Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair.

The challenges posed by delayed atrophic healing and nonunion stand as formidable obstacles in osteoporotic fracture treatment. The processes of type H angiogenesis and osteogenesis emerge as pivotal mechanisms during bone regeneration. Notably, the preconditioning of adipose-derived stem cell (ADSC) exosomes under hypoxic conditions has garnered attention for its potential to augment the secretion and functionality of these exosomes. In the present investigation, we embarked upon a comprehensive elucidation of the underlying mechanisms of hypo-ADSC-Exos within the milieu of osteoporotic bone regeneration. Our findings revealed that hypo-ADSC-Exos harboured a preeminent miRNA, namely, miR-21-5p, which emerged as the principal orchestrator of angiogenic effects. Through in vitro experiments, we demonstrated the capacity of hypo-ADSC-Exos to stimulate the proliferation, migration, and angiogenic potential of human umbilical vein endothelial cells (HUVECs) via the mediation of miR-21-5p. The inhibition of miR-21-5p effectively attenuated the proangiogenic effects mediated by hypo-ADSC-Exos. Mechanistically, our investigation revealed that exosomal miR-21-5p emanating from hypo-ADSCs exerts its regulatory influence by targeting sprouly1 (SPRY1) within HUVECs, thereby facilitating the activation of the PI3K/AKT signalling pathway. Notably, knockdown of SPRY1 in HUVECs was found to potentiate PI3K/AKT activation and, concomitantly, HUVEC proliferation, migration, and angiogenesis. The culminating stage of our study involved a compelling in vivo demonstration wherein GelMA loaded with hypo-ADSC-Exos was validated to substantially enhance local type H angiogenesis and concomitant bone regeneration. This enhancement was unequivocally attributed to the exosomal modulation of SPRY1. In summary, our investigation offers a pioneering perspective on the potential utility of hypo-ADSC-Exos as readily available for osteoporotic fracture treatment.

Synthesis and Biological Evaluation of PEGylated MWO4 Nanoparticles as Sonodynamic AID Inhibitors in Treating Diffuse Large B-Cell Lymphoma.

Sonodynamic therapy (SDT) triggered by ultrasound (US) has attracted increasing attention owing to its ability to overcome critical limitations, including low tissue-penetration depth and phototoxicity in photodynamic therapy (PDT). Biogenic metal oxide nanoparticles (NPs) have been used as anti-cancer drugs due to their biocompatibility properties with most biological systems. Here, sonosensitizer MWO4-PEG NPs (M = Fe Mn Co Ni) were synthesized as inhibitors to activation-induced cytidine deaminase (AID), thus neutralizing the extensive carcinogenesis of AID in diffuse large B-cell lymphoma (DLBCL). The physiological properties of these nanomaterials were examined using transmission electron microscopy (TEM). The inhibition of NPs to AID was primarily identified by the affinity interaction prediction between reactive oxygen species (ROS) and AID through molecular dynamics and molecular docking technology. The cell apoptosis and ROS generation in US-triggered NPs treated DLBCL cells (with high levels of AID) were also detected to indicate the sonosensitivity and toxicity of MWO4-PEG NPs to DLBCL cells. The anti-lymphoma studies using DLBCL and AID-deficient DLBCL cell lines indicated a concentration-dependent profile. The synthesized MWO4-PEG NPs in this study manifested good sonodynamic inhibitory effects to AID and well treatment for AID-positive hematopoietic cancers.

Novel laser positioning navigation to aid puncture during percutaneous nephrolithotomy: a preliminary report.

