The Dual Angiogenesis Effects via Nrf2/HO-1 Signaling Pathway of Melatonin Nanocomposite Scaffold on Promoting Diabetic Bone Defect Repair.

Purpose: Given the escalating prevalence of diabetes, the demand for specific bone graft materials is increasing, owing to the greater tendency towards bone defects and more difficult defect repair resulting from diabetic bone disease (DBD). Melatonin (MT), which is known for its potent antioxidant properties, has been shown to stimulate both osteogenesis and angiogenesis. Methods: MT was formulated into MT@PLGA nanoparticles (NPs), mixed with sodium alginate (SA) hydrogel, and contained within a 3D printing polycaprolactone/ß-Tricalcium phosphate (PCL/ß-TCP) scaffold. The osteogenic capacity of the MT nanocomposite scaffold under diabetic conditions was demonstrated via in vitro and in vivo studies and the underlying mechanisms were investigated. Results: Physicochemical characterization experiments confirmed the successful fabrication of the MT nanocomposite scaffold, which can achieve long-lasting sustained release of MT. The in vitro and in vivo studies demonstrated that the MT nanocomposite scaffold exhibited enhanced osteogenic capacity, which was elucidated by the dual angiogenesis effects activated through the NF-E2-related factor 2/Heme oxygenase 1 (Nrf2/HO-1) signaling pathway, including the enhancement of antioxidant enzyme activity to reduce the oxidative stress damage of vascular endothelial cells (VECs) and directly stimulating vascular endothelial growth factor (VEGF) production, which reversed the angiogenesis-osteogenesis uncoupling and promoted osteogenesis under diabetic conditions. Conclusion: This study demonstrated the research prospective and clinical implications of the MT nanocomposite scaffold as a novel bone graft for treating bone defect and enhancing bone fusion in diabetic individuals.

Apoptotic vesicles derived from human red blood cells promote bone regeneration via carbonic anhydrase 1.

Apoptotic vesicles (apoVs) are nanoscale vesicles derived from billions of apoptotic cells involved in the maintenance of the human body's homeostasis. Previous researches have shown that some apoVs, such as those derived from mesenchymal stem cells, contribute to bone formation. However, those apoVs cannot be extracted from patients in large quantities, and cell expansion is needed before apoV isolation, which limits their clinical translation. Mature RBCs, which have no nuclei or genetic material, are easy to obtain, showing high biological safety as a source of extracellular vesicles (EVs). Previous studies have demonstrated that RBC-derived EVs have multiple biological functions, but it is unknown whether RBCs produce apoVs and what effect these apoVs have on bone regeneration. In this study, we isolated and characterized RBC-derived apoVs (RBC-apoVs) from human venous blood and investigated their role in the osteogenesis of human bone mesenchymal stem cells (hBMSCs). We showed that RBCs could produce RBC-apoVs that expressed both general apoVs markers and RBC markers. RBC-apoVs significantly promoted osteogenesis of hBMSCs and enhanced bone regeneration in rat calvarial defects. Mechanistically, RBC-apoVs regulated osteogenesis by transferring carbonic anhydrase 1 (CA1) into hBMSCs and activating the P38 MAPK pathway. Our results indicated that RBC-apoVs could deliver functional molecules from RBCs to hBMSCs and promote bone regeneration, pointing to possible therapeutic use in bone tissue engineering.

Platelet-Derived Apoptotic Vesicles Promote Bone Regeneration via Golgi Phosphoprotein 2 (GOLPH2)-AKT Signaling Axis.

Apoptotic vesicles (apoVs) are apoptotic-cell-derived nanosized vesicles that take on dominant roles in regulating bone homeostasis. We have demonstrated that mesenchymal stem cell (MSC)-derived apoVs are promising therapeutic agents for bone regeneration. However, clinical translation of MSC-derived apoVs has been hindered due to cell expansion and nuclear substance. As another appealing source for apoV therapy, blood cells could potentially eliminate these limitations. However, whether blood cells can release apoVs during apoptosis is uncertain, and the detailed characteristics and biological properties of respective apoVs are not elucidated. In this study, we showed that platelets (PLTs) could rapidly release abundant apoVs during apoptosis in a short time. To recognize the different protein expressions between PLT-derived apoVs and PLTs, we established their precise protein landscape. Furthermore, we identified six proteins specifically enriched in PLT-derived apoVs, which could be considered as specific biomarkers. More importantly, PLT-derived apoVs promoted osteogenesis of MSCs and rescued bone loss via Golgi phosphoprotein 2 (GOLPH2)-induced AKT phosphorylation, therefore, leading to the emergence of their potential in bone regeneration. In summary, we comprehensively determined characteristics of PLT-derived apoVs and confirmed their roles in bone metabolism through previously unrecognized GOPLH2-dependent AKT signaling, providing more understanding for exploring apoV-based therapy in bone tissue engineering.

