and this effect was greater in direct contact. Surprisingly, biomineralization occurred even in the absence of mineralization medium. This differentiation was accompanied by expression of odontoblast-associated genes. Exposure by indirect contact did not stimulate the induction to such a level.These two biomaterials both seem to be bioactive and biocompatible, preserving DPSC proliferation, migration and adhesion. The observed strong mineralization induction through direct contact highlights the potential of these biomaterials for clinical application in dentin-pulp complex regeneration.
Project description:Pulpal revascularization is commonly used in the dental clinic to obtain apical closure of immature permanent teeth with thin dentinal walls. Although sometimes successful, stimulating bleeding from the periapical area of the tooth can be challenging and in turn may deleteriously affect tooth root maturation. Our objective here was to define reliable methods to regenerate pulp-like tissues in tooth root segments (RSs). G1 RSs were injected with human dental pulp stem cells (hDPSCs) and human umbilical vein endothelial cells (HUVECs) encapsulated in 5% gelatin methacrylate (GelMA) hydrogel. G2 RSs injected with acellular GelMA alone, and G3 empty RSs were used as controls. White mineral trioxide aggregate was used to seal one end of the tooth root segment, while the other was left open. Samples were cultured in vitro in osteogenic media (OM) for 13 d and then implanted subcutaneously in nude rats for 4 and 8 wk. At least 5 sample replicates were used for each experimental group. Analyses of harvested samples found that robust pulp-like tissues formed in G1, GelMA encapsulated hDPSC/HUVEC-filled RSs, and less cellularized host cell-derived pulp-like tissue was observed in the G2 acellular GelMA and G3 empty RS groups. Of importance, only the G1, hDPSC/HUVEC-encapsulated GelMA constructs formed pulp cells that attached to the inner dentin surface of the RS and infiltrated into the dentin tubules. Immunofluorescent (IF) histochemical analysis showed that GelMA supported hDPSC/HUVEC cell attachment and proliferation and also provided attachment for infiltrating host cells. Human cell-seeded GelMA hydrogels promoted the establishment of well-organized neovasculature formation. In contrast, acellular GelMA and empty RS constructs supported the formation of less organized host-derived vasculature formation. Together, these results identify GelMA hydrogel combined with hDPSC/HUVECs as a promising new clinically relevant pulpal revascularization treatment to regenerate human dental pulp tissues.
Project description:Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.
Project description:We previously described a novel tissue cryopreservation protocol to enable the safe preservation of various autologous stem cell sources. The present study characterized the stem cells derived from long-term cryopreserved dental pulp tissues (hDPSCs-cryo) and analyzed their differentiation into definitive endoderm (DE) and hepatocyte-like cells (HLCs) in vitro. Human dental pulp tissues from extracted wisdom teeth were cryopreserved as per a slow freezing tissue cryopreservation protocol for at least a year. Characteristics of hDPSCs-cryo were compared to those of stem cells from fresh dental pulps (hDPSCs-fresh). hDPSCs-cryo were differentiated into DE cells in vitro with Activin A as per the Wnt3a protocol for 6 days. These cells were further differentiated into HLCs in the presence of growth factors until day 30. hDPSCs-fresh and hDPSCs-cryo displayed similar cell growth morphology, cell proliferation rates, and mesenchymal stem cell character. During differentiation into DE and HLCs in vitro, the cells flattened and became polygonal in shape, and finally adopted a hepatocyte-like shape. The differentiated DE cells at day 6 and HLCs at day 30 displayed significantly increased DE- and hepatocyte-specific markers at the mRNA and protein level, respectively. In addition, the differentiated HLCs showed detoxification and glycogen storage capacities, indicating they could share multiple functions with real hepatocytes. These data conclusively show that hPDSCs-cryo derived from long-term cryopreserved dental pulp tissues can be successfully differentiated into DE and functional hepatocytes in vitro. Thus, preservation of dental tissues could provide a valuable source of autologous stem cells for tissue engineering.