PURPOSE: Accurate puncture of the renal collecting system is crucial to the success of percutaneous nephrolithotomy and presents a technical challenge for urologists. Here, we introduced the Surgical Approach Visualization and Navigation (SAVN) system, a novel navigation system to assist puncture and reduce intraoperative radiation. MATERIALS AND METHODS: Twenty kidneys of 10 cadavers were randomly divided into two groups for renal calyx puncture. In the control group, traditional fluoroscopy was used for guidance, while SAVN system was used in the experimental group. Puncture duration, number of puncture attempts, total number of intraoperative fluoroscopies, and number of fluoroscopies during the puncture procedure were recorded. RESULTS: The puncture duration was 14.2 ± 2.5 s in SAVN group and 48.3 ± 7.1 s in conventional group (P < 0.05). One puncture attempt was needed for successful puncture in SAVN group, while more than one in conventional group (P = 0.28). The total number of intraoperative fluoroscopies was 3.3 ± 1.0 in SAVN group and 14.5 ± 3.1 in control group (P < 0.05),while the number of fluoroscopies during the puncture procedure was 0 and 11.2 ± 2.4, respectively (P < 0.05). CONCLUSIONS: The novel SAVN system has a simplified structure and is easy to use. It can be used to successfully assist with puncture of the renal calyx, thus reducing puncture duration and radiation dose.

Treatment of open tibial diaphyseal fractures by external fixation combined with limited internal fixation versus simple external fixation: a retrospective cohort study.

BACKGROUND: The treatment of open tibial shaft fractures is challenging. External fixation (EF) is comparatively safe in treating these open injuries, meanwhile it has the advantages of easy application, minimal additional disruption, and convenient subsequent soft tissue repair. Nevertheless, its application is accompanied by a series of problems in alignment and bone healing. Therefore, limited internal fixation (LIF), such as cortical screws, has been used based on the external fixator for better therapeutic effect. The aim of this study is to compare the outcomes of EF combined with LIF and simple EF in the management of open tibial shaft fractures, evaluating the efficacy and safety of using the combined technique in treating such fractures. METHODS: From January 2012 to December 2016, patients with open tibial shaft fractures treated with EF with or without LIF augmentation were identified. A total of 152 patients were included in the analysis, and there were 85 patients in the simple external fixation group and 67 patients in the EF-LIF group. General assessment indicators included the direct cost of hospitalization and the times of first surgery, full weight bearing, and complete union. Infections and complications in union or limb alignment were compared as primary outcomes. Additionally, the number of patients who changed the fixation system for various reasons were analysed. RESULTS: Effective follow-up of all participants for statistical analysis was obtained. The follow-up time averaged 17.15 months (range: 12.00 to 24.00 months) in the EF group and 16.20 months (range: 12.00 to 19.00 months) in the EF-LIF group. Combined fixation provided shortened time to bear full weight and achieve complete bone union, while requiring additional first surgery time. No significant difference was found in infection rates or direct cost of hospitalization. Delayed union and non-union in the EF-LIF group were significantly decreased (20.9% versus 40.0, 1.5% versus 14.1%, p < 0.05). In limb alignment, patients with combined fixation exhibited reduced malreduction, loss of reduction, and malunion. In terms of secondary fixation, the EF-LIF group showed a markedly lower incidence (5.8% versus 34.1%, p < 0.001). CONCLUSION: Compared with simple EF, combined fixation is an effective and safe alternative for management of open tibial diaphyseal fractures. It provides superior initial reduction, better stability and decreases the risk of inferior alignment and delayed union without increasing the risk of infection.

Periostin contributes to the adventitial remodeling of atherosclerosis by activating adventitial fibroblasts.

