Mo2 C-Based Ceramic Electrode with High Stability and Catalytic Activity for Hydrogen Evolution Reaction at High Current Density.

Developing robust electrodes with high catalytic performance is a key step for expanding practical HER (hydrogen evolution reaction) applications. This paper reports on novel porous Mo2 C-based ceramics with oriented finger-like holes directly used as self-supported HER electrodes. Due to the suitable MoO3 sintering additive, high-strength (55 ± 6 MPa) ceramic substrates and a highly active catalytic layer are produced in one step. The in situ reaction between MoO3 and Mo2 C enabled the introduction of O in the Mo2 C crystal lattice and the formation of Mo2 C(O)/MoO2 heterostructures. The optimal Mo2 C-based electrode displayed an overpotential of 333 and 212 mV at 70 °C under a high current intensity of 1500 mA cm-2 in 0.5 m H2 SO4 and 1.0 m KOH, respectively, which are markedly better than the performance of Pt wire electrode; furthermore, its price is three orders of magnitude lower than Pt. The chronopotentiometric curves recorded in the 50 - 1500 mA cm-2 range, confirmed its excellent long-term stability in acidic and alkaline media for more than 260 h. Density functional theory (DFT) calculations showed that the Mo2 C(O)/MoO2 heterostructures has an optimum electronic structure with appropriate *H adsorption-free energy in an acidic medium and minimum water dissociation energy barrier in an alkaline medium.

[Influence of exogenous nitric oxide on chondrogenic differentiation of adipose derived stem cells].

OBJECTIVE: To determine the effects of exogenous nitric oxide (NO) donor sodium nitroprusside (SNP) on the proliferation and expression of related-gene of adipose-derived stem cells(ADSCs) and its role in chondrogenic differentiation of ADSCs. METHOD: Rat ADSCs were harvested and cultured, and then induced to osteogenic and adipogenic differentiations, detected with Alizarin red stained and Oil red O stain, respectively. The change of NO during chondrogenic differentiation of ADSCs was tested by NO detection kit. Cell counting kit-8 (CCK8) was used to detect the proliferation of ADSCs under different concentrations of SNP (0.25 mmol/L, 1.00 mmol/L, 4.00 mmol/L). Gene expression level of transformation growth factor (TGF-beta1) and specific gene of chondrogenic differentiation-signaling protein Smad3 and Collage II alPHA (Col-II alpha1), were detected by Real time- PCR (RT-PCR) method. RESULTS: Positive alizarin red staining and Oil red O staining were found after osteogenic and adipogenic induction of cultured ADSCs. Higher concentrations of NO were found in the supernatant of the experimental group with ADSCs chondrogenic differentiations compared with the controls (P<0.05). Low concentrations (0. 25 mmol/L, 1.00 mmol/L) of SNP showed no significant effects on cell proliferations (P>0.05), whereas high concentration (4. 00 mmol/L) of SNP inhibited cell proliferation (P<0.05). RT-PCR revealed that SNP inhibited the gene expression of TGF-beta1 mRNA and chondrogenic differentiation of specific gene Smad3 mRNA, Col-II alpha1 mRNA. CONCLUSION: SNP can inhibit chondrogenic differentiations by suppressing the production of TGF-beta1 and inhibiting downstream of TGF-beta1 signaling pathways, thereby inhibiting ADSCs differentiation into chondrocytes.

[Construction of beta-catenin miRNA-expressing vectors and test of silencing effect in adipose-derived stem cells from Sprague-Dawley rats].

UNLABELLED: OBEJECTIVE: To construct miRNA-expressing plasmid vector for interfering the expression of beta-catenin in adipose-derived stem cells from Sprague-Dawley rats. METHODS: The double strands oligonucleotides of miR-expressing cassette were ligated into plasmid pSWH to generate pSWH-miR. Then the PCR product of enhanced green fluorescence protein (EGFP) open reading frame (ORF) fragment was inserted into BamH I and Xbo I digested pSWH-miR to generate pSWH-EGFP-miR. The adipose-derived stemcells from rats (rADSCs) were transfected with pSWH-EGFP-miR respectively. The expression of beta-catenin was determined by Western blot at 72 h post-transfection. RESULTS: Five miRNA-expressing plasmid vectors were constructed (miR-780, miR-796, miR-1467, miR-1948, miR-1960). miR-780 and miR-796 had the best silencing effect on the expression of beta-catenin (P < 0.05), less did the miR-1960 (P < 0.05), but not the miR-1467 and the miR-1948 (P < 0.05). CONCLUSION: miR-780 and miR-796 could interference the beta-catenin in ADSCs with high efficiency.

