Functional diversity of apoptotic vesicle subpopulations from bone marrow mesenchymal stem cells in tissue regeneration.

Apoptosis releases numerous apoptotic vesicles that regulate processes such as cell proliferation, immunity, and tissue regeneration and repair. Now, it has also emerged as an attractive candidate for biotherapeutics. However, apoptotic vesicles encompass a diverse range of subtypes, and it remains unclear which specific subtypes play a pivotal role. In this study, we successfully isolated different apoptotic vesicle subtypes based on their sizes and characterized them using NTA and TEM techniques, respectively. We compared the functional variances among the distinct subtypes of apoptotic vesicles in terms of stem cell proliferation, migration, and differentiation, as well as for endothelial cell and macrophage function, effectively identifying subtypes that exhibit discernible functional differences. ApoSEV (with diameter <1000 nm) promoted stem cell proliferation, migration, and multi-potent differentiation, and accelerated skin wound healing of diabetes mouse model, while apoBD (with diameter >1000 nm) played the opposite effect on cell function and tissue regeneration. Lastly, employing protein analysis and gene sequencing techniques, we elucidated the intrinsic mechanisms underlying these differences between different subtypes of apoEVs. Collectively, this study identified that apoptotic vesicle subtypes possessed distinct bio-functions in regulating stem cell function and behaviour and modulating tissue regeneration, which primarily attribute to the distinct profiling of protein and mRNA in different subtypes. This comprehensive analysis of specific subtypes of apoEVs would provide novel insights for potential therapeutic applications in cell biology and tissue regeneration.

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.

Genistein mitigates senescence of bone marrow mesenchymal stem cells via ERRα-mediated mitochondrial biogenesis and mitophagy in ovariectomized rats.

Senescence of bone marrow mesenchymal stem cells (BMMSCs) induced by chronic oxidative stress is an important factor contributes to the postmenopausal osteoporosis (PMOP). Mitochondrial quality control takes a pivotal role in regulating oxidative stress and cell senescence. Genistein is a major isoflavone in soy products, which is best known for its ability to inhibit bone loss in both postmenopausal women and ovariectomized (OVX) rodents. Here we show that OVX-BMMSCs displayed premature senescence, elevated reactive oxygen species (ROS) level and mitochondria dysfunction, while genistein rescued these phenotypes. Using network pharmacology and molecular docking, we identified estrogen-related receptor α (ERRα) as the potential target of genistein. Knockdown of ERRα greatly abolished the anti-senescence effect of genistein on OVX-BMMSCs. Further, the mitochondrial biogenesis and mitophagy induced by genistein were inhibited by ERRα knockdown in OVX-BMMSCs. In vivo, genistein inhibited trabecular bone loss and p16INK4a expression, upregulated sirtuin 3 (SIRT3) and peroxisome proliferator-activated receptor gamma coactivator one alpha (PGC1α) expression in the trabecular bone area of proximal tibia in OVX rats. Together, this study revealed that genistein ameliorates senescence of OVX-BMMSCs through ERRα-mediated mitochondrial biogenesis and mitophagy, which provided a molecular basis for advancement and development of therapeutic strategies against PMOP.

Activating Wnt/ß-catenin signaling by autophagic degradation of APC contributes to the osteoblast differentiation effect of soy isoflavone on osteoporotic mesenchymal stem cells.

The functional role of autophagy in regulating differentiation of bone marrow mesenchymal stem cells (MSCs) has been studied extensively, but the underlying mechanism remains largely unknown. The Wnt/ß-catenin signaling pathway plays a pivotal role in the initiation of osteoblast differentiation of mesenchymal progenitor cells, and the stability of core protein ß-catenin is tightly controlled by the APC/Axin/GSK-3ß/Ck1α complex. Here we showed that genistein, a predominant soy isoflavone, stimulated osteoblast differentiation of MSCs in vivo and in vitro. Female rats were subjected to bilateral ovariectomy (OVX); four weeks after surgery the rats were orally administered genistein (50 mg·kg-1·d-1) for 8 weeks. The results showed that genistein administration significantly suppressed the bone loss and bone-fat imbalance, and stimulated bone formation in OVX rats. In vitro, genistein (10 nM) markedly activated autophagy and Wnt/ß-catenin signaling pathway, and stimulated osteoblast differentiation in OVX-MSCs. Furthermore, we found that genistein promoted autophagic degradation of adenomatous polyposis coli (APC), thus initiated ß-catenin-driven osteoblast differentiation. Notably, genistein activated autophagy through transcription factor EB (TFEB) rather than mammalian target of rapamycin (mTOR). These findings unveil the mechanism of how autophagy regulates osteogenesis in OVX-MSCs, which expands our understanding that such interplay could be employed as a useful therapeutic strategy for treating postmenopausal osteoporosis.

