Hypoxic dental pulp stem cells-released succinate promotes osteoclastogenesis and root resorption.

Introduction: Clinical studies have shown that endodontically-treated nonvital teeth exhibit less root resorption during orthodontic tooth movement. The purpose of this study was to explore whether hypoxic dental pulp stem cells (DPSCs) can promote osteoclastogenesis in orthodontically induced inflammatory root resorption (OIIRR). Methods: Succinate in the supernatant of DPSCs under normal and hypoxic conditions was measured by a succinic acid assay kit. The culture supernatant of hypoxia-treated DPSCs was used as conditioned medium (Hypo-CM). Bone marrow-derived macrophages (BMDMs) from succinate receptor 1 (SUCNR1)-knockout or wild-type mice were cultured with conditioned medium (CM), exogenous succinate or a specific inhibitor of SUCNR1 (4c). Tartrate-resistant acid phosphatase (TRAP) staining, Transwell assays, qPCR, Western blotting, and resorption assays were used to evaluate osteoclastogenesis-related changes. Results: The concentration of succinate reached a maximal concentration at 6 h in the supernatant of hypoxia-treated DPSCs. Hypo-CM-treated macrophages were polarized to M1 proinflammatory macrophages. Hypo-CM treatment significantly increased the formation and differentiation of osteoclasts and increased the expression of osteoclastogenesis-related genes, and this effect was inhibited by the specific succinate inhibitor 4c. Succinate promoted chemotaxis and polarization of M1-type macrophages with increased expression of osteoclast generation-related genes. SUCNR1 knockout decreased macrophage migration, M1 macrophage polarization, differentiation and maturation of osteoclasts, as shown by TRAP and NFATc1 expression and cementum resorption. Conclusions: Hypoxic DPSC-derived succinate may promote osteoclast differentiation and root resorption. The regulation of the succinate-SUCNR1 axis may contribute to the reduction in the OIIRR.

Enhanced Vascular-like Network Formation of Encapsulated HUVECs and ADSCs Coculture in Growth Factors Conjugated GelMA Hydrogels.

Tissue engineering primarily aimed to alleviate the insufficiency of organ donations worldwide. Nonetheless, the survival of the engineered tissue is often compromised due to the complexity of the natural organ architectures, especially the vascular system inside the organ, which allows food-waste transfer. Thus, vascularization within the engineered tissue is of paramount importance. A critical aspect of this endeavor is the ability to replicate the intricacies of the extracellular matrix and promote the formation of functional vascular networks within engineered constructs. In this study, human adipose-derived stem cells (hADSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured in different types of gelatin methacrylate (GelMA). In brief, pro-angiogenic signaling growth factors (GFs), vascular endothelial growth factor (VEGF165) and basic fibroblast growth factor (bFGF), were conjugated onto GelMA via an EDC/NHS coupling reaction. The GelMA hydrogels conjugated with VEGF165 (GelMA@VEGF165) and bFGF (GelMA@bFGF) showed marginal changes in the chemical and physical characteristics of the GelMA hydrogels. Moreover, the conjugation of these growth factors demonstrated improved cell viability and cell proliferation within the hydrogel construct. Additionally, vascular-like network formation was observed predominantly on GelMA@GrowthFactor (GelMA@GF) hydrogels, particularly on GelMA@bFGF. This study suggests that growth factor-conjugated GelMA hydrogels would be a promising biomaterial for 3D vascular tissue engineering.

The extracts of osteoblast developed from adipose-derived stem cell and its role in osteogenesis.

Cell-based therapy has become an achievable choice in regenerative medicines, particularly for musculoskeletal disorders. Adipose-derived stem cells (ASCs) are an outstanding resource because of their ability and functions. Nevertheless, the use of cells for treatment comes with difficulties in operation and safety. The immunological barrier is also a major limitation of cell therapy, which can lead to unexpected results. Cell-derived products, such as cell extracts, have gained a lot of attention to overcome these limitations. The goal of this study was to optimize the production of ASC-osteoblast extracts as well as their involvement in osteogenesis. The extracts were prepared using a freeze-thaw method with varying temperatures and durations. Overall, osteogenic-associated proteins and osteoinductive potential of the extracts prepared from the osteogenic-induced ASCs were assessed. Our results demonstrated that the freeze-thaw approach is practicable for cell extracts production, with minor differences in temperature and duration having no effect on protein concentration. The ASC-osteoblast extracts contain a significant level of essential specialized proteins that promote osteogenicity. Hence, the freeze-thaw method is applicable for extract preparation and ASC-osteoblast extracts may be beneficial as an optional facilitating biologics in bone anabolic treatment and bone regeneration.