Background and aims: Adventitial remodeling is an important pathological process of atherosclerosis, but cues implicated in adventitial remodeling are far from fully understood. Periostin (POSTN), a matricellular protein, has been demonstrated to have multiple roles in cardiovascular diseases. The aim of the study was to explore the function of POSTN in adventitial remodeling during atherosclerosis. Methods: An atherosclerosis model was constructed based on ApoE-/- mice fed a high-fat and high-cholesterol diet. The expression of POSTN in the adventitia of mouse atherosclerotic vascular specimens was detected by immunohistochemical staining. The roles of POSTN in regulating adventitial fibroblast activation were assessed by cell contractility and activation marker α-smooth muscle actin (α-SMA) expression evaluation in adventitial fibroblasts overexpressing POSTN. In addition, we performed quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting to examine the expression of the proinflammatory chemokines transforming growth factor-ß1 (TGF-ß1) and monocyte chemotactic protein 1 (MCP1), as well as some extracellular matrix (ECM)-related proteins, in POSTN-overexpressing adventitial fibroblasts. Finally, the integrin-related signaling pathway was detected upon POSTN overexpression in adventitial fibroblasts. Results: POSTN was highly expressed in the adventitia of atherosclerotic aortae in the mouse atherosclerosis model and promoted the activation and contraction of adventitial fibroblasts. Meanwhile, POSTN also induced adventitial fibroblasts to express TGF-ß1, monocyte chemotactic protein-1 (MCP1), and ECM-related proteins and activated the phosphorylation of focal adhesion kinase (FAK) and Src. Conclusions: Our results revealed that POSTN is elevated in adventitia during atherosclerosis and contributes to the adventitial remodeling of atherosclerosis by activating adventitial fibroblasts.

Possible causes of atherosclerosis: lncRNA COLCA1 induces oxidative stress in human coronary artery endothelial cells and impairs wound healing.

Background: Atherosclerosis is the most common cause of cardiovascular disease, accompanied by high mortality and poor prognosis. Low-density lipoprotein (LDL) and its oxidized form oxidized low-density lipoprotein (oxLDL) play an important role in atherosclerosis. This article will explore the role of the lncRNA COLCA1 (colorectal cancer associated 1)/hsa-miR-371a-5p/SPP1 (secreted phosphoprotein 1) pathway in oxLDL in causing human coronary artery endothelial cells (HCAECs) inflammation and related biological function changes. Methods: OxLDL was used to stimulate HCAECs. The inflammatory response and biological function changes of HCAECs were analyzed, total RNA-seq was performed on HCAECs before and after stimulation, and RT-Qpcr (real-time quantitative PCR) was used to verify the differential genes. Interference of the expression of COLCA1 in HCAECs was performed by siRNA interference technology to verify the role of COLCA1 in the biological function changes of HCAECs after oxLDL stimulation, and further prove that COLCA1 affects SPP1 through hsa-miR-371a-5p. Results: OxLDL can affect the oxidative stress response of HCAECs, which in turn affects the apoptosis and wound healing ability of HCAECs. COLCA1 and SPP1 were highly expressed after oxLDL stimulation, while hsa-miR-371a-5p was the opposite. After COLCA1 interference, the oxidative stress level of HCAECs stimulated by oxLDL decreased, the apoptosis level also significantly decreased, and the wound healing ability was enhanced. After simultaneous COLCA1 interference and recovery of the expression of hsa-miR-371a-5p, these improved functions disappeared. The dual-luciferase assay confirmed that hsa-miR-371a-5p and COLCA1, hsa-miR-371a-5p and SPP1 has binding targets. Conclusions: OxLDL can up-regulate the expression of COLCA1 in HCAECs, which in turn affects the intracellular COLCA1/hsa-miR-371a-5p/SPP1 pathway to regulate the level of oxidative stress in cells. This in turn affects the level of apoptosis and wound healing ability, which causes cells to produce a continuous inflammatory response.

Umbilical Mesenchymal Stem Cell-Derived Exosome-Encapsulated Hydrogels Accelerate Bone Repair by Enhancing Angiogenesis.

