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Chun-Yan Zheng,1,* Xiao-Yang Chu,2,* Chun-Yan Gao,1 Hua-Ying Hu,3 Xin He,1 Xu Chen,1 Kai Yang,4 Dong-Liang Zhang1 1Department of Orthodontics, Beijing Stomatological Hospital, Capital Medical University School of Stomatology, Capital Medical University, Beijing, People’s Republic of China; 2Department of Stomatology, Fifth Medical Center of Chinese PLA General Hospital, Beijing, People’s Republic of China; 3Birth Defects Prevention and Control Technology Research Center, Medical Innovation Research Division of Chinese PLA General Hospital, Beijing, People’s Republic of China; 4Prenatal Diagnosis Center, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, People’s Republic of China*These authors contributed equally to this workCorrespondence: Dong-Liang Zhang, Department of Orthodontics, Beijing Stomatological Hospital, Capital Medical University School of Stomatology, Capital Medical University, 11 Xilahutong Road, Beijing, 100040, People’s Republic of China, Email zhangdongliang@mail.ccmu.edu.cnBackground: Naringin is a naturally occurring flavanone that promotes osteogenesis. Owing to the high lipophilicity, poor in vivo bioavailability, and extensive metabolic alteration upon administration, the clinical efficacy of naringin is understudied. Additionally, information on the molecular mechanism by which it promotes osteogenesis is limited.Methods: In this study, we prepared TAT & RGD peptide-modified naringin-loaded nanoparticles (TAT-RGD-NAR-NPs), evaluated their potency on the osteogenic differentiation of human dental pulp stem cells (hDPSCs), and studied its mechanism of action through metabolomic analysis.Results: The particle size and zeta potential of TAT-RGD-NAR-NPs were 160.70± 2.05 mm and – 20.77± 0.47mV, respectively. The result of cell uptake assay showed that TAT-RGD-NAR-NPs could effectively enter hDPSCs. TAT-RGD-NAR-NPs had a more significant effect on cell proliferation and osteogenic differentiation promotion. Furthermore, in metabolomic analysis, naringin particles showed a strong influence on the glycerophospholipid metabolism pathway of hDPSCs. Specifically, it upregulated the expression of PLA2G3 and PLA2G1B (two isozymes of phospholipase A2, PLA2), increased the biosynthesis of lysophosphatidic acid (LPA).Conclusion: These results suggested that TAT-RGD-NPs might be used for transporting naringin to hDPSCs for modulating stem cell osteogenic differentiation. The metabolomic analysis was used for the first time to elucidate the mechanism by which naringin promotes hDPSCs osteogenesis by upregulating PLA2G3 and PLA2G1B.Keywords: nanoparticles, TAT, RGD, hDPSCs, osteogenic differentiation

Background Unresolved inflammation and tissue destruction are considered to underlie the failure of dental pulp repair. As key mediators of the injury response, dental pulp stem cells (DPSCs) play a critical role in pulp tissue repair and regeneration. Resolvin E1 (RvE1), a major dietary omega-3 polyunsaturated fatty-acid metabolite, is effective in resolving inflammation and activating wound healing. However, whether RvE1 facilitates injured pulp-tissue repair and regeneration through timely resolution of inflammation and rapid mobilization of DPSCs is unknown. Therefore, we established a pulp injury model and investigated the effects of RvE1 on DPSC-mediated inflammation resolution and injured pulp repair. Methods A pulp injury model was established using 8-week-old Sprague-Dawley rats. Animals were sacrificed on days 1, 3, 7, 14, 21, and 28 after pulp capping with a collagen sponge immersed in PBS with RvE1 or PBS. Hematoxylin-eosin and Masson’s trichrome staining, immunohistochemistry, and immunohistofluorescence were used to evaluate the prohealing properties of RvE1. hDPSCs were incubated with lipopolysaccharide (LPS) to induce an inflammatory response, and the expression of inflammatory factors after RvE1 application was measured. Effects of RvE1 on hDPSC proliferation, chemotaxis, and odontogenic differentiation were evaluated by CCK-8 assay, transwell assay, alkaline phosphatase (ALP) staining, alizarin red staining, and quantitative PCR, and possible signaling pathways were explored using western blotting. Results In vivo, RvE1 reduced the necrosis rate of damaged pulp and preserved more vital pulps, and promoted injured pulp repair and reparative dentin formation. Further, it enhanced dentin matrix protein 1 and dentin sialoprotein expression and accelerated pulp inflammation resolution by suppressing TNF-α and IL-1β expression. RvE1 enhanced the recruitment of CD146 + and CD105 + DPSCs to the damaged molar pulp mesenchyme. Isolated primary cells exhibited the mesenchymal stem cell immunophenotype and differentiation. RvE1 promoted hDPSC proliferation and chemotaxis. RvE1 significantly attenuated pro-inflammatory cytokine (TNF-α, IL-1β, and IL-6) release and enhanced ALP activity, nodule mineralization, and especially, expression of the odontogenesis-related genes DMP1 , DSPP , and BSP in LPS-stimulated DPSCs. RvE1 regulated AKT, ERK, and rS6 phosphorylation in LPS-stimulated DPSCs. Conclusions RvE1 promotes pulp inflammation resolution and dentin regeneration and positively influences the proliferation, chemotaxis, and differentiation of LPS-stimulated hDPSCs. This response is, at least partially, dependent on AKT, ERK, and rS6-associated signaling in the inflammatory microenvironment. RvE1 has promising application potential in regenerative endodontics.

