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.

Disclosing phototransformation mechanisms of decabromodiphenyl ether (BDE-209) in different media by simulated sunlight: Implication by compound-specific stable isotope analysis.

As one of the typical brominated flame retardants, decabromodiphenyl ether (BDE-209) has been widely detected in environment. However, scarce information was available on BDE-209 phototransformation mechanisms in various media. In this study, compound-specific stable isotope analysis was first applied to investigate BDE-209 phototransformation in n-hexane, MeOH:H2O (v:v, 8:2), and simulated seawater by simulated sunlight. BDE-209 transformation followed pseudo-first-order kinetic, with degradation rate in the following of n-hexane (2.66 × 10-3 min-1) > simulated seawater (1.83 × 10-3 min-1) > MeOH:H2O (1.41 × 10-3 min-1). Pronounced carbon isotope fractionation was first observed for BDE-209 phototransformation, with carbon isotope enrichment factors (εC) of -1.01 ± 0.14‰, -1.77 ± 0.26‰, -2.94 ± 0.38‰ in n-hexane, MeOH:H2O and simulated seawater, respectively. Combination analysis of products and stable carbon isotope, debromination with cleavage of C-Br bonds as rate-limiting step was the main mechanism for BDE-209 phototransformation in n-hexane, debromination and hydroxylation with cleavage of C-Br bonds as rate-limiting steps in MeOH:H2O, and debromination, hydroxylation and chlorination in simulated seawater. This present study confirmed that stable carbon isotope analysis was a robust method to discovery the underlying phototransformation mechanisms of BDE-209 in various solutions.

Hierarchically patterned triple-layered gelatin-based electrospun membrane functionalized by cell-specific extracellular matrix for periodontal regeneration.

OBJECTIVES: Regenerating the periodontium poses a critical challenge in oral medicine. To repair various periodontal defects, it is necessary to adopt a bio-scaffold that provides both the architecture and bioactive cues for local stem cells to migrate, reside, proliferate, and differentiate. The objective of this study is to combine a cell-specific decellularized extracellular matrix (ECM) and a biomimetic electrospinning scaffold to regenerate severely destructed periodontium. METHODS: SEM, water contact angle (WCA), live/dead staining, swelling ratio, tensile test and immune-fluorescent staining were used to define the suitable topography for certain dental stem cells seeding and culturing. Transwell assay, CCK-8, Alizarin Red staining and PCR immune-fluorescent staining were used to determine ideal cell-specific ECM for PDLSCs/BMSCs migration, viability, and oriented differentiation. A biodegradable triple-layered electrospun scaffold (TLS) was fabricated by electrospinning with aligned fibers on both surfaces and a polyporous structure in the middle. The morphology and inter-porous structure of the TLS were characterized by SEM and mercury intrusion porosimetry (MIP). The surface of the TLS was functionalized with cell-specific ECM (Bi-ECM-TLS) through decellularization of the cell sheets cultured on the scaffold. The regenerative outcome of Bi-ECM-TLS was assessed by an in-situ rat periodontal defect model. Micro-CT, HE-staining, Masson's trichome staining, Sirius Red staining and Immunofluorescent staining were used for histological analysis. RESULTS: Aligned Gelatin/PCL fibrous membrane (GPA) was most effective for both PDLSCs and BMSCs in culture with WCA around 50 degrees and better mechanical strength than the rest. MSCs favored the same type of ECM (cell-specific ECM), and their regenerative properties were effectively induced with better chemotaxis, proliferative and differentiating behaviors. TLS characterization showed that TLS possessed aligned-random-aligned structure and inter-porous structure. In a rat model of periodontal defects, the TLS functionalized by BMSC-specific ECM for bone regeneration and PDLSC-specific ECM demonstrated highest BV/TV ratio, best bone structure and ligament fiber orientation and blood vessel formation, suggesting optimal performance in regenerating both alveolar bone and periodontal ligaments over TLS, single-ECM loaded TLS and r-Bi-ECM-TLS. SIGNIFICANCE: This study highlights the importance of combining a cell-specific decellularized ECM and a biomimetic electrospinning scaffold for targeted periodontal tissue regeneration, with potential implications for periodontal tissue engineering and improved patient outcomes.

ApoSEVs-Mediated Modulation of Versatile Target Cells Promotes Diabetic Wound Healing: Unveiling a Promising Strategy.

Background: Diabetic chronic wounds present a formidable challenge in clinical management, lacking effective treatment options. Mesenchymal stem cell (MSC) transplantation has emerged as a promising therapy for tissue repair and regeneration. However, transplanted MSCs often undergo rapid apoptosis, giving rise to heterogeneous extracellular vesicles (EVs), including apoptotic bodies (apoBDs) and apoptotic small extracellular vesicles (apoSEVs). The potential stimulatory role of these EVs in diabetic wound healing remains unknown. Methods: In this study, we investigated the effects of apoSEVs derived from adipose-derived mesenchymal/stromal cells (ADSCs) on the recovery of diabetic wounds by modulating the function of versatile target cells. First, we characterized the apoSEVs and apoBDs derived from apoptotic ADSCs. Subsequently, we evaluated the effects of apoSEVs and apoBDs on macrophages, endothelial cells, and fibroblasts, three essential cell types in wound healing, under high-glucose conditions. Furthermore, we developed a gelatin methacryloyl (GelMA) hydrogel for the sustained release of apoSEVs and investigated its therapeutic effects on wound healing in type 2 diabetic mice in vivo. Results: apoSEVs facilitated the polarization of M1 phenotype macrophages to M2 phenotype, promoted proliferation, migration, and tube formation of endothelial cells, and enhanced fibroblast proliferation and migration. However, apoBDs failed to improve the function of endothelial cells and fibroblasts. In vivo, the apoSEVs-loaded GelMA effectively promoted wound healing by facilitating collagen fiber deposition, angiogenesis, and immune regulation. Conclusion: Our study elucidates the beneficial effects of apoSEVs on wound recovery in diabetes and introduces a novel strategy for diabetic wound treatment based on apoSEVs.

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.

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.

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.

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.

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.

Extracellular Vesicles Derived From Apoptotic Cells: An Essential Link Between Death and Regeneration.

Apoptosis is a universal and continuous event during tissue development, restoration, repair, and regeneration. Mounting evidence has demonstrated that apoptosis is essential for the activation of tissue regeneration. However, the underlying mechanism remains elusive. A striking development in recent years comes from research on extracellular vesicles (EVs) derived from apoptotic cells. During apoptosis, cells secrete vesicles of various sizes containing various components. Apoptotic cell-derived EVs (ApoEVs) have been found to transit to neighboring cells or cells in distant tissues through the circulation. These vesicles could act as containers to transmit the nucleic acid, protein, and lipid signals to target cells. ApoEVs have been shown to promote regeneration in the cardiovascular system, skin, bone, muscle, kidney, etc. Moreover, several specific signaling pathways mediating the anabolic effects of ApoEVs have been classified. In this review, we comprehensively discussed the latest findings on the function of ApoEVs in tissue regeneration and disease prevention. These findings may reveal unexpected clues regarding the regulatory network between cell death and tissue regeneration and suggest novel targets for regenerative medicine. The findings discussed here also raise the question whether and to what extent ApoEVs contribute to embryonic development. This question is all the more urgent because the exact functions of apoptotic events during numerous developmental processes are still largely unclear.