Repair of large bone defects represents a major challenge for orthopedic surgeons. The newly formed microvessels inside grafts play a crucial role in successful bone tissue engineering. Previously, an active role for mesenchymal stem cell (MSC)-derived exosomes in blood vessel development and progression was suggested in the repair of multiple tissues. However, the reports on the application of MSC-derived exosomes in the repair of large bone defects are sparse. In this study, we encapsulated umbilical MSC-derived exosomes (uMSCEXOs) in hyaluronic acid hydrogel (HA-Gel) and combined them with customized nanohydroxyapatite/poly-ε-caprolactone (nHP) scaffolds to repair cranial defects in rats. Imaging and histological evaluation indicated that the uMSCEXOs/Gel/nHP composites markedly enhanced bone regeneration in vivo, and the uMSCEXOs might play a key role in this process. Moreover, the in vitro results demonstrated that uMSCEXOs promoted the proliferation, migration, and angiogenic differentiation of endothelial progenitor cells (EPCs) but did not significantly affect the osteogenic differentiation of BMSCs. Importantly, mechanistic studies revealed that exosomal miR-21 was the potential intercellular messenger that promoted angiogenesis by upregulating the NOTCH1/DLL4 pathway. In conclusion, our findings exhibit a promising exosome-based strategy in repairing large bone defects through enhanced angiogenesis, which potentially regulated by the miR-21/NOTCH1/DLL4 signaling axis.

Locally delivered adjuvant biofilm-penetrating antibiotics rescue impaired endochondral fracture healing caused by MRSA infection.

Infection is a devastating complication following an open fracture. We investigated whether local rifampin-loaded hydrogel can combat infection and improve healing in a murine model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10-12 weeks and stabilized with an intramedullary pin. A total of 1 × 106 colony-forming units (CFU) of MRSA was inoculated. A collagen-based hydrogel containing low-dose (60 µg) and high-dose (300 µg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (µCT), immunohistochemistry, and histological analyses. All untreated MRSA-infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin-1ß stain were associated with poor callus formation. High-dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low-dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with high-dose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). µCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampin-loaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin-loaded hydrogels is required to correlate these findings with clinical efficacy.

Smoking Alters Inflammation and Skeletal Stem and Progenitor Cell Activity During Fracture Healing in Different Murine Strains.

Smokers are at a higher risk of delayed union or nonunion after fracture repair. Few specific interventions are available for prevention because the molecular mechanisms that result in these negative sequelae are poorly understood. Murine models that mimic fracture healing in smokers are crucial in further understanding the local cellular and molecular alterations during fracture healing caused by smoking. We exposed three murine strains, C57BL/6J, 129X1/SvJ, and BALB/cJ, to cigarette smoke for 3 months before the induction of a midshaft transverse femoral osteotomy. We evaluated fracture healing 4 weeks after the osteotomy using radiography, micro-computed tomography (µCT), and biomechanical testing. Radiographic analysis demonstrated a significant decrease in the fracture healing capacity of smoking 129X1/SvJ mice. µCT results showed delayed remodeling of fracture calluses in all three strains after cigarette smoke exposure. Biomechanical testing indicated the most significant impairment in the functional properties of 129X1/SvJ in comparison with C57BL/6J and BALB/cJ mice after cigarette smoke exposure. Thus, the 129X1/SvJ strain is most suitable in simulating smoking-induced impaired fracture healing. Furthermore, in smoking 129X1/SvJ murine models, we investigated the molecular and cellular alterations in fracture healing caused by cigarette smoking using histology, flow cytometry, and multiplex cytokine/chemokine analysis. Histological analysis showed impaired chondrogenesis in cigarette smoking. In addition, the important reparative cell populations, including skeletal stem cells and their downstream progenitors, demonstrated decreased expansion after injury as a result of cigarette smoking. Moreover, significantly increased pro-inflammatory mediators and the recruitment of immune cells in fracture hematomas were demonstrated in smoking mice. Collectively, our findings demonstrate the significant cellular and molecular alterations during fracture healing impaired by smoking, including disrupted chondrogenesis, aberrant skeletal stem and progenitor cell activity, and a pronounced initial inflammatory response. © 2020 American Society for Bone and Mineral Research (ASBMR).