Lyophilized apoptotic vesicle-encapsulated adhesive hydrogel sponge as a rapid hemostat for traumatic hemorrhage in coagulopathy.

Rapid hemostasis of uncontrolled bleeding following traumatic injuries, especially accompanied by coagulopathies, remains a significant clinical challenge. Extracellular vesicles (EVs) show therapeutic effects for fast clotting. However, low yield, specific storage conditions, and lack of proper carriers have hindered EVs' clinical application. Herein, we establish an optimized procedure method to generate lyophilized mesenchymal stem cell-derived apoptotic vesicles (apoVs) with adhesive hydrogel sponge to show superior procoagulant activity for traumatic hemorrhage. Mechanistically, apoVs' procoagulant ability stems from their high tissue factor (TF) and phosphatidylserine (PS) expression independent of hemocytes and circulating procoagulant microparticles (cMPs). Their stable hemostatic capability was maintained after 2-month room temperature storage. Subsequently, we mixed apoVs with both phenylboronic acid grafted oxidized hyaluronic acid (PBA-HA) and poly(vinyl alcohol) (PVA) simultaneously, followed by lyophilization to construct a novel apoV-encapsulated hydrogel sponge (apoV-HS). Compared to commercial hemostats, apoV-HS exhibits rapid procoagulant ability in liver-laceration and femoral artery hemorrhage in rat and rabbit models of coagulopathies. The combination of high productivity, physiological stability, injectability, plasticity, excellent adhesivity, biocompatibility, and rapid coagulant property indicates that apoV-HS is a promising therapeutic approach for heavy hemorrhage in civilian and military populations.

Apoptotic Vesicles Regulate Bone Metabolism via the miR1324/SNX14/SMAD1/5 Signaling Axis.

Mesenchymal stem cells (MSCs) are widely used in the treatment of diseases. After their in vivo application, MSCs undergo apoptosis and release apoptotic vesicles (apoVs). This study investigates the role of apoVs derived from human bone marrow mesenchymal stem cells (hBMMSCs) in bone metabolism and the molecular mechanism of the observed effects. The results show that apoVs can promote osteogenesis and inhibit osteoclast formation in vitro and in vivo. ApoVs may therefore attenuate the bone loss caused by primary and secondary osteoporosis and stimulate bone regeneration in areas of bone defect. The mechanisms responsible for apoV-induced bone regeneration include the release of miR1324, which inhibit expression of the target gene Sorting Nexin 14 (SNX14) and thus activate the SMAD1/5 pathway in target cells. Given that MSC-derived apoVs are easily obtained and stored, with low risks of immunological rejection and neoplastic transformation, The findings suggest a novel therapeutic strategy to treat bone loss, including via cell-free approaches to bone tissue engineering.

Robot-assisted subretinal injection system: development and preliminary verification.

BACKGROUND: To design and develop a surgical robot capable of assisting subretinal injection. METHODS: A remote center of motion (RCM) mechanical design and a master-slave teleoperation were used to develop and manufacture the assisted subretinal surgery robot (RASR). Ten fresh isolated porcine eyes were divided into the Robot Manipulation (RM) group and Manual Manipulation (MM) group (5 eyes for each group), and subretinal injections were performed by the robot and manual manipulation methods, respectively. A preliminary verification of the robot was performed by comparing the advantages and disadvantages of the robot manipulation and manual manipulation by using optical coherent tomography (OCT), fundus photography, and video motion capture analysis after the surgery. RESULTS: Both the robot and the manual manipulation were able to perform subretinal injections with a 100% success rate. The OCT results showed that the average subretinal area was 1.548 mm2 and 1.461 mm2 in the RM and MM groups, respectively (P > 0.05). Meanwhile the volume of subretinal fluid obtained using the retinal map mode built in OCT was not statistically different between the RM and MM groups (P > 0.05). By analyzing the surgical video using Kinovea, a motion capture and analysis software, the results suggest that the mean tremor amplitude of the RM group was 0.3681 pixels (x direction), which was significantly reduced compared to 18.8779 pixels (x direction) in the MM group (P < 0.0001). CONCLUSION: Robot-assisted subretinal injection system (RASR) is able to finish subretinal injection surgery with better stability and less fatigue than manual manipulation.