Dental pulp stem cells (DPSCs) are suitable for use in regenerative medicine. Cryopreserved human DPSCs (hDPSCs) ameliorate diabetic polyneuropathy, and the effects of hDPSC transplantation are related to VEGF and NGF secretion. This study evaluated the long-term effects of a single transplantation of hDPSCs on diabetic polyneuropathy. hDPSCs were obtained from human third molars extracted for orthodontic treatment, which were then transplanted into the unilateral hindlimb skeletal muscles 8 weeks after streptozotocin injection in nude mice. The effects of hDPSC transplantation were analyzed at 16 weeks post-transplantation. DPSC transplantation significantly improved delayed nerve conduction velocity, decreased blood flow, and increased sensory perception thresholds. Furthermore, the hDPSC-conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons. In conclusion, the therapeutic effects of hDPSC transplantation with a single injection last for prolonged periods and may be beneficial in treating long-term diabetic polyneuropathy.

Background Microcapsule is considered as a promising 3D microenvironment for Bone Tissue Engineering (BTE) applications. Microencapsulation of cells in an appropriate scaffold not only protected the cells against excess stress but also promoted cell proliferation and differentiation. Through the current study, we aimed to microcapsulate the human Dental Pulp Stem Cells (hDPSCs) and evaluated the proliferation and osteogenic differentiation of those cells by using MTT assay, qRT-PCR, Alkaline phosphatase, and Alizarine Red S. Results The SEM results revealed that Alg/Gel microcapsules containing nHA showed a rough and more compact surface morphology in comparison with the Alg/Gel microcapsules. Moreover, the microencapsulation by Alg/Gel/nHA could improve cell proliferation and induce osteogenic differentiation. The cells cultured in the Alg/Gel and Alg/Gel/nHA microcapsules showed 1.4-fold and 1.7-fold activity of BMP-2 gene expression more in comparison with the control group after 21 days. The mentioned amounts for the BMP-2 gene were 2.5-fold and 4-fold more expression for the Alg/Gel and Alg/Gel/nHA microcapsules after 28 days. The nHA, addition to hDPSCs-laden Alg/Gel microcapsule, could up-regulate the bone-related gene expressions of osteocalcin, osteonectin, and RUNX-2 during the 21 and 28 days through the culturing period, too. Calcium deposition and ALP activities of the cells were observed in accordance with the proliferation results as well as the gene expression analysis. Conclusion The present study demonstrated that microencapsulation of the hDPSCs inside the Alg/Gel/nHA hydrogel could be a potential approach for regenerative dentistry in the near future. Graphical abstract

Bone tissue engineering using different scaffolds is a new therapeutic approach in regenerative medicine. This study explored the osteogenic potential of human dental pulp stem cells (hDPSCs) grown on a hydrolytically modified poly(L-lactide-co-caprolactone) (PLCL) electrospun scaffold and a non-woven hyaluronic acid (HYAFF-11™) mesh. The adhesion, immunophenotype, and osteogenic differentiation of hDPSCs seeded on PLCL and HYAFF-11™ scaffolds were analyzed. The results showed that PLCL and HYAFF-11™ scaffolds significantly supported hDPSCs adhesion; however, hDPSCs’ adhesion rate was significantly higher on PLCL than on HYAFF-11™. SEM analysis confirmed good adhesion of hDPSCs on both scaffolds before and after osteogenesis. Alizarin red S staining showed mineral deposits on both scaffolds after hDPSCs osteogenesis. The mRNA levels of runt-related transcription factor 2 (Runx2), collagen type I (Coll-I), osterix (Osx), osteocalcin (Ocn), osteopontin (Opn), bone sialoprotein (Bsp), and dentin sialophosphoprotein (Dspp) gene expression and their proteins were higher in hDPSCs after osteogenic differentiation on both scaffolds compared to undifferentiated hDPSCs on PLCL and HYAFF-11™. These results showed that PLCL scaffolds provide a better environment that supports hDPSCs attachment and osteogenic differentiation than HYAFF-11™. The high mRNA of early osteogenic gene expression and mineral deposits observed after hDPSCs osteogenesis on a PLCL mat indicated its better impact on hDPSCs’ osteogenic potential than that of HYAFF-11™, and hDPSC/PLCL constructs might be considered in the future as an innovative approach to bone defect repair.