Inflammatory conversion of quiescent osteoblasts by metastatic breast cancer cells through pERK1/2 aggravates cancer-induced bone destruction.

Disruption of bone homeostasis caused by metastatic osteolytic breast cancer cells increases inflammatory osteolysis and decreases bone formation, thereby predisposing patients to pathological fracture and cancer growth. Alteration of osteoblast function induces skeletal diseases due to the disruption of bone homeostasis. We observed increased activation of pERK1/2 in osteolytic breast cancer cells and osteoblasts in human pathological specimens with aggressive osteolytic breast cancer metastases. We confirmed that osteolytic breast cancers with high expression of pERK1/2 disrupt bone homeostasis via osteoblastic ERK1/2 activation at the bone-breast cancer interface. The process of inflammatory osteolysis modulates ERK1/2 activation in osteoblasts and breast cancer cells through dominant-negative MEK1 expression and constitutively active MEK1 expression to promote cancer growth within bone. Trametinib, an FDA-approved MEK inhibitor, not only reduced breast cancer-induced bone destruction but also dramatically reduced cancer growth in bone by inhibiting the inflammatory skeletal microenvironment. Taken together, these findings suggest that ERK1/2 activation in both breast cancer cells and osteoblasts is required for osteolytic breast cancer-induced inflammatory osteolysis and that ERK1/2 pathway inhibitors may represent a promising adjuvant therapy for patients with aggressive osteolytic breast cancers by altering the shared cancer and bone microenvironment.

Application of a novel shape-memory alloy concentrator in displaced olecranon fractures: a report of the technique and mid-term clinical results.

PURPOSE: Olecranon fracture is a common upper limb fracture, and several surgical approaches have been advocated for its fixation. To overcome the complications associated with common techniques, we present a novel shape-memory alloy concentrator, an alternative for tension band compression, to fix olecranon fracture. METHODS: Fifty-seven patients (26 men and 31 women) with olecranon fracture, with a mean age of 45 years, were included in this study. Each patient had undergone open reduction and internal fixation using the Nitinol (Ni-Ti) arched shape-memory connector (ASC). The clinical assessments were performed using the Disability of the Arm, Shoulder, and Hand (DASH) questionnaire and the Mayo Elbow Performance (MEP) score, which were both recorded at the final follow-up visit. RESULTS: The patients were followed up for 44 months on average (range, 31 to 56 months). No patients were lost to follow-up, and all of the olecranon fractures healed in an average of 15 weeks (range, 10 to 34 weeks). The mean DASH score was 8.6 (range, 0 to 32.4), and the mean MEP score was 92.5 (range, 74 to 100). Nine patients showed postoperative complications: prominent hardware (2), infection (1), loss of the range of functional motion (5), and heterotopic ossification (1). CONCLUSION: The ASC may serve as a favorable device for multi-fragmented and comminuted fractures with rare hardware irritation and may also provide continuous concentrative compression to accelerate osseous healing, thereby aiding the restoration and permitting an early rehabilitation with a low incidence of postoperative complications.

Combination of mini locking plate and nitinol arched shape-memory connector for purely lateral malleolus fractures: technique and clinical results.

BACKGROUND: Lateral malleolus fractures occur frequently. The common techniques for fixing purely lateral malleolus fractures are often challenging, owing to the extent of soft tissue damage and fracture non-union. Herein, we report a new treatment that entails minimally invasive insertion and continuous compression of the broken ends as a novel technique, and evaluate its clinical results. METHODS: This study enrolled 21 patients (13 males and 8 females; mean age 32.06±3.45 years, range 23-69 years) with purely lateral malleolus fractures. Each patient underwent open reduction treatment with a mini locking plate for internal fixation and compression of the fracture end with an Arched Shape-Memory Connector (ASC). The clinical assessments were made using the American Orthopedic Foot and Ankle Society (AOFAS) scores, which were recorded at the final follow-up visit. RESULTS: The patients were followed for an average of 14.7±1.2 months (range, 12-18 months). None of the patients showed surgical failure, and all of the purely lateral malleolus fractures healed in an average of 12.6±1.5 weeks (range, 10-16 weeks). The mean AOFAS score was 88.94 (range, 83-90). CONCLUSIONS: The new treatment had beneficial outcomes for purely lateral malleolus fractures. Mini locking plates are minimally invasive for surgical intervention, and combined with continuous concentrated compression with an ASC to accelerate osseous healing, they aid in restoration of function and enable early rehabilitation with a low incidence of postoperative complications.

