Boron Neutron Capture Therapy-Derived Extracellular Vesicles via DNA Accumulation Boost Antitumor Dendritic Cell Vaccine Efficacy.

Radiated tumor cell-derived extracellular vesicles (RT-EVs) encapsulate abundant DNA fragments from irradiated tumor cells, in addition to acting as integrators of multiple tumor antigens. Accumulating evidence indicates these DNA fragments from damaged cells are involved in downstream immune responses, but most of them are degraded in cells before incorporation into derived RT-EVs, thus the low abundance of DNA fragments limits immune responses of RT-EVs. Here, this study found that different radiations affected fates of DNA fragments in RT-EVs. Boron neutron capture therapy (BNCT) induced DNA accumulation in RT-EVs (BEVs) by causing more DNA breaks and DNA oxidation resisting nuclease degradation. This is attributed to the high-linear energy transfer (LET) properties of alpha particles from the neutron capture reaction of 10B. When being internalized by dendritic cells (DCs), BEVs activated the DNA sensing pathway, resulting in functional enhancements including antigen presentation, migration capacity, and cytokine secretion. After vaccination of the BEVs-educated DCs (BEV@BMDCs), the effector T cells significantly expanded and infiltrated into tumors, suggesting robust anti-tumor immune activation. BEV@BMDCs not only effectively inhibited the primary tumor growth and metastasis formation but also elicited long-term immune memory. In conclusion, a successful DC vaccine is provided as a promising candidate for tumor vaccine.

Boron-Containing Mesoporous Silica Nanoparticles with Effective Delivery and Targeting of Liver Cancer Cells for Boron Neutron Capture Therapy.

Nanocarriers have been researched comprehensively for the development of novel boron-containing agents in boron neutron capture therapy (BNCT). We designed and synthesized a multifunctional mesoporous silica nanoparticle (MSN)-based boron-containing agent. The latter was coated with a lipid bilayer (LB) and decorated with SP94 peptide (SFSIIHTPILPL) on the surface as SP94-LB@BA-MSN. The latter incorporated boric acid (BA) into hydrophobic mesopores, coated with an LB, and modified with SP94 peptide on the LB. SP94-LB@BA-MSN enhanced nano interface tumor-targeting ability but also prevented the premature release of drugs, which is crucial for BNCT because adequate boron content in tumor sites is required. SP94-LB@BA-MSN showed excellent efficacy in the BNCT treatment of HepG-2 cells. In animal studies with tumor-bearing mice, SP94-LB@BA-MSN exhibited a satisfactory accumulation at the tumor site. The boron content reached 40.18 ± 5.41 ppm in the tumor site 4 h after injection, which was 8.12 and 15.51 times higher than those in mice treated with boronated phenylalanine and those treated with BA. For boron, the tumor-to-normal tissue ratio was 4.41 ± 1.13 and the tumor-to-blood ratio was 5.92 ± 0.45. These results indicated that nanoparticles delivered boron to the tumor site effectively while minimizing accumulation in normal tissues. In conclusion, this composite (SP94-LB@BA-MSN) shows great promise as a boron-containing delivery agent for the treatment of hepatocellular carcinoma using BNCT. These findings highlight the potential of MSNs in the field of BNCT.

Boron-Containing MOF Nanoparticles with Stable Metabolism in U87-MG Cells Combining Microdosimetry To Evaluate Relative Biological Effectiveness of Boron Neutron Capture Therapy.

Accurate prediction of the relative biological effectiveness (RBE) of boron neutron capture therapy (BNCT) is challenging. The therapy is different from other radiotherapy; the dynamic distribution of boron-containing compounds in tumor cells affects the therapeutic outcome considerably and hampers accurate measurement of the neutron-absorbed dose. Herein, we used boron-containing metal-organic framework nanoparticles (BMOFs) with high boron content to target U87-MG cells and maintain the concentration of the 10B isotope in cells. The content of boron in the cells could maintain 90% (60 ppm) within 20 min compared with that at the beginning; therefore, the accurate RBE of BNCT can be acquired. The effects of BNCT upon cells after neutron irradiation were observed, and the neutron-absorbed dose was obtained by Monte Carlo simulations. The RBE of BMOFs was 6.78, which was 4.1-fold higher than that of a small-molecule boron-containing agent (boric acid). The energy spectrum of various particles was analyzed by Monte Carlo simulations, and the RBE was verified theoretically. Our results suggested that the use of nanoparticle-based boron carriers in BNCT may have many advantages and that maintaining a stable boron distribution within cells may significantly improve the efficiency of BNCT.

3D culture boosting fullerenol nanoparticles to induce calreticulin exposure on MCF-7 cells for enhanced macrophage-mediated cell removal.