Microvessels derived from hiPSCs are a novel source for angiogenesis and tissue regeneration.

The establishment of effective vascularization represents a key challenge in regenerative medicine. Adequate sources of vascular cells and intact vessel fragments have not yet been explored. We herein examined the potential application of microvessels induced from hiPSCs for rapid angiogenesis and tissue regeneration. Microvessels were generated from human pluripotent stem cells (iMVs) under a defined induction protocol and compared with human adipose tissue-derived microvessels (ad-MVs) to illustrate the similarity and differences of the alternative source. Then, the therapeutic effect of iMVs was detected by transplantation in vivo. The renal ischemia-reperfusion model and skin damage model were applied to explore the potential effect of vascular cells derived from iMVs (iMVs-VCs). Besides, the subcutaneous transplantation model and muscle injury model were established to explore the ability of iMVs for angiogenesis and tissue regeneration. The results revealed that iMVs had remarkable similarities to natural blood vessels in structure and cellular composition, and were potent for vascular formation and self-organization. The infusion of iMVs-VCs promoted tissue repair in the renal and skin damage model through direct contribution to the reconstruction of blood vessels and modulation of the immune microenvironment. Moreover, the transplantation of intact iMVs could form a massive perfused blood vessel and promote muscle regeneration at the early stage. The infusion of iMVs-VCs could facilitate the reconstruction and regeneration of blood vessels and modulation of the immune microenvironment to restore structures and functions of damaged tissues. Meanwhile, the intact iMVs could rapidly form perfused vessels and promote muscle regeneration. With the advantages of abundant sources and high angiogenesis potency, iMVs could be a candidate source for vascularization units for regenerative medicine.

Characterization of Mesenchymal Stem Cells Derived from Bisphosphonate-Related Osteonecrosis of the Jaw Patients' Gingiva.

Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a clinical condition that specifically occurs in the oral cavity, characterized by retarded wound healing in oral mucosa accelerating the exposure of bone. Moreover, the pathological mechanism remains poorly understood. Gingival mesenchymal stem cells (GMSCs) play a critical role in gingival healing and soft tissue regeneration. Although previous studies have showed that bisphosphonates (BPs) are highly toxic to healthy GMSC, there is overall lack of direct evidence demonstrating the characterization of GMSCs derived from BRONJ patients. In present study, we isolated GMSCs for the first time from the central area of BRONJ patients' gingiva (center-BRONJ GMSCs) and the peripheral area (peri-BRONJ GMSCs), and found that they exhibited decreased proliferation, adhesion, migration capacities and underwent early apoptosis in vitro compared control GMSCs. Notably, the central and peripheral BRONJ GMSCs transplantation in a mice excisional skin model also displayed lower cell survival rate and poor healing effects than that of controls. Mechanistically, TGF-ß1 signaling pathway was suppressed not only in BRONJ patients' gingival lesions but also in BRONJ GMSCs transplantation animal model. The results above suggested that under the microenvironment of BRONJ patients, the dysfunction of GMSCs and the suppressed TGF-ß1 signaling pathway may be the vital factors in impaired gingival healing, thus contributing to persistent exposure of underlying bone and development of BRONJ. This study provides new insights into the prevention for BRONJ by improving the functions of GMSCs and upregulating TGF-ß1 in accelerating gingival wound healing. Schematic illustration of the dysfunction of BRONJ GMSCs in vitro and BRONJ GMSCs transplantation in a mice skin model delaying cutaneous wound healing mainly via suppressing TGF-ß1 signaling pathway.