Effects of Novel Nanoparticulate Bioceramic Endodontic Material on Human Dental Pulp Stem Cells In Vitro.

OBJECTIVES: This study aimed to investigate the in vitro effects of root canal filling and repair paste (nRoot BP) on human dental pulp stem cells (hDPSCs). METHODS: The effects of nRoot BP and iRoot BP Plus on the adhesion, proliferation, migration, and differentiation of hDPSCs were examined in vitro for 72 hours. The adhesion of cells was observed using immunofluorescence rhodamine ghost pen cyclic peptide staining and scanning electron microscopy (SEM). Cell density and changes in migration area were measured under a fluorescence inverted microscope. Fluorescent quantitative PCR was performed to detect genes related to odontogenesis and osteogenesis. RESULTS: Cells adhering to the surfaces of nRoot BP and iRoot BP Plus exhibited similar irregular polygonal morphologies, with cells extending irregular pseudopods to adhere to the materials. CCK-8 results indicated that the density of living cells for nRoot BP and iRoot BP Plus was lower than that of the blank control group at 3 and 5 days of culture. There was no significant difference in cell migration between the groups (P > .05). The migration ability of iRoot BP Plus and nRoot BP was similar to that of the control group. Both nRoot BP and iRoot BP Plus increased the expression of the RUNX2 gene, but there was no significant difference between the groups (P < .05). Furthermore, both nRoot BP and iRoot BP Plus downregulated the expression of the DSPP gene, with no significant difference between them (P > .05). CONCLUSIONS: nRoot BP exhibited a slight inhibition of hDPSC proliferation but did not affect the adhesion and migration of hDPSCs. The impact of nRoot BP on the osteogenic and odontogenic differentiation of hDPSCs was similar to that of iRoot BP Plus.

Effect of simultaneous and sequential use of TGF-ß1 and TGF-ß3 with FGF-2 on teno/ligamentogenic differentiation of periodontal ligament stem cells.

OBJECTIVE: The periodontal ligament is a crucial part of the periodontium, and its regeneration is challenging. This study compares the effect of simultaneous and sequential use of FGF-2 and TGF-ß1 with FGF-2 and TGF-ß3 on the periodontal ligament stem cells (PDLSCs) teno/ligamentogenic differentiation. DESIGN: This study comprises ten different groups. A control group with only PDLSCs; FGF-2 group containing PDLSCs with a medium culture supplemented with FGF-2 (50 ng/mL). In other experimental groups, different concentrations (5 ng/mL or 10 ng/mL) of TGF-ß1&-ß3 simultaneously or sequentially were combined with FGF-2 on the cultured PDLSCs. TGF-ß was added to the medium after day 3 in the sequential groups. Methyl Thiazolyl Tetrazolium (MTT) assay on days 3, 5, and 7 and Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) analysis after day 7 were conducted to investigate PLAP1, SCX, and COL3A1, RUNX2 genes. All experiments were conducted in a triplicate. The One-way and Two-way ANOVA with Tukey post hoc were utilized to analyze the results of the MTT and RT-qPCR tests, respectively. A p-value less than 0.05 is considered significant. RESULTS: The proliferation of cells on days 3, 5, and 7 was not significantly different among different experimental groups (P > 0.05). A higher expression of the PLAP1, SCX, and COL3A1 have been seen in groups with sequential use of growth factors; among these groups, the group using 5 ng/mL of TGF-ß3 led other groups with the most amount of significant upregulation in PLAP1(17.69 ± 1.11 fold; P < 0.0001), SCX (5.71 ± 0.38 fold; P < 0.0001), and COL1A3 (6.35 ± 0.39 fold; P < 0.0001) expression, compared to the control group. The expression of the RUNX2 decreased in all groups compared to the control group; this reduction was more in groups with sequential use of growth factors. CONCLUSION: The sequential use of growth factors can be more effective than simultaneous use in teno/ligamentogenic differentiation of PDLSCs. Moreover, treatment with 5 ng/mL TGF-ß3 after FGF-2 was more effective than TGF-ß1.