Avoiding Limb-Length Discrepancy with Reconstruction of a Massive Tibial Defect Using a Bone Allograft and a Minimally Invasive Lengthening System in a Pediatric Patient: A Case Report.

CASE: A 7-year-old boy was found to have Ewing sarcoma of the left tibia. The sarcoma was resected, and the defect was reconstructed using a humeral head allograft and intramedullary limb-lengthening nail. CONCLUSIONS: Limb-salvage reconstruction in children can be complicated by the sacrifice of epiphyseal plates and limb-length discrepancies and thus requires techniques tailored to each case.

Systemic Parathyroid Hormone Enhances Fracture Healing in Multiple Murine Models of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a multisystemic disease that afflicts more than 415 million people globally-the incidence and prevalence of T2DM continues to rise. It is well-known that T2DM has detrimental effects on bone quality that increase skeletal fragility, which predisposes subjects to an increased risk of fracture and fracture healing that results in non- or malunion. Diabetics have been found to have perturbations in metabolism, hormone production, and calcium homeostasis-particularly PTH expression-that contribute to the increased risk of fracture and decreased fracture healing. Given the perturbations in PTH expression and the establishment of hPTH (1-34) for use in age-related osteoporosis, it was determined logical to attempt to ameliorate the bone phenotype found in T2DM using hPTH (1-34). Therefore, the present study had two aims: (i) to establish a suitable murine model of the skeletal fragility present in T2DM because no current consensus model exists; and (ii) to determine the effects of hPTH (1-34) on bone fractures in T2DM. The results of the present study suggest that the polygenic mouse of T2DM, TALLYHO/JngJ, most accurately recapitulates the diabetic osteoporotic phenotype seen in humans and that the intermittent systemic administration of hPTH (1-34) increases fracture healing in T2DM murine models by increasing the proliferation of mesenchymal stem cells. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Application of a Ni-Ti arched shape-memory connector in unstable lateral malleolus fractures: A retrospective study.

PURPOSE: To compare the outcomes of the arched shape-memory connector (ASC) only fixation and the lateral one-third tubular plate fixation in managing unstable Type A or B lateral malleolus fractures according to the Weber (AO) classification, and to evaluate the feasibility and reliability of ASC only fixation in treating these fractures. METHODS: From January 2010 to January 2015, 148 patients with Type A or B (Weber (AO) classification) lateral malleolus fractures treated with the arched shape-memory connector (ASC) only fixation or lateral plate fixation were included. There were 66 patients in the ASC only fixation group and 82 patients in the lateral plate group. Intergroup differences were absent regarding patient and fracture characteristics. The incision length, complete-union time, major complications and complaints, incidence of hardware removal, and final radiographic and functional evaluations were compared. RESULTS: The follow-up time averaged 18.2 months in the ASC fixation group and 17.2 months in the lateral plate group. The ASC only fixation group had significantly decreased wound infection (4.55% versus 14.63%) and skin necrosis (none versus 7.32%). Of patients who underwent ASC only fixation 3.03% reported lateral ankle pain, 7.58% received palpable hardware, and 3.03% reported hardware irritation, while the corresponding observations in the lateral plate group were 19.51%, 54.88%, and 14.63%, respectively, representing a statistical difference. Furthermore, compared with the lateral plate group, the incidence of hardware removal was markedly lower in the ASC fixation group (12.12% versus 30.49%). In terms of reduction accuracy, complete-union time, and AOFAS scores, no appreciable differences were observed. CONCLUSIONS: ASC only fixation is a reliable alternative for managing Type A or B lateral malleolus fractures, leading to fewer soft tissue complications, fewer hardware complaints, and a reduced need for hardware removal, and a reduced need for hardware removal. In addition, ASC can be used for augmented plate fixation in certain comminuted fracture patterns.