Eliminating senescent cells by white adipose tissue-targeted senotherapy alleviates age-related hepatic steatosis through decreasing lipolysis.

Cellular senescence is an important risk factor in the development of hepatic steatosis. Senolytics present therapeutic effects on age-related hepatic steatosis without eliminating senescent hepatocytes directly. Therefore, it highlights the need to find senolytics' therapeutic targets. Dysfunction of adipose tissue underlies the critical pathogenesis of lipotoxicity in the liver. However, the correlation between adipose tissue and hepatic steatosis during aging and its underlying molecular mechanism remains poorly understood. We explored the correlation between white adipose tissue (WAT) and the liver during aging and evaluated the effect of lipolysis of aged WAT on hepatic steatosis and hepatocyte senescence. We screened out the ideal senolytics for WAT and developed a WAT-targeted delivery system for senotherapy. We assessed senescence and lipolysis of WAT and hepatic lipid accumulation after treatment. The results displayed that aging accelerated cellular senescence and facilitated lipolysis of WAT. Free fatty acids (FFAs) generated by WAT during aging enhanced hepatic steatosis and induced hepatocyte senescence. The combined usage of dasatinib and quercetin was screened out as the ideal senolytics to eliminate senescent cells in WAT. To minimize non-specific distribution and enhance the effectiveness of senolytics, liposomes decorated with WAT affinity peptide P3 were constructed for senotherapy in vivo. In vivo study, WAT-targeted treatment eliminated senescent cells in WAT and reduced lipolysis, resulting in the alleviation of hepatic lipid accumulation and hepatocyte senescence when compared to non-targeted treatment, providing a novel tissue-targeted, effective and safe senotherapy for age-related hepatic steatosis.

Skeletal muscle-derived extracellular vesicles transport glycolytic enzymes to mediate muscle-to-bone crosstalk.

Identification of cues originating from skeletal muscle that govern bone formation is essential for understanding the crosstalk between muscle and bone and for developing therapies for degenerative bone diseases. Here, we identified that skeletal muscle secreted multiple extracellular vesicles (Mu-EVs). These Mu-EVs traveled through the bloodstream to reach bone, where they were phagocytized by bone marrow mesenchymal stem/stromal cells (BMSCs). Mu-EVs promoted osteogenic differentiation of BMSCs and protected against disuse osteoporosis in mice. The quantity and bioactivity of Mu-EVs were tightly correlated with the function of skeletal muscle. Proteomic analysis revealed numerous proteins in Mu-EVs, some potentially regulating bone metabolism, especially glycolysis. Subsequent investigations indicated that Mu-EVs promoted the glycolysis of BMSCs by delivering lactate dehydrogenase A into these cells. In summary, these findings reveal that Mu-EVs play a vital role in BMSC metabolism regulation and bone formation stimulation, offering a promising approach for treating disuse osteoporosis.

Bone-targeted delivery of senolytics to eliminate senescent cells increases bone formation in senile osteoporosis.

Systemic elimination of senescent cells using senolytic drugs presents therapeutic effects on age-related diseases, including senile osteoporosis. However, low bioavailability and potential side effects of senolytics restrict clinical application. Therefore, we developed a bone-targeted delivery system for senolytics to effective treatment of senile osteoporosis. In this study, quercetin was screened out as the ideal senolytics for eliminating senescent BMSCs. Treatment of quercetin efficiently decreased the senescence markers in senescent BMSCs models. After treatment with quercetin in vitro, cell mitosis and calcification staining assay confirmed that the proliferation and osteogenesis of the senescent BMSCs populations were enhanced. To enhance the effectiveness and minimize the side effect of treatment, liposomes decorated with bone affinity peptide (DSS)6 were constructed for bone-targeted delivery of quercetin. After administration of liposomes loading quercetin in two aged mice models, histological and cellular analysis confirmed that bone-targeted treatment with quercetin efficiently eliminated senescent cells in bone, restored the function of BMCSs, and promoted bone formation in aged mice models when compared to non-targeted treatment. Taken together, the bone-targeted delivery of senolytics efficiently eliminates senescent cells to recover bone mass and microarchitecture, showing an effective treatment for senile osteoporosis. STATEMENT OF SIGNIFICANCE: Senile osteoporosis, a common and hazardous chronic disease, has been still lacking effective therapy. How to effectively eliminate the hazards of senescent cells in skeleton to bone formation remains challenge. In this study, quercetin was screened out as the ideal senolytic drug for senescent BMSCs and could effectively eliminated senescent BMSCs to restore the cellular functions of senescent BMSCs models in vitro. Then, the bone-targeted liposomes were designed to encapsulate and deliver senolytics efficiently to senile bone tissue. Based on two aged mice models, we confirmed that bone-targeted delivery of quercetin efficiently eliminated senescent cells in skeleton and enhanced bone formation in vivo, suggesting the bone-targeted elimination of senescent cells is an effective treatment for senile osteoporosis.