ECM-based bioadhesive hydrogel for sutureless repair of deep anterior corneal defects.

Corneal transplantation is the gold standard treatment for corneal-related blindness; however, this strategy faces challenges such as limited donor cornea, graft rejection, suture-related complications, and the need for specialized equipment and advanced surgical skills. Development of tissue adhesives for corneal regeneration is of great clinical value. However, currently available corneal tissue sealants pose challenges, such as lack of safety, biocompatibility, and desired mechanical properties. To meet these requirements simultaneously, a bovine stromal corneal extracellular matrix (dCor) was used to design a bioadhesive photocurable hydrogel based on gelatin methacrylate (GelMA) and polyethylene glycol diacrylate (PEGDA) hydrogels (dCor/Gel-PEG). Integration of dCor into the dual networks of GelMA and PEGDA (Gel-PEG) led to a bioadhesive hydrogel for curing corneal defects, which could be crosslinked by Irgacure 2959 within 5 min ultraviolet irradiation. The viability of corneal stromal stem cells (CSSCs) was improved on the dCor/Gel-PEG hydrogel in comparison to the Gel-PEG hydrogel. The gene expression profile supported the keratocyte differentiation of CSSCs seeded on dCor/Gel-PEG via increased KERA and ALDH, with inhibited myofibroblast transdifferentiation via decreased α-SMA due to the presence of dCor. Interestingly, the dCor/Gel-PEG hydrogel exhibited favorable mechanical performance in terms of elasticity and bioadherence to the host corneal stroma. Ex vivo and in vivo examinations proved the feasibility of this hydrogel for the sutureless reconstruction of deep anterior corneal defects with promising histopathological results.

Cordyceps sinensis accelerates stem cell recruitment to human skeletal muscle after exercise.

Cordyceps sinensis is a parasitic fungus known to induce immune responses. The impact of Cordyceps supplementation on stem cell homing and expansion to human skeletal muscle after exercise remains unexplored. In this study, we examined how pre-exercise Cordyceps supplementation influences cell infiltration, CD34+ cell recruitment, and Pax7+ cell expansion in human skeletal muscle after high-intensity interval exercise (HIIE) on a cycloergometer. A randomized, double-blind, placebo-controlled crossover study was conducted with 14 young adults (age: 24 ± 0.8 years). A placebo (1 g cornstarch) and Cordyceps (1 g Cordyceps sinensis) were administered before exercise (at 120% maximal aerobic power). Multiple biopsies were taken from the vastus lateralis for muscle tissue analysis before and after HIIE. This exercise regimen doubled the VEGF mRNA in the muscle at 3 h post-exercise (P = 0.006). A significant necrotic cell infiltration (+284%, P = 0.05) was observed 3 h after HIIE and resolved within 24 h. This response was substantially attenuated by Cordyceps supplementation. Moreover, we observed increases in CD34+ cells at 24 h post-exercise, notably accelerated by Cordyceps supplementation to 3 h (+51%, P = 0.002). This earlier response contributed to a four-fold expansion in Pax7+ cell count, as demonstrated by immunofluorescence double staining (CD34+/Pax7+) (P = 0.01). In conclusion, our results provide the first human evidence demonstrating the accelerated resolution of exercise-induced muscle damage by Cordyceps supplementation. This effect is associated with earlier stem cell recruitment into the damaged sites for muscle regeneration.

Curcumin Improves Functional Recovery of Ruptured Tendon by Promoting Tenogenesis via PI3K/Akt Signaling.

