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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)

Schematic illustration of cell-laden microfiber aggregates for pulp regeneration. (A) The fabrication process of DPSC-laden and HUVEC-laden microfibers. (B) Cell-laden microfiber aggregates were filled into tooth roots for ectopic 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.

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.

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.

Jian Yang,1,2 Xin Gao,1,2 Xiaotao Xing,1,2 Haisen Huang,1,2 Qi Tang,1,3 Shixing Ma,1,2 Xun Xu,1,2 Cheng Liang,1,2 Maojiao Li,1,2 Li Liao,1,2 Weidong Tian1,2 1State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Engineering Research Center of Oral Translational Medicine, Ministry of Education & National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of China; 3West China School of Public Health & West China Fourth Hospital, Sichuan University, Chengdu, 610041, People’s Republic of ChinaCorrespondence: Li Liao; Weidong TianState Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Engineering Research Center of Oral Translational Medicine, Ministry of Education & National Engineering Laboratory for Oral Regenerative Medicine, West China Hospital of Stomatology, Sichuan University, Chengdu, 610041, People’s Republic of ChinaTel/ Fax +86-28-85503499Email lliao@scu.edu.cn; drtwd@sina.comPurpose: 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.Keywords: extracellular vesicles, size-exclusion chromatography, ultracentrifugation, total exosomes isolation, combination methods

Background: 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.Results: Firstly, we use Sepharose-4B to build a self-made size exclusion chromatography (SEC) column and perform separation and identification. 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.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.

The transplantation of bone marrow mesenchymal stem cells (BMSCs) promotes bone repair and regeneration. However, it has been shown that the majority of BMSCs die within a short period after transplantation. During apoptosis, BMSCs generate a large number of apoptotic cell-derived extracellular vesicles (ApoEVs). This study aims to understand the potential role of ApoEVs in bone defect repair and regeneration. We confirm that BMSCs undergo apoptosis 2 days after transplantation into the defect of the cranium. In vitro, we find that abundant ApoEVs were generated by apoptotic BMSCs and can be engulfed by BMSCs and promote the proliferation, migration, and osteogenic differentiation of recipient cells. ApoEVs from cells in the middle stage of apoptosis were the most efficient at enhancing the regeneration capacity of BMSCs. In vivo, transplantation of ApoEVs in the calvarial defect region significantly promoted bone regeneration in both mouse and rat models. Mechanistically, ApoEVs promote new bone formation by upregulating the Reactive Oxygen Species (ROS) and activating the JNK signaling. This study reveals a previously unknown role of the dying transplanted BMSCs in promoting the viability of endogenous BMSCs and repairing the bone defects. Since it could avoid several adverse effects and limits of BMSCs cytotherapy, treatment of ApoEVs might be a promising strategy in bone repair and regeneration.

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.

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.