A single-cell interactome of human tooth germ from growing third molar elucidates signaling networks regulating dental development.

BACKGROUND: Development of dental tissue is regulated by extensive cell crosstalk based on various signaling molecules, such as bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) pathways. However, an intact network of the intercellular regulation is still lacking. RESULT: To gain an unbiased and comprehensive view of this dental cell interactome, we applied single-cell RNA-seq on immature human tooth germ of the growing third molar, discovered refined cell subtypes, and applied multiple network analysis to identify the central signaling pathways. We found that immune cells made up over 80% of all tooth germ cells, which exhibited profound regulation on dental cells via Transforming growth factor-ß, Tumor necrosis factor (TNF) and Interleukin-1. During osteoblast differentiation, expression of genes related to extracellular matrix and mineralization was continuously elevated by signals from BMP and FGF family. As for the self-renewal of apical papilla stem cell, BMP-FGFR1-MSX1 pathway directly regulated the G0-to-S cell cycle transition. We also confirmed that Colony Stimulating Factor 1 secreted from pericyte and TNF Superfamily Member 11 secreted from osteoblast regulated a large proportion of genes related to osteoclast transformation from macrophage and monocyte. CONCLUSIONS: We constructed the intercellular signaling networks that regulated the essential developmental process of human tooth, which served as a foundation for future dental regeneration engineering and the understanding of oral pathology.

Quantitative Segmentation Analysis of the Radiological Changes by Using ITK-SNAP: Risk Assessment of the Severity and Recurrence of Medication-related Osteonecrosis of the Jaw.

Background and purpose: Medication-related osteonecrosis of the jaw (MRONJ) severely impairs patients' quality of life and is remarkably refractory to treatment. There are lots of studies about identification of the radiographic features of MRONJ, yet reports about quantitative radiographic analysis for the risk assessment of the severity and recurrence of MRONJ are rarely heard. The aim of this study was to investigate the volumes of osteolytic lesions and radiodensity values of osteosclerotic lesions in MRONJ patients by using ITK-SNAP for severity prediction and prognosis evaluation. Materials and methods: Of 78 MRONJ patients (78 lesions) involved in this retrospective study, 53 were presented as osteolytic lesions and 25 were presented as osteosclerotic changes alone. Comprehensive CBCT images, demographics and clinical data of patients were investigated. The volumetric analysis and radiodensity measurement were performed by ITK-SNAP. SPSS 25.0 were used for statistical analysis. Results: The osteolytic lesion volumes in MRONJ patients receiving intravenous bisphosphonates (P=0.004) and patients without osteoporosis (P=0.027) were significantly large. No significant correlation between the volumes and bisphosphonates duration was found (P=0.094). The radiodensity values of osteosclerotic lesions was significantly correlated with bisphosphonates duration (P=0.040). The surrounding area of post-surgical lesions in MRONJ patients with recurrence showed significantly great radiodensity values (P=0.025). No significant correlation between the radiodensity values and the transformation from osteosclerotic lesions to osteolytic lesions was observed (P=0.507). Conclusion: MRONJ patients receiving intravenous bisphosphonates develop into large volumes of osteolytic lesions more easily. Long-term bisphosphonates duration is possibly related with higher bone density of osteosclerotic lesions, while higher density is not associated with the transformation from osteosclerotic lesions to osteolytic lesions. A rise of bone mineral density nearby post-surgical lesions is probably a predictor for MRONJ recurrence.

Activation of mesenchymal stem cells promotes new bone formation within dentigerous cyst.