OBJECTIVE: In our previous study, we found that local release of curcumin from nanomicelles prevents peritendinous adhesion during Achilles tendon healing. The aim of this study is to further investigate the signaling integrated by curcumin to direct the tenogenetic program of tendon stem cells contributing to tendon healing. METHODS: A surgical model of tendon rupture and repair (TRR) was established in rats. Peritendinous adhesion and inflammation, biomechanical function, and expression of ß-catenin and epithelial cellular adhesion molecule (EpCAM) were determined. A dataset was analyzed to investigate differentially expressed genes and enriched genes related to the signaling pathways. Tendon stem cells were treated with curcumin to investigate the cellular and molecular events as well as the signaling pathway. RESULTS: In rat TRR model, curcumin treatment resulted in not only significantly decreased peritendinous inflammatory but also improved tendon functional recovery along with significantly increased expressions of EpCAM and ß-catenin. Analysis of the dataset indicated that the enriched genes were positively related to differentiation pathways but negatively related to proliferation pathways. In rat tendon stem cells, curcumin treatment inhibited proliferation but promoted differentiation. Curcumin's antioxidative activity was associated with tenogenesis. The upregulated expression of tendon lineage-specific markers was dependent on phosphatidylinositol 3'-kinase/Akt (PI3K/Akt) pathway which could be a potential mechanism of tenogenesis of curcumin treatment. CONCLUSION: Curcumin could improve tendon functional recovery via promoting tenogenesis in addition to its antioxidant and anti-inflammatory activities. Curcumin induced differentiation of tendon stem/progenitor cell into tenocytes via PI3K/Akt signaling pathway. This finding provided evidence for the application of curcumin to prevent adhesion during tendon repair.

Ligand-Selective Targeting of Macrophage Hydrogel Elicits Bone Immune-Stem Cell Endogenous Self-Healing Program to Promote Bone Regeneration.

Targeting macrophages can facilitate the site-specific repair of critical bone defects. Herein, a composite hydrogel, gelatin-Bletilla striata polysaccharide-mesoporous bioactive glass hydrogel (GBMgel), is constructed via the self-assembly of mesoporous bioactive glass on polysaccharide structures, through the Schiff base reaction. GBMgel can efficiently capture macrophages and drive the recruitment of seed stem cells and vascular budding required for regeneration in the early stages of bone injury, and the observed sustained release of inorganic silicon ions further enhances bone matrix deposition, mineralization, and vascular maturation. Moreover, the use of macrophage-depleted rat calvarial defect models further confirms that GBMgel, with ligand-selective macrophage targeting, increases the bone regeneration area and the proportion of mature bone. Mechanistic studies reveal that GBMgel upregulates the TLR4/NF-κB and MAPK macrophage pathways in the early stages and the JAK/STAT3 pathway in the later stages; thus initiating macrophage polarization at different time points. In conclusion, this study is based on the endogenous self-healing properties of bone macrophages, which enhances stem cell homing, and provides a research and theoretical basis upon which bone tissue can be reshaped and regenerated using the body's immune power, providing a new strategy for the treatment of critical bone defects.

The preliminary study of exosomes derived from thymosin beta 4-treated adipose-derived stem cells in fat grafting.

BACKGROUND: The retention rate in autologous fat grafting is an increasing concern for surgeons and patients. Our previous research verified that thymosin beta 4 (Tß4) positively affected fat survival, while the mechanism was unknown. The endothelial cells (ECs) and exosomes derived from adipose-derived stem cells (ADSCs) were regarded to play a critical role in fat transplantation. OBJECTIVE: This study aimed to evaluate the effect of exosomes derived from Tß4-treated ADSCs on EC proliferation and to identify the exosomal microRNA (miRNA) profile compared with the Tß4-untreated group. Additionally, this research intended to recognize the related molecules and signaling pathways in the Tß4-treated group with potential roles in fat transplants. METHODS: ADSCs were collected from patients who underwent liposuction surgery. Depending on whether the medium was supplemented with exogenous Tß4 or not, exosomes derived from cultured ADSCs were divided into the Tß4-Exos group and Con-Exos group. Exosome uptake and cell counting kit-8 (CCK-8) assays assessed the influence of Tß4-Exos on EC proliferation. The exosomal miRNAs of the two groups were analyzed by next-generation sequencing. With the criteria at the |log2 (fold change)| ≥ 1 and p-value < 0.05, up-regulated and down-regulated differentially expressed miRNAs (DEMs) were obtained. Prediction databases were used to predict the downstream mRNAs for DEMs. And then, overlapping genes for the up-regulated DEMs and the down-regulated were screened out, followed by enrichment analysis, protein-protein interaction network construction, and the gene cluster and hub gene identification. RESULTS: ADSCs were obtained from four female patients. The exosome uptake and CCK-8 assays showed that the Tß4-Exos could increase cell growth rate compared with the control group (DMEM-H + PBS). In Tß4-Exos and Con-Exos groups, 2651 exosomal miRNAs were recognized, with 80 up-regulated and 99 down-regulated DEMs according to the criteria. After the prediction, 621 overlapping genes for the up-regulated and 572 for the down-regulated DEMs were screened. The subsequent bioinformatics analysis found specific molecules and pathways related to the positive effect on fat survival. CONCLUSIONS: The exosomes derived from Tß4-treated ADSCs probably positively affect EC proliferation. Compared with the Con-Exos group, several exosomal DEMs, genes, and pathways were distinguished. These findings of this exploratory study provide the potential direction for future in-depth research on fat grafting.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

