Delivery of affordable and scalable encapsulated allogenic/autologous mesenchymal stem cells in coagulated platelet poor plasma for dental pulp regeneration.
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ABSTRACT:
SUBMITTER: Angelopoulos I
PROVIDER: S-EPMC8748942 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
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Project description: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.
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Project description:Regenerative endodontic procedures using autologous platelet-rich plasma (PRP) can improve the biologic outcome of treatment. However, its mechanism of action on improving pulp regeneration is not fully elucidated. Autophagy was recently shown to be related to tissue repair and osteogenesis. Therefore, the objective of this study was to investigate the effect of PRP in dental pulp regeneration and to elucidate the role of autophagy involved in this process. Human dental pulp cells (hDPCs) were isolated from healthy dental pulp and co-cultured with an increasing concentration of PRP. Cellular migration and proliferation were determined by scratch assay, transwell assay, and cell-counting kit 8 assay. Osteogenic differentiation was clarified by using alkaline phosphatase staining, alizarin red staining, and real-time polymerase chain reaction (RT-PCR) to measure the gene expression levels of alkaline phosphatase, collagen-1, osteocalcin, dentin matrix protein 1, and dentin sialophosphoprotein. Autophagic bodies were observed by transmission electron microscopy and the expression of autophagy marker light chain 3B (LC3B) was determined by immunofluorescence staining. The mRNA and protein expression level of LC3B and Beclin-1 were quantified by qRT-PCR and western blotting. The effect of PRP on cellular migration, proliferation, and osteogenic differentiation was further investigated in the milieu of autophagy activator, rapamycin, and inhibitor, 3-methyladenine. Results showed that PRP promoted cell migration, proliferation, and osteogenic differentiation. Autophagic bodies were strongly activated and the expression level of LC3B and Beclin-1 was significantly promoted by PRP. Autophagy inhibition suppressed PRP-induced hDPCs migration, proliferation, and osteogenic differentiation, whereas autophagy activator substantially augmented PRP-stimulated migration, proliferation, and differentiation. Taken together, these findings suggested that PRP could effectively promote regenerative potentials associated with autophagy.
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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.
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Project description:The function of the dental pulp is closely connected to the extracellular matrix (ECM) structure, and ECM has received significant attention due to its biological functions for regulating cells. As such, the interaction between the ECM niche and cells is worth exploring for potential clinical uses. In this study, dental pulp stem cell (DPSC)-derived ECM (DPM) was prepared through cell culture and decellularization to function as the cell niche, and changes in DPSC behaviour and histological analysis of dental pulp tissue regeneration were evaluated following the DPM culture. DPM promoted the replication of DPSCs and exhibited retention of their mineralization. Then, the DPM-based culture strategy under odontogenic culture medium was further investigated, and the mineralization-related markers showed that DPSCs were regulated towards odontogenic differentiation. Dental pulp-like tissue with well-arranged ECM was harvested after a 2-month subcutaneous implantation in nude mice with DPM application. Additionally, DPSCs cultured on the plastic culture surface showed the up-regulation of mineralization makers in vitro, but there was a disorder in matrix formation and mineralization when the cells were cultured in vivo. DPM-based cultivation could serve as a cell niche and modulate DPSC behaviour, and this method also provided an alternative to harvest tissue-specific ECM and provided a strategy for ECM-cell interaction.
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Project description:The present challenge in dental pulp tissue engineering scaffold materials lies in the development of tissue-specific scaffolds that are conducive to an optimal regenerative microenvironment and capable of accommodating intricate root canal systems. This study utilized porcine dental pulp to derive the decellularized extracellular matrix (dECM) via appropriate decellularization protocols. The resultant dECM was dissolved in an acid pepsin solution to form dECM hydrogels. The analysis encompassed evaluating the microstructure and rheological properties of dECM hydrogels and evaluated their biological properties, including in vitro cell viability, proliferation, migration, tube formation, odontogenic, and neurogenic differentiation. Gelatin methacrylate (GelMA) hydrogel served as the control. Subsequently, hydrogels were injected into treated dentin matrix tubes and transplanted subcutaneously into nude mice to regenerate dental pulp tissue in vivo. The results showed that dECM hydrogels exhibited exceptional injectability and responsiveness to physiological temperature. It supported the survival, odontogenic, and neurogenic differentiation of dental pulp stem cells in a 3D culture setting. Moreover, it exhibited a superior ability to promote cell migration and angiogenesis compared to GelMA hydrogel in vitro. Additionally, the dECM hydrogel demonstrated the capability to regenerate pulp-like tissue with abundant blood vessels and a fully formed odontoblast-like cell layer in vivo. These findings highlight the potential of porcine dental pulp dECM hydrogel as a specialized scaffold material for dental pulp regeneration.