Calreticulin (CRT) on the cell surface that acts as an "eat me" signal is vital for macrophage-mediated programmed cell removal. The polyhydroxylated fullerenol nanoparticle (FNP) has appeared as an effective inducer to cause CRT exposure on cancer cell surface, but it failed in treating some cancer cells such as MCF-7 cells based on previous findings. Here, we carried out the 3D culture of MCF-7 cells, and interestingly found that the FNP induced CRT exposure on cells in 3D spheres via re-distributing CRT from endoplasmic reticulum (ER) to cell surface. Phagocytosis experiments in vitro and in vivo illustrated the combination of FNP and anti-CD47 monoclonal antibody (mAb) further enhanced macrophage-mediated phagocytosis to cancer cells. The maximal phagocytic index in vivo was about three times higher than that of the control group. Moreover, in vivo tumorigenesis experiments in mice proved that FNP could regulate the progress of MCF-7 cancer stem-like cells (CSCs). These findings expand the application of FNP in tumor therapy of anti-CD47 mAb and 3D culture can be used as a screening tool for nanomedicine.

Fabricated AIE-Based Probe to Detect the Resistance to Anoikis of Cancer Cells Detached from Tumor Tissue.

(1) Background: Resisting anoikis is a vital and necessary characteristic of malignant cancer cells, but there is no existing quantification method. Herein, a sensitive probe for assessing anoikis resistance of cancer cells detached from the extracellular matrix was developed based on the aggregation-induced emission (AIE) of AIEgens. It has been reported that detached cancer cell endocytose activated integrin clusters, and in the endosome these clusters recruit and activate phosphorylate focal adhesion kinase (pFAK) in the cytoplasm to induce signaling that supports the growth of detached cancer cells. (2) Methods: We established a lost nest cell model of cancer cells and determined their ability to resist anoikis. The colocalization of the activated integrin, pFAK, and endosomes in model cells was observed and calculated. (3) Results: The fluorescence signal intensity of the probe was significantly higher than that of the integrin antibody in the model cells and the fluorescence signal of probe signal was better overlapped with labeled pFAK by fluorescence in endosomes in model cells. (4) Conclusions: We developed a quantitative multi-parametric image analysis program to calculate fluorescent intensity of the probe and antibodies against pFAK and Rab5 in the areas of colocalization. A positive correlation of fluorescence signal intensity between the probe and pFAK on the endosome was observed. Therefore, the probe was used to quantitatively evaluate resisting anoikis of different cancer cell lines under the lost nest condition.

Oral Administration of Omega-3 Fatty Acids Attenuates Lung Injury Caused by PM2.5 Respiratory Inhalation Simply and Feasibly In Vivo.

For developing an effective interventional approach and treatment modality for PM2.5, the effects of omega-3 fatty acids on alleviating inflammation and attenuating lung injury induced by inhalation exposure of PM2.5 were assessed in murine models. We found that daily oral administration of the active components of omega-3 fatty acids, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) effectively alleviated lung parenchymal lesions, restored normal inflammatory cytokine levels and oxidative stress levels in treating mice exposed to PM2.5 (20 mg/kg) every 3 days for 5 times over a 14-day period. Especially, CT images and the pathological analysis suggested protective effects of DHA and EPA on lung injury. The key molecular mechanism is that DHA and EPA can inhibit the entry and deposition of PM2.5, and block the PM2.5-mediated cytotoxicity, oxidative stress, and inflammation.

The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au25 Probes.

Triple-negative breast cancer (TNBC) accounts for nearly one-quarter of all breast cancer cases, but effective targeted therapies for this disease remain elusive because TNBC cells lack the expression of the most common three receptors seen in other subtypes of breast cancers. The medium-term diagnosis of breast cancers is essential for development and prognosis. According to reports, patients with TNBC may be converted to a positive epidermal growth factor receptor 2(HER-2) after chemotherapy, and trastuzumab treatment will have a better prognosis. Therefore, it is important to accurately quantify the expression of HER-2 in breast cancer cells. Herein, we design a red fluorescent Au25 probe synthesized with BSA-biotin as the ligand, which is accurately quantified by HER-2 primary antibody-biotin using the avidin system. The quantitative detection of the expression of HER-2 in breast cancers is helpful for the companion diagnostic of breast cancer treatment and provides follow-up treatment.

Cyclic Peptide Modified Gold Clusters Induce Lung Tumor Cell Apoptosis via Generating Intracellular Oxidative Stress.