Antioxidant Effect of Melatonin on Proliferation, Apoptosis, and Oxidative Stress Variables in Frozen-Thawed Neonatal Mice Spermatogonial Stem Cells.

Cryopreservation of spermatogonial stem cells (SSCs) is an important method to restore and maintain fertility in preadolescent children suffering from cancer. For protection of SSCs from cryoinjury, various antioxidant agents have been used. The aim of this study was to assess the antiapoptotic and antioxidant effects of melatonin in frozen-thawed SSCs. SSCs were isolated from testes of neonatal mice (3-6 days old) and their purities were measured by flow cytometry with promyelocytic leukemia zinc finger protein. After culturing, the cells were frozen in two groups (1) control and (2) melatonin (100 µM) and stored for 1 month. Finally, the cell viability, colonization rate, expression of Bcl-2 and BAX gene, and intracellular reactive oxygen species (ROS) were evaluated after freezing-thawing. Melatonin increased the viability and colonization of SSCs and Bcl-2 gene expression. It also diminished BAX gene expression and intracellular ROS. The results of this study show that melatonin with antioxidant and antiapoptotic effects can be used as an additive for freezing and long-term storage of cells and infertility treatment in the clinic.

[Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro].

OBJECTIVE: To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition. METHODS: SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for ß-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture. RESULTS: The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and ß-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05). CONCLUSION: In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

Fucoidan (Undaria pinnatifida)/Polydopamine Composite-Modified Surface Promotes Osteogenic Potential of Periodontal Ligament Stem Cells.

Fucoidan, a marine-sulfated polysaccharide derived from brown algae, has been recently spotlighted as a natural biomaterial for use in bone formation and regeneration. Current research explores the osteoinductive and osteoconductive properties of fucoidan-based composites for bone tissue engineering applications. The utility of fucoidan in a bone tissue regeneration environment necessitates a better understanding of how fucoidan regulates osteogenic processes at the molecular level. Therefore, this study designed a fucoidan and polydopamine (PDA) composite-based film for use in a culture platform for periodontal ligament stem cells (PDLSCs) and explored the prominent molecular pathways induced during osteogenic differentiation of PDLSCs through transcriptome profiling. Characterization of the fucoidan/PDA-coated culture polystyrene surface was assessed by scanning electron microscopy and X-ray photoelectron spectroscopy. The osteogenic differentiation of the PDLSCs cultured on the fucoidan/PDA composite was examined through alkaline phosphatase activity, intracellular calcium levels, matrix mineralization assay, and analysis of the mRNA and protein expression of osteogenic markers. RNA sequencing was performed to identify significantly enriched and associated molecular networks. The culture of PDLSCs on the fucoidan/PDA composite demonstrated higher osteogenic potency than that on the control surface. Differentially expressed genes (DEGs) (n = 348) were identified during fucoidan/PDA-induced osteogenic differentiation by RNA sequencing. The signaling pathways enriched in the DEGs include regulation of the actin cytoskeleton and Ras-related protein 1 and phosphatidylinositol signaling. These pathways represent cell adhesion and cytoskeleton organization functions that are significantly involved in the osteogenic process. These results suggest that a fucoidan/PDA composite promotes the osteogenic potential of PDLSCs by activation of critical molecular pathways.