Background Concentrated growth factor (CGF), as a natural biomaterial, is known to contain platelets, cytokines, and growth factors to facilitate the healing process, but there has been little information acquired in regenerative endodontics. The purpose of this study was to investigate the effects of CGF on proliferation, migration, and differentiation in human dental stem pulp cells (hDPSCs) exposed to lipopolysaccharide (LPS) in vitro and its potential role in pulp regeneration of the immature teeth in vivo . Methods In vitro experiments: CGF-conditioned medium were extracted by freeze-dried method. hDPSCs were isolated and identified. The proliferative potential of hDPSCs with different concentration of CGF and LPS was evaluated by Cell Counting Kit-8. Migration capacity was analyzed by Transwell assays, odonto/osteoblastic differentiation was determined by measuring alkaline phosphatase (ALP) activity using ALP staining, and the extent of mineralization was evaluated by using Alizarin red S staining. The mRNA expression level of DMP-1, DSPP, OPN, Runx2, and OCN were determined by quantitative polymerase chain reaction (qPCR). In vivo experiments: CGF were used as root canal filling agent of the immature single-rooted teeth in the beagle dogs. The teeth were then radiographed, extracted, fixed, demineralized, and subjected to histologic analyses at 8 weeks. The newly formed dentine-pulp complex and the development of apical foramen were evaluated by the hematoxylin-eosin (HE) and Masson trichrome technique. Soft tissues were analyzed by immunohistochemical staining of vascular endothelial growth factor (VEGF) and Nestin. Results In vitro experiments: The cultured cells exhibited the characteristics of mesenchymal stem cell. The treatment of LPS significantly increased the expression of TNF-α, IL-1β, IL-6, and IL-8 in hDPSCs, and CGF inhibited the mRNA expression of IL-8 in LPS-stimulated hDPSCs. The proliferation values of the CGF group in LPS-stimulated hDPSCs were significantly higher than that of the control group from day 3 to day 7 ( P  < 0.05). In addition, the number of migratory cells of the CGF group was greater than that of the control group at 24 h with or without LPS treatment. ALP activities increased gradually in both groups from day 4 to day 7. The mineralized nodules and the expression of odontogenesis-related genes DMP-1 and DSPP, osteogenesis-related genes OPN, Runx2, and OCN were dramatically enhanced by CGF in LPS-stimulated hDPSCs at days 21 and 28. In vivo experiments: In CGF treated group, the results of radiograph, HE, and Masson trichrome staining showed a continuing developed tooth of the immature teeth in the beagle dogs (i.e., the ingrowth of soft tissues into the root canal, the thickened internal root dentin walls, and the closed apex), which resembled the normal tooth development in the positive control group. The immunohistochemical staining showed that VEGF and Nestin were both moderately expressed in the regenerated pulp-like tissues which indicating the vascularization and innervation. Conclusions CGF has a positive effect on the proliferation, migration, and differentiation of hDPSCs exposed to LPS in vitro, and it can also promote the regeneration of dentine-pulp complex of the immature teeth in the beagle dogs in vivo. Therefore, CGF could be a promising alternative biomaterial in regenerative endodontics.

Dental pulp stem cells (DPSCs) show potential for treating neurodegenerative and traumatic diseases due to their neural crest origin. Melatonin (MT), an endogenous neurohormone with well-documented anti-inflammatory and antioxidant properties, has shown promising results with MSCs in terms of engraftment, proliferation, and neuronal differentiation in animal SCI models. However, the effects of melatonin preconditioning on human dental pulp stem cells (hDPSCs) for SCI treatment remain unclear. This study investigates the impact of melatonin preconditioning on hDPSCs engraftment, neural differentiation, and neurological function in rats with SCI. Forty-two male Sprague–Dawley rats were divided into six groups: Control, Sham, Model, Vehicle, Lesion Treatment A (SCI + hDPSCs), and Lesion Treatment B (SCI + MT-hDPSCs). After obtaining hDPSCs, stem cells were evaluated using flow cytometry. Cell viability was assessed using the MTT assay. SCI was induced in the Model, Vehicle, Lesion Treatment A, and Lesion Treatment B groups. The Lesion Treatment A and B groups received hDPSCs and hDPSCs pretreated with melatonin, respectively, 1 week after SCI, while the Vehicle group received only an intravenous injection of DMEM to simulate treatment. The other groups were used for behavioral testing. Immunohistochemistry (IHC) was employed to assess hDPSCs engraftment and differentiation at the SCI site. Motor function across the six groups was evaluated using the Basso, Beattie, and Bresnahan (BBB) score. Histological studies and cell counts confirmed hDPSCs implantation at the injury site, with a significantly higher presence in the MT-hDPSCs compared to hDPSCs ( p  < 0.01). IHC revealed that hDPSCs and MT-hDPSCs differentiated into neurons and astrocytes, with greater differentiation observed in the MT-hDPSCs compared to the hDPSCs ( p  < 0.01 and p  < 0.05, respectively). Functional improvement was noted in both SCI + hDPSCs and SCI + MT-hDPSCs groups compared to SCI and Vehicle groups from Week 4 onward ( p  < 0.001). Significant differences were also observed between the SCI + hDPSCs and SCI + MT-hDPSCs groups starting from Week 7 ( p  < 0.01). Preconditioning hDPSCs with melatonin enhances engraftment, neuronal differentiation, and greater performance improvement compared to hDPSCs alone in the SCI animal model.