Exosomes from human umbilical cord mesenchymal stem cells enhance fracture healing through HIF-1α-mediated promotion of angiogenesis in a rat model of stabilized fracture.

OBJECTIVES: Exosomes, as important players in intercellular communication due to their ability to transfer certain molecules to target cells, are believed to take similar effects in promoting bone regeneration with their derived stem cells. Studies have suggested that umbilical cord mesenchymal stem cells (uMSCs) could promote angiogenesis. This study investigated whether exosomes derived from uMSCs (uMSC-Exos) could enhance fracture healing as primary factors by promoting angiogenesis. MATERIALS AND METHODS: uMSCs were obtained to isolate uMSC-Exos by ultrafiltration, with exosomes from human embryonic kidney 293 cells (HEK293) and phosphate-buffered saline (PBS) being used as control groups. NanoSight, laser light scattering spectrometer, transmission electron microscopy and Western blotting were used to identify exosomes. Next, uMSC-Exos combined with hydrogel were transplanted into the fracture site in a rat model of femoral fracture. Bone healing processes were monitored and evaluated by radiographic methods on days 7, 14, 21 and 31 after surgery; angiogenesis of the fracture sites was assessed by radiographic and histological strategies on post-operative day 14. In vitro, the expression levels of osteogenesis- or angiogenesis-related genes after being cultured with uMSC-Exos were identified by qRT-PCR. The internalization ability of exosomes was determined using the PKH67 assay. Cell cycle analysis, EdU incorporation and immunofluorescence staining, scratch wound assay and tube formation analysis were also used to determine the altered abilities of human umbilical vein endothelial cells (HUVECs) administered with uMSC-Exos in proliferation, migration and angiogenesis. Finally, to further explore the underlying molecular mechanisms, specific RNA inhibitors or siRNAs were used, and the subsequent effects were observed. RESULTS: uMSC-Exos had a diameter of approximately 100 nm, were spherical, meanwhile expressing CD9, CD63 and CD81. Transplantation of uMSC-Exos markedly enhanced angiogenesis and bone healing processes in a rat model of femoral fracture. In vitro, other than enhancing osteogenic differentiation, uMSC-Exos increased the expression of vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1α (HIF-1α). uMSC-Exos were taken up by HUVECs and enhanced their proliferation, migration and tube formation. Finally, by using specific RNA inhibitors or siRNAs, it has been confirmed that HIF-1α played an important role in the uMSC-Exos-induced VEGF expression, pro-angiogenesis and enhanced fracture repair, which may be one of the underlying mechanisms. CONCLUSIONS: These results revealed a novel role of exosomes in uMSC-mediated therapy and suggested that implanted uMSC-Exos may represent a crucial clinical strategy to accelerate fracture healing via the promotion of angiogenesis. HIF-1α played an important role in this process.

[Comparison of biological characteristics between bone marrow mesenchymal stem cells and anterior cruciate ligament derived mesenchymal stem cells in rats].