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Project description:Dental pulp-derived stem cells (DPSCs) have the potential to regenerate dentin and dental pulp tissue because of their differentiation capacity and angiogenic properties. However, for regenerative approaches to gain regulatory and clinical acceptance, protocols are needed to determine more feasible ways to cultivate DPSCs, namely, without the use of xenogeneic-derived components (animal sera) and exogenous growth factors.In this study, human DPSCs were isolated from third molars and expanded in standard culture conditions containing fetal bovine serum (DPSCs-FBS) or conditions containing human serum (DPSCs-HS). After cell characterization and evaluation of their angiogenic secretome, DPSCs were seeded in tooth slice/scaffolds and implanted subcutaneously into immunodeficient mice. After 30 days, tooth slices were retrieved and evaluated for dental pulp tissue regeneration. Immunohistochemistry and confocal microscopy were used to quantify blood vessel formation and evaluate predentin and dentin formation.After culture, DPSCs-HS produced concentrations of angiogenic growth factors equivalent to DPSCs-FBS. Additionally, in DPSCs-HS, several angiogenic factors were produced in at least 1-fold higher concentrations than in DPSCs-FBS. In vivo, it was determined that DPSCs-HS produced a robust angiogenic response and regeneration of dentin equivalent to DPSCs-FBS.These findings show that DPSCs can be isolated and expanded to clinical scale numbers in media devoid of animal serum or exogenous growth factors and still maintain their pulp regenerative properties. The implications of these findings are significant for further development of clinical protocols using DPSCs in cell therapies.
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Project description:The vitality of the pulp is fundamental to the functional life of the tooth. For this aim, active and living biomaterials are required to avoid the current drastic treatment, which is the removal of all the cellular and molecular content regardless of its regenerative potential. The regeneration of the pulp tissue is the dream of many generations of dental surgeons and will revolutionize clinical practices. Recently, the potential of the regenerative medicine field suggests that it would be possible to achieve such complex regeneration. Indeed, three crucial steps are needed: the control of infection and inflammation and the regeneration of lost pulp tissues. For regenerative medicine, in particular for dental pulp regeneration, the use of nano-structured biomaterials becomes decisive. Nano-designed materials allow the concentration of many different functions in a small volume, the increase in the quality of targeting, as well as the control of cost and delivery of active molecules. Nanomaterials based on extracellular mimetic nanostructure and functionalized with multi-active therapeutics appear essential to reverse infection and inflammation and concomitantly to orchestrate pulp cell colonization and differentiation. This novel generation of nanomaterials seems very promising to meet the challenge of the complex dental pulp regeneration.
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Project description:Platelet-rich fibrin (PRF) provides a scaffold for cell migration and growth factors for promoting wound healing and tissue regeneration. Here, we report using PRF in periodontal healing after open flap debridement (OFD) in canine periodontitis. A split-mouth design was performed in twenty dogs. Forty periodontitis surgical sites were randomly categorized into 2 groups; OFD alone and OFD with PRF treatment. Clinical parameters of periodontal pocket depth, gingival index, and the cemento-enamel junction-alveolar bone levels/root length ratio were improved in the OFD + PRF group. The OFD + PRF group also demonstrated a dramatically decreased inflammatory score compared with the OFD group. Collagen accumulation was improved in the OFD + PRF group at later time points compared with baseline. PRF application also significantly reduced inflammatory cytokine expression (TNFA and IL1B), and promoted the expression of collagen production-related genes (COL1A1, COL3A1, and TIMP1) and growth factors (PDGFB, TGFB1, and VEGFA). These findings suggest that PRF combined with OFD provides a new strategy to enhance the overall improvement of canine periodontitis treatment outcomes, especially in terms of inflammation and soft tissue healing. Therefore, PRF use in treating periodontitis could play an important role as a regenerative material to improve canine periodontitis treatment.
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Project description: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.
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