Muscle-derived extracellular vesicles improve disuse-induced osteoporosis by rebalancing bone formation and bone resorption.

Osteoporosis is a highly prevalent skeletal bone disorder worldwide with characteristics of reduced bone mass and increased risk of osteoporotic fractures. It has been predicted to become a global challenge with the aging of the world population. However, the current therapy based on antiresorptive drugs and anabolic drugs has unwanted side effects. Although cell-based treatments have shown therapeutic effects for osteoporosis, there are still some limitations inhibiting the process of clinical application. In the present study, we developed EVs derived from skeletal muscle tissues (Mu-EVs) as a cell-free therapy to treat disuse-induced osteoporosis. Our results showed that Mu-EVs could be prepared easily and abundantly from skeletal muscle tissues, and that these Mu-EVs had typical features of extracellular vesicles. In vitro studies demonstrated that Mu-EVs from normal skeletal muscles could be phagocytized by bone marrow stromal/stem cells (BMSCs) and osteoclasts (OCs), and promoted osteogenic differentiation of BMSCs while inhibited OCs formation. Correspondingly, Mu-EVs from atrophic skeletal muscles attenuated the osteogenesis of BMSCs and strengthened the osteoclastogenesis of monocytes. In vivo experiments revealed that Mu-EVs could efficiently reverse disuse-induced osteoporosis by enhancing bone formation and suppressing bone resorption. Collectively, our results suggest that Mu-EVs may be a potential cell-free therapy for osteoporosis treatment. STATEMENT OF SIGNIFICANCE: Osteoporosis is a highly prevalent skeletal bone disorder worldwide and has become a global health concern with the aging of the world population. The current treatment for osteoporosis has unwanted side effects. Extracellular veiscles (EVs) from various cell sources are a promising candidate for osteoporosis treatment. In the present study, our team established protocols to isolate EVs from culture supernatant of skeletal muscles (Mu-EVs). Uptake of Mu-EVs by BMSCs and osteoclasts influences the balance of bone remodeling via promoting the osteogenic differentiation of BMSCs and inhibiting the osteoclasts formation of monocytes. In addition, exogenous Mu-EVs from normal skeletal muscles are proved to reverse the disuse-induced osteoporosis. We provide experimental evidence that Mu-EVs therapy is a potential cell-free platform for osteoporosis treatment towards clinical application.

Vascularized dental pulp regeneration using cell-laden microfiber aggregates.

Regeneration of dental pulp via the transplantation of dental pulp stem cells (DPSCs) has emerged as a novel therapy for dental pulp necrosis after inflammation and injury. However, providing sufficient oxygen and nutrients to support stem cell survival, self-renewal, and differentiation in the narrow root canal remains a great challenge. In this study, we explored a novel strategy based on cell-laden microfibers for dental pulp regeneration. Firstly, we fabricated suitable GelMA hydrogels that facilitate the survival and proliferation of DPSCs and human umbilical vein endothelial cells (HUVECs) and possess satisfactory biomechanical properties to generate microfibers. Two kinds of GelMA microfibers were fabricated with DPSCs and HUVECs via a silicone-tube-based coagulant bath-free method. Live/dead and Ki-67 immunofluorescence staining assays identified that these two cell lines maintained high survival rate and proliferation ability in GelMA microfibers. Immunofluorescence staining confirmed that DPSCs fully spread in the microfibers and highly expressed CD90 and laminin. HUVECs positively express CD31 and VE-cad in microfibers and could migrate well in the GelMA hydrogel. In vitro permeation experiments confirmed the superiority of microfiber aggregates (MAs) in liquid permeation compared to GelMA hydrogel blocks. We further adopted an ectopic pulp regeneration assay in nude mice to validate the regeneration of the aggregates of mixed DPSC-microfibers and HUVEC-microfibers in vivo. Compared to a conventional mixture of DPSCs and HUVECs in GelMA hydrogel blocks, the aggregates of cell-laden microfibers generated more pulp-like tissue, blood vessels, and odontoblast-like cells that positively express DMP-1 and DSPP. To our knowledge, this is the first attempt to apply cell-laden MAs for pulp regeneration. Our study proposes a new solution to the challenge of pulp regeneration, which might promote the clinical translation and application of stem cell-based therapy.