Metastatic lung cancer is the leading cause of death for cancer patients. Although many chemical drugs were developed for cancer treatment, metastatic cancer mortality did not decrease significantly. In this article, we designed an Au clusters (AuCs) modified by cyclic RGD peptides which well target the integrin of human lung carcinoma cells (A549). The RGD-AuCs could well induce A549 cells apoptosis, but have no cytotoxicity on the human bronchial epithelial cells (16HBE), which are normal cells support respiratory system. The AuCs could be internalized and localized in the lysosomes of A549 tumor cells and further release into cytoplasma. We found the ROS level was increased by AuCs, and such high ROS level finally leads to depolarization of mitochondria. Eventually, the AuCs stimulating mitochondria related apoptosis pathway to induce A549 tumor cells apoptosis. We deduce the gold clusters would be an effective therapeutic candidate to against metastatic lung tumor in the future studies.

The Aggregation Induced Emission Probe of Detecting Enhanced Permeation and Retention Effects is Structured for Evaluating the Applicability of Nanotherapy to Different Tumor Individuals.

Enhanced permeation and retention (EPR) effect, the mechanism by which nanodrugs accumulate in tumors and acquire superior curative effect. The questions of these mechanisms occur because of limited clinical transformation of engineered nanomaterials after 30 years. The difference of EPR limits the therapeutic effect of nanodrugs in the individual patient. Evaluation of the EPR effect in the individual patient will aid in selecting patients who will accumulate higher amounts of nanotherapeutics and show better therapeutic efficacy. Based on varied TIMP1/MMP-9 in serum, an aggregation-induced emission luminogen probe was designed and constructed to detect and evaluate the EPR effect in model mouse. The result showed that the ratio of TIMP1/MMP-9 (in the range 0.2-1.2) and fluorescence intensity of the probe were negative linear correlation and the effects of BSA-rhodamine accumulation in tumor were individualized differences as well as correlated with the relative ratio of TIMP-1/MMP-9 in serum. Our data support the development of these biomarkers probes based on the personalized nanotherapy of tumor.

Trp2 Peptide-Assembled Nanoparticles with Intrinsically Self-Chelating 64Cu Properties for PET Imaging Tracking and Dendritic Cell-Based Immunotherapy against Melanoma.

Dendritic cell-based immunotherapy, in which the antigen is effectively delivered to dendritic cells and then the dendritic cells stimulated by the antigen migrate to draining lymph nodes (DLNs) to induce the CD8+ T-cell immune response, shows great promise for tumor immunotherapy. In this study, we used coassembled nanoparticles formed by Trp2 antigen and the conjugates of short-chain poly(ethylene glycol) (PEG) and pyropheophorbide-A (PPa) (Trp2/PPa-PEGm) to deliver Trp2 to DCs. Intrinsically self-chelating 64Cu of coassemblies could be used to sensitively image the migration of DCs in vivo by positron emission tomography (PET) imaging. The coassemblies of the Trp2 antigen were efficiently engulfed by DCs without causing DC cytotoxicity in vitro and induced DC maturation. After injection of DCs labeled by coassemblies of the Trp2 antigen, the homing of DCs to DLNs in vivo could be sensitively observed by PET imaging. The C57BL/6 mice injected with DCs containing the Trp2/PPa-PEGm NP showed antigen-specific immune responses including enhanced interferon-γ (IFN-γ) production, splenocyte proliferation, and percentage of IFN-γ-secreting CD8+ T cells. In addition, C57BL/6 mice inoculated with B16-F10 tumor cells showed delayed tumor growth after immunization with the Trp2/PPa-PEGm NP-labeled DC vaccine and enhanced infiltration of CD8+ T cells in tumors.

Intrinsic Biotaxi Solution Based on Blood Cell Membrane Cloaking Enables Fullerenol Thrombolysis In Vivo.

We report the construction of blood cell membrane cloaked mesoporous silica nanoparticles for delivery of nanoparticles [fullerenols (Fols)] with fibrinolysis activity which endows the active Fol with successful thrombolysis effect in vivo. In vitro, Fols present excellent fibrinolysis activity, and the Fol with the best fibrinolysis activity is screened based on the correlation between Fols' structure and their fibrinolysis activity. However, the thrombolytic effect in vivo is not satisfactory. To rectify the unsatisfactory situation and avoid the exogenous stimuli, a natural blood cell membrane cloaking strategy with loading the active Fol is chosen to explore as a novel thrombolysis drug. After cloaking, the therapeutic platform prolongs blood circulation time and enhances the targeting effect. Interestingly, compared with platelet membrane cloaking, red blood cell (RBC) membrane cloaking demonstrates stronger affinity with fibrin and more enrichment at the thrombus site. The Fol with RBC cloaking shows quick and efficient thrombolysis efficacy in vivo with less bleeding risk, more excellent blood compatibility, and better biosafety when compared with the clinical drug urokinase (UK). These findings not only validate the blood cell membrane cloaking strategy as an effective platform for Fol delivery on thrombolysis treatment, but also hold a great promising solution for other active nanoparticle deliveries in vivo.