Towards Induction of Angiogenesis in Dental Pulp Stem Cells Using Chitosan-Based Hydrogels Releasing Basic Fibroblast Growth Factor.

INTRODUCTION: Chitosan is a natural biopolymer that attracted enormous attention in biomedical fields. The main components of regenerative endodontic procedures (REPs), as well as tissue engineering, are scaffolds, stem cells, and growth factors. As one of the basic factors in the REPs is maintaining vascularization, this study was aimed at developing basic fibroblast growth factor- (bFGF-) loaded scaffolds and investigating their effects on the angiogenic induction in human dental pulp stem cells (hDPSCs). METHODS: Poly (ε-caprolactone) (PCL)/chitosan- (CS-) based highly porous scaffold (PCL/CS) was prepared and evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) analyses. The adhesion and survival potency of seeded cells were assessed by SEM and MTT assays, respectively. The amount of angiogenic markers was investigated in gene and protein levels by real-time PCR and western blotting assays, respectively. RESULTS: Based on our findings, the SEM and FTIR tests confirmed the appropriate structure of synthesized scaffolds. Besides, the adhesion and survival rate of cells and the levels of VEGFR-2, Tie2, and Angiopoietin-1 genes were increased significantly in the PCL/CS/bFGF group. Also, the western blotting results showed the upregulation of these markers at protein levels, which were considerably higher at the PCL/CS/bFGF group (P < 0.05). CONCLUSIONS: On a more general note, this study demonstrates that the bFGF-loaded PCL/CS scaffolds have the potential to promote angiogenesis of hDPSCs, which could provide vitality of dentin-pulp complex as the initial required factor for regenerative endodontic procedures.

Methotrexate mediates the integrity of intestinal stem cells partly through nitric oxide-dependent Wnt/ß-catenin signaling in methotrexate-induced rat ileal mucositis.

This study aimed to elucidate the role of nitric oxide (NO) in intestinal stem cells in methotrexate-induced ileal mucositis in rats. Methotrexate induced the mRNA expressions of the Wnt/ß-catenin target genes Wnt3a, Sox9, and Lgr5 and the Wnt-antagonist gene sFRP-1 and the protein expressions of Lgr5 and sFRP-1. Methotrexate also induced Lgr5+ cells and lysozyme+ cells. A non-selective NO inhibitor inhibited the methotrexate induction of Wnt/ß-catenin target genes and Lgr5+ cells but enhanced that of sFRP-1 expression. Thus, methotrexate mediates the integrity of intestinal stem cells partly through NO-dependent Wnt/ß-catenin signaling and may enhance tolerability to methotrexate-induced injury.

Epidermal growth factor-like domain protein 6 recombinant protein facilitates osteogenic differentiation in adipose stem cells via bone morphogenetic protein 2/recombinant mothers against decapentaplegic homolog 4 signaling pathway.

Adipose-derived mesenchymal stem cells (ADSCs) are a class of pluripotent stem cells isolated from the adipose tissue; they can differentiate into osteoblasts after induction and play an important role in bone repair. EGFL6 protein is secreted by adipocytes and osteoblasts and can promote endothelial cell migration and angiogenesis. This study aimed to explore the effect of recombinant EGFL6 protein on the osteogenic differentiation of ADSCs. The cells were incubated with fluorescein isothiocyanate-conjugated antibodies and analyzed by flow cytometry. Alizarin red staining and alkaline phosphatase staining were used to detect the osteogenic differentiation ability. mRNA expression was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Protein expression was determined using Western blotting. The osteogenic differentiation ability of ADSCs isolated from the adipose tissue was significantly weakened after EGFL6 knockdown; this ability was restored upon the addition of EGFL6 recombinant protein. BMP2 knockdown inhibited the effect of EGFL6 recombinant protein on osteogenic differentiation. EGFL6 recombinant protein promoted osteogenic differentiation of ADSCs through the BMP2/SMAD4 signaling pathway. This may provide a potential target for the osteogenic differentiation of ADSCs.