Local Elimination of Senescent Cells Promotes Bone Defect Repair during Aging.

Due to the declined function of bone marrow mesenchymal stem cells (BMSCs), the repair of bone defects in the elderly is retarded. Elimination of senescent cells emerges as a promising strategy for treating age-related diseases. However, whether the local elimination of senescent BMSCs can promote bone regeneration in the elderly remains elusive. To tackle the above issue, we first screened out the specific senolytics for BMSCs and confirmed their effect of eliminating senescent BMSCs in vitro. Treatment with quercetin, which is determined the best senolytics for senescent BMSCs, efficiently removed senescent cells in the population. Moreover, the self-renewal capacity was restored as well as osteogenic ability of BMSCs after treatment. We then designed a microenvironment-responsive hydrogel based on the MMPs secreted by senescent cells. This quercetin-encapsulated hydrogel exhibited a stable microstructure and responsively released quercetin in the presence of senescence in vitro. In vivo, the quercetin-loaded hydrogel effectively cleared the local senescent cells and reduced the secretion of MMPs in the bone. Due to the removal of local senescent cells, the hydrogel significantly accelerated the repair of bone defects in the femur and skull of old rats. Taken together, our study revealed the role of removing senescent cells in bone regeneration and provided a novel therapeutic approach for bone defects in aged individuals.

Bone morphogenetic protein 7 mediates stem cells migration and angiogenesis: therapeutic potential for endogenous pulp regeneration.

Pulp loss is accompanied by the functional impairment of defense, sensory, and nutrition supply. The approach based on endogenous stem cells is a potential strategy for pulp regeneration. However, endogenous stem cell sources, exogenous regenerative signals, and neovascularization are major difficulties for pulp regeneration based on endogenous stem cells. Therefore, the purpose of our research is to seek an effective cytokines delivery strategy and bioactive materials to reestablish an ideal regenerative microenvironment for pulp regeneration. In in vitro study, we investigated the effects of Wnt3a, transforming growth factor-beta 1, and bone morphogenetic protein 7 (BMP7) on human dental pulp stem cells (h-DPSCs) and human umbilical vein endothelial cells. 2D and 3D culture systems based on collagen gel, matrigel, and gelatin methacryloyl were fabricated to evaluate the morphology and viability of h-DPSCs. In in vivo study, an ectopic nude mouse model and an in situ beagle dog model were established to investigate the possibility of pulp regeneration by implanting collagen gel loading BMP7. We concluded that BMP7 promoted the migration and odontogenic differentiation of h-DPSCs and vessel formation. Collagen gel maintained the cell adhesion, cell spreading, and cell viability of h-DPSCs in 2D or 3D culture. The transplantation of collagen gel loading BMP7 induced vascularized pulp-like tissue regeneration in vivo. The injectable approach based on collagen gel loading BMP7 might exert promising therapeutic application in endogenous pulp regeneration.

An Isolation System to Collect High Quality and Purity Extracellular Vesicles from Serum.

PURPOSE: Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles that function as carriers and play a role in intercellular communication. There are a large number of EVs in the blood and serve as an indicator of pathophysiological conditions. Studies on the basics and application of EVs are hampered by the limitations of current protocols to isolate EVs from blood. However, current isolation methods are difficult to achieve a balance between yield and purity. METHODS: Firstly, we use Sepharose-4B to build a self-made size exclusion chromatography (SEC) column and perform separation and characteristics. Then, we use the SEC column to systematically compare the efficiency with the most common EV isolation methods: Ultracentrifugation (UC) and total exosomes isolation commercial kit (TEI). The EVs isolated through different methods were characterized the yield and size of EVs, analyzed their protein profiles, the morphology and purity were observed under the transmission electron microscope. To further improve the quality and purity, we combined SEC and UC methods and established a two-steps method to isolated EVs from serum. RESULTS: Self-made SEC column can well separate EVs from complex serum protein, and EVs enriched in the 8-13 fractions with good morphology and yield. By systematically compare SEC with the commonly used UC and TEI kit, SEC is outstanding in all aspects and balances both isolation purity and yield. However, using the SEC method alone still has certain limitations and residual impurities. The SEC+UC combined method can cleverly solve the shortcomings of SEC and optimize the quality and purity of EVs from serum, which is much better than using one method alone. CONCLUSION: Our study presents the combination of size-exclusion chromatography and ultracentrifugation as a feasible and time-saving method to isolate high-quality and purity extracellular vesicles from serum.

Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Dental Pulp Regeneration.

INTRODUCTION: The transplantation of dental pulp stem cells (DPSCs) has emerged as a novel strategy for the regeneration of lost dental pulp after pulpitis and trauma. Dental pulp regeneration of the young permanent tooth with a wide tooth apical foramen has achieved significant progress in the clinical trials. However, because of the narrow apical foramen, dental pulp regeneration in adult teeth using stem cells remains difficult in the clinic. Finding out how to promote vascular reconstitution is essential for the survival of stem cells and the regeneration of dental pulp after transplantation into the adult tooth. METHODS: Adipose tissue-derived microvascular fragments (ad-MVFs) were isolated from human adipose tissues. The apoptosis and senescence of DPSCs cultured in conditioned media were evaluated to explore the effects of ad-MVFs on DPSCs. DPSCs combined with ad-MVFs were inserted into the human tooth root segments and implanted subcutaneously into immunodeficient mice. Regenerated pulplike tissues were analyzed by hematoxylin and eosin and immunohistochemistry. The vessels in regenerated tissues were analyzed by Micro-CT and immunofluorescence. RESULTS: The isolated ad-MVFs contained endothelial cells and pericytes. ad-MVFs effectively prevented the apoptosis and senescence of the transplanted DPSCs both in vivo and in vitro. Combined with DPSCs, ad-MVFs obviously facilitated the formation of vascular networks in the transplants. DPSCs combined with ad-MVFs formed dental pulp-like tissues with abundant cells and matrix after 4 weeks of implantation. The supplementation of ad-MVFs led to more odontoblastlike cells and increased the formation of mineralized substance around the root canal. CONCLUSIONS: Cotransplantation with ad-MVFs promotes the angiogenesis and revascularization of transplanted DPSC aggregates, leading to robust regeneration of dental pulp.

Human chorionic plate-derived mesenchymal stem cells transplantation restores ovarian function in a chemotherapy-induced mouse model of premature ovarian failure.

BACKGROUND: Previous studies have reported that transplantation of mesenchymal stem cells (MSCs) from many human tissues could ameliorate ovarian dysfunction. However, no study has revealed the therapeutic efficiency of MSCs derived from the chorionic plate (CP-MSCs) for premature ovarian failure (POF). METHODS: We investigated the restorative effects of CP-MSCs on cyclophosphamide (CTX)-induced POF. The POF mouse models were established via intraperitoneal injection of 50 mg/kg CTX into female mice for 15 consecutive days. After that, CP-MSCs were intravenously transplanted into the mice once a week for 4 weeks. The serum estradiol (E2) and follicle-stimulating hormone (FSH) levels in the mouse models were detected using enzyme-linked immunosorbent assay (ELISA) before and after treatment. Ovarian function was evaluated through counting the follicles, estrous cycles, and oocytes. RESULTS: CP-MSC transplantation restored the serum hormone level and ovarian function of the mice in the mouse model of POF induced by CTX. The levels of serum E2 and FSH in the POF model group was 232.33 ± 17.16 pg/mL and 4.48 ± 0.29 mIU/mL, respectively, after 6 weeks of treatment, which were similar to the values in the wild-type (WT) group. The superovulation demonstrated that ovarian function was significantly improved compared with nontreated POF model mice. The CP-MSC transplantation could restore CTX-induced ovarian dysfunction. CONCLUSIONS: Our results offer a potential application for human CP-MSCs in POF treatment.

Biological characteristics and karyotiping of a new isolation method for human adipose mesenchymal stem cells in vitro.

OBJECTIVE: A new method was presented to prepare clinical-grade human adipose-derived stromal stem cells (ASCs) and its safety in vitro, such as biological characteristics and genetic features alteration were investigated. METHODS: The morphology of the ASCs which were cultured in vitro using serum-free medium was observed. Cell cycle and CD markers profile were tested by flow cytometry, while karyotype was analyzed by the chromosome G-banding technology. Growth factors expression was tested by ELISA and tumor-related genes were analyzed by the real-time PCR, respectively. RESULTS: ASCs were adult stem cells with spindle shape. The proliferation ratio of ASCs began to slow down after 10 passages, and was significant after 15 passages. Cell cycle analysis revealed that the percentage of G2 phase and S phase cells was stable. There was no obvious missing, translocation or dislocation in terms of karyotype. Expression level of tumor relevant genes and cytokines at different passages had no significant difference. CONCLUSIONS: The clinical-grade ASCs prepared with this new method, less than ten passages, was safe for clinical trials.