Objective: To compare the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and anterior cruciate ligament derived mesenchymal stem cells (ACL-MSCs) from rats in vitro. Methods: Ten male SPF-level BN rats, weighing 200-220 g, were selected to obtain anterior cruciate ligaments and bone marrows, and ACL-MSCs and BMSCs were isolated for passage culture respectively under sterile condition. The cell morphology was observed, and the cells at passage 3 were used to detect the surface markers of CD34, CD45, CD90, and CD29 by flow cytometry, the ability of cell proliferation by cell counting kit 8 (CCK-8), and colony formation ability by clone forming test. The mRNA levels of differentiation related genes [alkaline phosphatas (ALP), bone gamma-carboxyglutamate protein, runt related transcription factor 2, bone morphogenetic protein 2 (BMP-2), secreted phosphoprotein 1 (Spp1), collagen type II α1 (Col2α1), Aggrecan (Acan), Sox9, peroxisome proliferator activated receptor γ2 (PPARγ2), and CCAAT-enhancer-binding protein-α] were also determined by real-time fluorescent quantitative PCR. Results: BMSCs and ACL-MSCs had similar morphology, adherent cells displaying long fusiform. The immunoprofile of ACL-MSCs and BMSCs at passage 3 was positive for CD29 and CD90 and was negative for CD45 and CD34. The absorbance ( A) value of ACL-MSCs (1.11±0.08) was significantly higher than that of BMSCs (0.78±0.05) ( t=3.599, P=0.023); the number of colonies of ACL-MSCs [(53.00±5.51)/hole] was significantly more than that of BMSCs [(30.67±4.84)/hole] ( t=3.045, P=0.038). The results of toluidine blue staining, alizarin red staining, and oil red O staining were positive in BMSCs and ACL-MSCs at 21 days after osteogenic, chondrogenic, and adipogenic induction. The mRNA expressions of BMP-2, Spp1, Col2α1, Acan, Sox9, and PPARγ2 in ACL-MSCs were significantly higher than those in BMSCs ( P<0.01). Conclusion: The proliferation potential of ACL-MSCs is greater than that of BMSCs, and the former is apt to differentiate into chondrocytes. ACL-MSCs are promising cells to promote tendon-bone healing.

Stem cell-derived exosomes: A promising strategy for fracture healing.

OBJECTIVES: To describe the biological characteristics of exosomes and to summarize the current status of stem cell-derived exosomes on fracture healing. Meanwhile, future challenges, limitations and perspectives are also discussed. METHODS: Search and analyze the related articles in pubmed database through the multi-combination of keywords like "stem cells","exosomes","bone regeneration" and "fracture healing". CONCLUSION: Stem cell-derived exosome therapy for fracture healing has been enjoying popularity and is drawing increasing attention. This strategy helps to promote proliferation and migration of cells, as well as osteogenesis and angiogenesis, in the process of bone formation. Although the exact mechanisms remain elusive, exosomal miRNAs seem to play vital roles. Future studies are required to solve multiple problems before clinical application, including comprehensive and thorough understanding of exosomes, the exact roles of exosomes in regulating bone formation, and the optimal source, dose and frequency of treatment, as well as technical and safety issues. Moreover, studies based on fracture models of large animals are could offer guidance and are in demand.

Stem cell therapy: a promising biological strategy for tendon-bone healing after anterior cruciate ligament reconstruction.

Tendon-bone healing after anterior cruciate ligament (ACL) reconstruction is a complex process, impacting significantly on patients' prognosis. Natural tendon-bone healing usually results in fibrous scar tissue, which is of inferior quality compared to native attachment. In addition, the early formed fibrous attachment after surgery is often not reliable to support functional rehabilitation, which may lead to graft failure or unsatisfied function of the knee joint. Thus, strategies to promote tendon-bone healing are crucial for prompt and satisfactory functional recovery. Recently, a variety of biological approaches, including active substances, gene transfer, tissue engineering and stem cells, have been proposed and applied to enhance tendon-bone healing. Among these, stem cell therapy has been shown to have promising prospects and draws increasing attention. From commonly investigated bone marrow-derived mesenchymal stem cells (bMSCs) to emerging ACL-derived CD34+ stem cells, multiple stem cell types have been proven to be effective in accelerating tendon-bone healing. This review describes the current understanding of tendon-bone healing and summarizes the current status of related stem cell therapy. Future limitations and perspectives are also discussed.