Outcome of keratolimbal allograft transplantation with deep anterior lamellar keratoplasty for bilateral limbal stem cell deficiency.

Objectives: To evaluate and compare the outcome of keratolimbal allograft (KLAL) transplantation with or without deep anterior lamellar keratoplasty (DALK) for bilateral severe limbal stem cell deficiency (LSCD). Methods: This retrospective review included 49 eyes of 46 patients who underwent KLAL transplantation at the Department of Ophthalmology of Chinese PLA general hospital, 2009-2020, for bilateral severe LSCD were examined for corneal clarity and corneal scarring to determine whether to combine DALK with KLAL transplantation. Preoperative information, surgical decision tree, surgical procedures, and postoperative data were collected for each eye. Results: All patients had preoperative severe or total LSCD. Twenty-four eyes underwent KLAL transplantation only, 25 KLAL transplantation plus DALK. The mean follow-up was 46.80 ± 31.22 months (18-158 months). Overall KLAL survival (with or without DALK) was 71.43% at the final follow-up (KLAL-only 66.67%, KLAL-DALK 76%). Kaplan-Meier survival analysis showed that the 3-year survival probability of all grafts was 70.53 ± 10.89% (KLAL-only 64.86 ± 10.11%, KLAL-DALK 75.79 ± 8.62%). The proportion of BCVA ≥ 20/200 eyes among all KLAL transplantations increased from 11 eyes (22.45%) preoperatively to 25 eyes (51.02%) after 1 year and 24 eyes (48.98%) at the last follow-up (P = 0.01). The proportion of BCVA ≥ 20/200 eyes in the KLAL-DALK group increased significantly (P = 0.04), from 16.0% at baseline to 48.0% after 1 year to 44.0% at the last follow-up. Seventeen eyes (34.69%) had postoperative complications. Conclusion: KLAL-DALK is an effective option to restore a stable ocular surface and visual acuity rapidly in patients with bilateral, late-stage, severe LSCD.

Macrophage-derived apoptotic vesicles regulate fate commitment of mesenchymal stem cells via miR155.

BACKGROUND: In tissue engineering, mesenchymal stem cells (MSCs) are common seed cells because of abundant sources, strong proliferation ability and immunomodulatory function. Numerous researches have demonstrated that MSC-macrophage crosstalk played a key role in the tissue engineering. Macrophages could regulate the differentiation of MSCs via different molecular mechanisms, including extracellular vesicles. Apoptotic macrophages could generate large amounts of apoptotic vesicles (apoVs). ApoVs are rich in proteins, RNA (microRNAs, mRNAs, ncRNAs, etc.) and lipids, and are a key intercellular communication mediator that can exert different regulatory effects on recipient cells. MiRNAs account for about half of the total RNAs of extracellular vesicles, and play important roles in biological processes such as cell proliferation and differentiation, whereas the functions of macrophage-derived apoVs remain largely unknown. There was no research to clarify the role of macrophage-derived apoVs in MSC fate choices. In this study, we aimed to characterize macrophage-derived apoVs, and investigate the roles of macrophage-derived apoVs in the fate commitment of MSCs. METHODS: We characterized macrophage-derived apoVs, and investigated their role in MSC osteogenesis and adipogenesis in vitro and in vivo. Furthermore, we performed microRNA loss- and gain-of-function experiments and western blot to determine the molecular mechanism. RESULTS: Macrophages could produce a large number of apoVs after apoptosis. MSCs could uptake apoVs. Then, we found that macrophage-derived apoVs inhibited osteogenesis and promoted adipogenesis of MSCs in vitro and in vivo. In mechanism, apoVs were enriched for microRNA155 (miR155), and apoVs regulated osteogenesis and adipogenesis of MSCs by delivering miR155. Besides, miR155 regulated osteogenesis and adipogenesis of MSCs cultured with macrophage-derived apoVs via the SMAD2 signaling pathway. CONCLUSIONS: Macrophage-derived apoVs could regulate the osteogenesis and adipogenesis of MSCs through delivering miR155, which provided novel insights for MSC-mediated tissue engineering.