BACKGROUND: Dentigerous cyst (DC) is a bone destructive disease and remains a challenge for clinicians. Marsupialization enables the bone to regenerate with capsule maintaining, making it a preferred therapeutic means for DC adjacent to vital anatomical structures. Given that capsules of DC are derived from odontogenic epithelium remnants at the embryonic stage, we investigated whether there were mesenchymal stem cells (MSCs) located in DC capsules and the role that they played in the bone regeneration after marsupialization. METHODS: Samples obtained before and after marsupialization were used for histological detection and cell culture. The stemness of cells isolated from fresh tissues was analyzed by morphology, surface marker, and multi-differentiation assays. Comparison of proliferation ability between MSCs isolated from DC capsules before (Bm-DCSCs) and after (Am-DCSCs) marsupialization was evaluated by Cell Counting Kit-8 (CCK-8), fibroblast colony-forming units (CFU-F), and 5'-ethynyl-2'-deoxyuridine (EdU) assay. Their osteogenic capacity in vitro was detected by alkaline phosphatase (ALP) and Alizarin Red staining (ARS), combined with real-time polymerase chain reaction (RT-PCR) and immunofluorescence (IF) staining. Subcutaneous ectopic osteogenesis as well as cranial bone defect model in nude mice was performed to detect their bone regeneration and bone defect repairability. RESULTS: Bone tissue and strong ALP activity were detected in the capsule of DC after marsupialization. Two types of MSCs were isolated from fibrous capsules of DC both before (Bm-DCSCs) and after (Am-DCSCs) marsupialization. These fibroblast-like, colony-forming cells expressed MSC markers (CD44+, CD90+, CD31-, CD34-, CD45-), and they could differentiate into osteoblast-, adipocyte-, and chondrocyte-like cells under induction. Notably, Am-DCSCs performed better in cell proliferation and self-renewal. Moreover, Am-DCSCs showed a greater osteogenic capacity both in vitro and in vivo compared with Bm-DCSCs. CONCLUSIONS: There are MSCs residing in capsules of DC, and the cell viability as well as the osteogenic capacity of them is largely enhanced after marsupialization. Our findings suggested that MSCs might play a crucial role in the healing process of DC after marsupialization, thus providing new insight into the treatment for DC by promoting the osteogenic differentiation of MSCs inside capsules.

Combined Administration of Bisphosphonates, Chemotherapeutic Agents, and/or Targeted Drugs Increases the Risk for Stage 3 Medication-Related Osteonecrosis of the Jaw: A 4-Year Retrospective Study.

OBJECTIVES: Patients with stage 3 medication-related osteonecrosis of the jaw (MRONJ) suffer from severe complications. Chemotherapeutic agents and targeted drugs are considered to be associated with the development of MRONJ. However, little is known regarding the association of those agents with stage 3 MRONJ. The purpose of this study is to analyze the comprehensive medication history of patients with advanced-stage MRONJ (stage 2 and stage 3) and evaluate the possible risk factors for stage 3 MRONJ. Patients and Methods. Sixty patients with advanced-stage MRONJ were involved in this retrospective study. Patients with developmental maxillofacial anomalies, previous radiation in the head and neck areas, and jaw bone tumors were excluded from the study. All patients were divided into two groups by their MRONJ stage (stage 2 or stage 3). Demographic and clinical characteristics, comprehensive medication data (bisphosphonates, chemotherapeutic agents, targeted drugs, and immunosuppressive agents), and results of serological biomarkers were recorded and compared between two groups. Univariate and multivariate logistic regressions were performed by SPSS 25.0 for evaluating risk factors of stage 3 MRONJ. RESULTS: Our results indicate that chemotherapy (adjusted OR = 3.43; 95% CI: 1.03 to 11.38), targeted drugs (adjusted OR = 3.69; 95% CI: 1.06 to 12.80), and maxillary lesions (adjusted OR = 4.26; 95% CI: 1.19 to 15.23) increase the risk of stage 3 MRONJ. CONCLUSION: The outcome of this study justifies that chemotherapeutic agents and targeted drugs are probably risk factors for stage 3 MRONJ. In addition, the osteonecrosis in maxilla is more easily to develop into stage 3 MRONJ. Intense clinical observation is recommended in MRONJ patients with maxillary osteonecrosis and in those who concurrently administered bisphosphonates, chemotherapeutic agents, and/or targeted drugs. This trial is registered with ChiCTR2000032428.

Stress response in periodontal ligament stem cells may contribute to bisphosphonate­associated osteonecrosis of the jaw: A gene expression array analysis.