Melatonin and the Programming of Stem Cells.

Melatonin interacts with various types of stem cells, in multiple ways that comprise stimulation of proliferation, maintenance of stemness and self-renewal, protection of survival, and programming toward functionally different cell lineages. These various properties are frequently intertwined but may not be always jointly present. Melatonin typically stimulates proliferation and transition to the mature cell type. For all sufficiently studied stem or progenitor cells, melatonin's signaling pathways leading to expression of respective morphogenetic factors are discussed. The focus of this article will be laid on the aspect of programming, particularly in pluripotent cells. This is especially but not exclusively the case in neural stem cells (NSCs) and mesenchymal stem cells (MSCs). Concerning developmental bifurcations, decisions are not exclusively made by melatonin alone. In MSCs, melatonin promotes adipogenesis in a Wnt (Wingless-Integration-1)-independent mode, but chondrogenesis and osteogenesis Wnt-dependently. Melatonin upregulates Wnt, but not in the adipogenic lineage. This decision seems to depend on microenvironment and epigenetic memory. The decision for chondrogenesis instead of osteogenesis, both being Wnt-dependent, seems to involve fibroblast growth factor receptor 3. Stem cell-specific differences in melatonin and Wnt receptors, and contributions of transcription factors and noncoding RNAs are outlined, as well as possibilities and the medical importance of re-programming for transdifferentiation.

ANKRD22 is a novel therapeutic target for gastric mucosal injury.

Ankyrin repeat domain 22 (ANKRD22) is a nuclear-encoded mitochondrial membrane protein that is highly expressed in normal gastric mucosal epithelial cells and activated macrophages. As a regulator of mitochondrial Ca2+, ANKRD22 could help repair damaged gastric mucosa by promoting the mobilization of LGR5+ gastric epithelial cells via the upregulation of Wnt/ß-catenin pathway activity in a mouse model. Furthermore, the inhibition of ANKRD22 alleviated the macrophage activation-mediated inflammatory response by reducing the phosphorylation of nuclear factor of activated T cells (NFAT). ANKRD22 plays a significant role in the repair of gastric mucosal damage and may become an ideal novel target for the treatment of gastric mucosal injury. However, there is no systematic introduction to ANKRD22 targeting. Therefore, we wrote this review to elaborate the functional mechanism of ANKRD22 in gastric mucosal injury and to analyze its potential application value in clinical therapy.

Prolonged oral antimicrobial administration prevents doxorubicin-induced loss of active intestinal stem cells.

Acute intestinal mucositis is a common off-target effect of chemotherapy, leading to co-morbidities such as vomiting, diarrhea, sepsis, and death. We previously demonstrated that the presence of enteric bacteria modulates the extent of jejunal epithelial damage induced by doxorubicin (DXR) in mice. Despite conventional thinking of the crypt as a sterile environment, recent evidence suggests that bacterial signaling influences aISC function. In this study, we labeled aISCs using transgenic Lgr5-driven fluorescence or with immunostaining for OLFM4. We examined the effect of DXR in both germ free (GF) mice and mice depleted of microbiota using an established antimicrobial treatment protocol (AMBx). We found differences in DXR-induced loss of aISCs between GF mice and mice treated with AMBx. aISCs were decreased after DXR in GF mice, whereas AMBx mice retained aISC expression after DXR. Neither group of mice exhibited an inflammatory response to DXR, suggesting the difference in aISC retention was not due to differences in local tissue inflammation. Therefore, we suspected that there was a protective microbial signal present in the AMBx mice that was not present in the GF mice. 16S rRNA sequencing of jejunal luminal contents demonstrated that AMBx altered the fecal and jejunal microbiota. In the jejunal contents, AMBx mice had increased abundance of Ureaplasma and Burkholderia. These results suggest pro-survival signaling from microbiota in AMBx-treated mice to the aISCs, and that this signaling maintains aISCs in the face of chemotherapeutic injury. Manipulation of the enteric microbiota presents a therapeutic target for reducing the severity of chemotherapy-associated mucositis.