Methodological study of directed differentiation of pluripotent stem cells into corneal endothelial cells.

Background: Corneal transplantation is the most effective clinical treatment for irreversible corneal endothelial decompensation. However, while visual rehabilitation can be achieved by corneal transplantation, transplant rejection, poor postoperative visual acuity, and lack of suitable donor tissue are currently the greatest obstacles to corneal transplantation. As a result, endothelial cell-based therapy has emerged as an alternative to corneal transplantation treatment. Human induced pluripotent stem cells (hiPSCs) were induced to differentiate into human corneal endothelial cell (hCEC)-like cells in our study, which aimed to provide an experimental basis for studying the clinical translation and application of induced PSC (iPSC) to corneal endothelial cell (CEC) differentiation. Methods: A two-step method to convert hiPSCs into hCEC-like cells was applied in the study. First, the transforming growth factor beta (TGF-ß) and Wnt signaling pathways were regulated by adding SB4315542 and CHIR99021, and hiPSCs were induced to differentiate into neural crest cells (NCCs) by a chemically defined and serum-free in vitro induction method. Subsequently, NCCs were induced to differentiate into hCEC-like cells by adding B27, platelet-derived growth factor (PDGF)-BB, and XAV939 (Wnt pathway inhibitor) to CEC culture medium. Results: IPSCs were directionally differentiated into NCCs. During the process of differentiation, iPSCs gradually lost the monoclonal morphology of PSCs. Moreover, ß-catenin and SRY-related HMG-box gene 10 (SOX10) proteins were immunohistochemically expressed on the 7th day of differentiation. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) results demonstrated that the cells expressed SOX9, SOX10, nerve growth factor receptor (NGFR), human natural killer-1 (HNK-1), and ß-catenin, indicating that they were successfully directionally differentiated from iPSCs to NCCs. After 5-7 days of differentiation, the cells demonstrated a hexagonal morphology of monolayer tight junctions. Immunofluorescence results demonstrated that the cells expressed CEC indicator zonula occludens-1 (ZO-1) protein. QRT-PCR results demonstrated that the cells expressed collagen type IV alpha 1 (COL4A1), COL8A2, COL8A1, and ZO-1, which indicated that they were successfully directionally differentiated from NCC to CEC. Conclusions: Our study successfully provided a simple and efficient method with clear chemical composition and serum-free media to directionally differentiate hiPSCs into hCEC-like cells, thereby advancing the results of previous studies on directional transformation into epithelial cells.

Proteomic analysis of MSC-derived apoptotic vesicles identifies Fas inheritance to ameliorate haemophilia a via activating platelet functions.

Apoptotic vesicles (apoVs) are apoptotic cell-derived nanosized vesicles that play a crucial role in multiple pathophysiological settings. However, their detailed characteristics, specific surface markers, and biological properties are not fully elucidated. In this study, we compared mesenchymal stem cell (MSC)-derived apoVs and exosomes from three different types of MSCs including human bone marrow MSCs (hBMSCs), human adipose MSCs (hASCs), and mouse bone marrow MSCs (mBMSCs). We established a unique protein map of MSC-derived apoVs and identified the differences between apoVs and exosomes in terms of functional protein cargo and surface markers. Furthermore, we identified 13 proteins specifically enriched in apoVs compared to exosomes, which can be used as apoV-specific biomarkers. In addition, we showed that apoVs inherited apoptotic imprints such as Fas to ameliorate haemophilia A in factor VIII knockout mice via binding to the platelets' FasL to activate platelet functions, and therefore rescuing the blood clotting disorder. In summary, we systemically characterized MSC-derived apoVs and identified their therapeutic role in haemophilia A treatment through a previously unknown Fas/FasL linkage mechanism.