Gene expression alterations in periodontal ligament stem cells (PDLSCs) during bisphosphonate (BP) usage and the transcriptomic mechanism underlying BP­related osteonecrosis of the jaw have not been fully elucidated. In the present study, human PDLSCs were isolated from adults with no history of periodontal disease, and subsequently incubated and treated with zoledronate on days 3 and 5. Subsequently, PDLSCs from all timepoints were screened using an Affymetrix Gene Expression Array. Limma differential expression analysis was performed on a normalized gene expression matrix, followed by cluster analysis, pathway and network analyses. Overall, 906 genes (352 upregulated and 554 downregulated) exhibited differential expression levels between days 0 and 5, and these were termed slow­response genes. These slow­response genes were enriched in cellular stress response signaling pathways (upregulated genes), as well as proliferation­ and ossification­associated signaling pathways (downregulated genes). Furthermore, 168 (day 3 vs. 0) and 105 (day 5 vs. 3) genes were differentially expressed between adjacent timepoints. These genes were also enriched in stress response­ and proliferation­associated signaling pathways, but not in ossification­associated signaling pathways. Poly(ADP­ribose) polymerase 1 (PARP1) and CYLD lysine 63 deubiquitinase (CYLD) had the most protein­protein interaction partners among the slow­response genes and were connected with both stress­ (e.g. caspase­1) and ossification­associated genes [e.g. secreted phosphoprotein 1 and collagen type I α1 chain (COL1A1)]. BP treatment induced stress response­like transcriptional alterations in PDLSCs, followed by inhibition of proliferation and ossification. These alterations may contribute to the onset of jaw osteonecrosis. PARP1 and CYLD may be two key genes involved in this pathological procedure.

Decreased Osteogenic Ability of Periodontal Ligament Stem Cells Leading to Impaired Periodontal Tissue Repair in BRONJ Patients.

Bisphosphonate-related osteonecrosis of the jaws (BRONJ) is a severe adverse reaction, which results in progressive bone destruction in the maxillofacial region of patients. To date, the pathological mechanisms remain largely unclear. Recently, we found that BRONJ patient had significantly deep periodontal pockets and severe periodontal bone defects before the exposed necrotic bone. Human periodontal ligament stem cells (hPDLSCs) play key roles in physiological maintenance and regeneration of periodontal tissues. However, the activities of hPDLSCs derived from BRONJ lesions and the role of hPDLSCs in BRONJ periodontal defect repair remain poorly understood. The aim of the present study was to elucidate the role of hPDLSCs in BRONJ. In this study, we found that the capacities of cell proliferation, adhesion, and migration of hPDLSCs derived from BRONJ lesions (BRONJ-hPDLSCs) were significantly decreased compared with control-hPDLSCs. BRONJ-hPDLSCs underwent early apoptosis compared with control-hPDLSCs. Importantly, we first demonstrated that BRONJ-hPDLSCs exhibited impaired osteogenic differentiation abilities in ectopic osteogenesis of nude mice. The above results suggested that the impaired BRONJ-hPDLSCs may be an important factor in deficient periodontal repair of BRONJ lesions and provide new insight into the underlying mechanism of BRONJ.

Dental Resin Composites Reinforced by Rough Core-Shell SiO2 Nanoparticles with a Controllable Mesoporous Structure.

A porous structure within filler particles may improve interfacial bonding between the resin matrix and fillers for the preparation of dental resin composites (DRCs). In this study, rough core-shell SiO2 (rSiO2) nanoparticles with controllable mesoporous structures were synthesized via an oil-water biphase reaction system and characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and N2 adsorption-desorption measurements. The influence of the mesoporous shell thickness of rSiO2 and mass ratio between rSiO2 and smooth SiO2 (sSiO2) on the physical and mechanical properties of DRCs was studied. The rSiO2 with a thin mesoporous shell could form a strong physical interlocking with the resin matrix, which improved the mechanical properties with the exception of flexural modulus. The mechanical properties were further optimized by mixing rSiO2 and sSiO2. The flexural strength and compressive strength of the DRC at a mass ratio of 5:5 increased by 24.3% and 16.8%, respectively, compared with the DRC filled with sSiO2 alone. There is no statistically significant difference in the flexural modulus between these two DRCs (p > 0.05). The DRCs in this study showed excellent biocompatibility on the human dental pulp cells (HDPCs) as demonstrated by the cytotoxicity tests. The use of rSiO2 provides a promising approach to develop strong, durable, and biocompatible DRCs.