We compared the therapeutic effects and mechanism of transplanted human dental pulp stem cells (hDPSCs) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in a rat stroke model and an in vitro model of ischemia. Rats were intravenously injected with hDPSCs or hBM-MSCs 24 h after middle cerebral artery occlusion (MCAo), and both groups showed improved functional recovery and reduced infarct volume versus control rats, but the hDPSC group showed greater reduction in infarct volume than the hBM-MSC group. The positive area for the endothelial cell marker was greater in the lesion boundary areas in the hDPSC group than in the hBM-MSC group. Administration of hDPSCs to rats with stroke significantly decreased reactive gliosis, as evidenced by the attenuation of MCAo-induced GFAP+/nestin+ and GFAP+/Musashi-1+ cells, compared with hBM-MSCs. In vivo findings were confirmed by in vitro data illustrating that hDPSCs showed superior neuroprotective, migratory, and in vitro angiogenic effects in oxygen–glucose deprivation (OGD)-injured human astrocytes (hAs) versus hBM-MSCs. Comprehensive comparative bioinformatics analyses from hDPSC- and hBM-MSC-treated in vitro OGD-injured hAs were examined by RNA sequencing technology. In gene ontology and KEGG pathway analyses, significant pathways in the hDPSC-treated group were the MAPK and TGF-β signaling pathways. Thus, hDPSCs may be a better cell therapy source for ischemic stroke than hBM-MSCs.

Human dental pulp stem cells (hDPSCs) senescence impairs their proliferation and osteogenic differentiation, critical for dental stem cell therapy. This study evaluated the effects of Resveratrol on the senescence of hDPSCs to explore new therapeutic strategies. Metabolomic analysis identified age-related metabolic differences in dental pulp tissues, with enriched pathways linked to Resveratrol. In vitro, Resveratrol improved proliferation, delayed senescence, promoted osteogenic differentiation, and enhanced mitochondrial autophagy, function, and biogenesis in senescent hDPSCs, reducing mitochondrial damage and oxidative stress. Mechanistically, silencing PINK1 or PGC-1α reversed Resveratrol-mediated promotion of proliferation, osteogenesis, and senescence suppression. Blocking SIRT1 abrogated its effects on mitochondrial quality control. These findings highlight Resveratrol’s potential to mitigate hDPSCs senescence via SIRT1-dependent mitochondrial regulation, offering insights for age-related dental regenerative therapies.

Background/Aim: Human dental pulp mesenchymal stem cells (hDPSCs) are considered to be a good cell source for cell-based clinical therapy, due to the advantages of high proliferation capacity, multilineage differentiation potential, immune regulation abilities, less ethnic concerns and non-invasive access. However, hDPSCs were traditionally isolated and expanded in medium containing fetal bovine serum (FBS), which is a barrier for clinical application due to the safety issues (virus transmission and allergy). Although many studies make efforts to screen out a suitable culture medium, the results are not promising so far. Therefore, a standard good manufacturing practice (GMP) compliant culture system is urgently required for the large-scale cell production. This study aimed to find suitable culture conditions for producing clinical grade hDPSCs to meet the requirements for clinical cell-based therapy and further to promote the application of hDPSCs into tissue regeneration or disease cure. Materials and Methods: We derived hDPSCs from nine orthodontic teeth expanded in two different media: a GMP compliant and xenogeneic serum-free medium (AMMS) and a serum containing medium (SCM). Cell propterties including morphology, proliferation, marker expression, differentiation, stemness, senescence and cytokine secretion between these two media were systematically compared. Results: hDPSCs cultured in both media exhibited the typical characteristics of mesenchymal stem cells (MSCs). However, we found that more cell colonies formed in the primary culture in AMMS, and the hDPSCs displayed higher proliferation capacity, differentiation potential and better stemness maintenance during sub-culturing in AMMS. Conclusion: Cell properties of hDPSCs could be improved when they were isolated and expanded in AMMS, which might provide a good candidate of culture medium for large-scale cell manufacturing.