Microfluidic Analysis for Separating and Measuring the Deformability of Cancer Cell Subpopulations.

Increased deformability and softness endow tumor cells with highly invasive and metastatic capabilities. We exploited these characteristics to fabricate a high-throughput microfluidic device to measure cell deformability and separate cancer cells. Driven by hydrodynamic forces, the cells with better deformability passed through the chip faster, whereas stiffer cells passed through the device over a longer time period. The MDA-MB-231 and MCF-7 cell lines were used to evaluate the device because their metastatic potentials were known. We found that MDA-MB-231 cells, which were softer and exhibited stronger deformability, passed through the device more quickly. HeLa cells were also successfully separated into softer and stiffer subpopulations, whose distinct mechanical properties were confirmed by atomic force microscopy. We also measured the expression of metastasis-associated proteins (epidermal growth factor receptor and integrin ß 1) and found that subpopulations with varied deformabilities had different expression levels. Our results suggested that this high-throughput microfluidic device could be used to screen and evaluate the curative effects of drug and cancer progression by simultaneously testing cell deformability and expression levels of metastasis-associated proteins in separated cell subpopulations.

Exploring the Interaction of Fullerenol with Key Digestive Proteases Using Raman-Based Frequency-Shift Sensing and Molecular Simulation Analysis.

Fullerenol has been demonstrated to be a potential anticancer nanodrug thanks to its excellent antioxidant properties. In this work, surface-enhanced Raman scattering (SERS)-based frequency shift method is employed to explore the interaction between fullerenol and the key digestive proteases (pepsin and trypsin) in the gastrointestinal tract. A dynamic adsorption process of fullerenol on pepsin/trypsin has been monitored by our detection platform. The binding sites of fullerenol to trypsin and pepsin are evidenced by introducing their inhibitors to the detection system, and the binding configurations/modes of fullerenol to pepsin and trypsin are precisely determined through computational simulations. The permeability of fullerenol through intestinal epithelial cells monolayer is also investigated and the results show that it can reach 6% in 6 h and 13% in 48 h, indicating its high oral bioavailability in human body. This work demonstrates the activity of fullerenol in the gastrointestinal tract and provides potential guidance for appropriate oral medicine design with the aid of a molecular docking method.

Au Nanoparticles Attenuate RANKL-Induced Osteoclastogenesis by Suppressing Pre-Osteoclast Fusion.

Osteoclasts are multinucleated terminal cells that originate from a hematopoietic monocyte/macrophage lineage. Excessive osteoclast formation in vivo can lead to bone metabolic diseases such as postmenopausal osteoporosis, multiple myeloma, rheumatoid arthritis, and lytic bone metastases of cancer cells. Au nanoparticles (AuNPs) are inorganic nanoparticles with outstanding biocompatibility. We assessed their effect on osteoclastogenesis and found that pre-osteoclast fusion induced by receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colonystimulating factor (M-CSF) was suppressed by AuNPs. Cell migration and actin ring formation were also significantly inhibited. Finally, AuNPs reduced osteoclast bone absorption function. Interestingly, we observed altered fusogenic gene expression in treated pre-osteoclasts. Our results suggest that AuNPs have potential as a therapeutic agent for osteoclast-related bone metabolism diseases.

Nanoparticles with High-Surface Negative-Charge Density Disturb the Metabolism of Low-Density Lipoprotein in Cells.

Endocytosis is an important pathway to regulate the metabolism of low-density lipoprotein (LDL) in cells. At the same time, engineering nanoparticles (ENPs) enter the cell through endocytosis in biomedical applications. Therefore, a crucial question is whether the nanoparticles involved in endocytosis could impact the natural metabolism of LDL in cells. In this study, we fabricated a series of gold nanoparticles (AuNPs) (13.00 ± 0.69 nm) with varied surface charge densities. The internalized AuNPs with high-surface negative-charge densities (HSNCD) significantly reduced LDL uptake in HepG-2, HeLa, and SMMC-7721 cells compared with those cells in control group. Notably, the significant reduction of LDL uptake in cells correlates with the reduction of LDL receptors (LDL-R) on the cell surface, but there is no change in protein and mRNA of LDL-Rs. The cyclic utilization of LDL-R in cells is a crucial pathway to maintain the homoeostasis of LDL uptake. The release of LDL-Rs from LDL/LDL-R complexes in endosomes depended on reduction of the pH in the lumen. AuNPs with HSNCD hampered vacuolar-type H⁺-ATPase V1 (ATPaseV1) and ATPaseV0 binding on the endosome membrane, blocking protons to enter the endosome by the pump. Hence, fewer freed LDL-Rs were transported into recycling endosomes (REs) to be returned to cell surface for reuse, reducing the LDL uptake of cells by receptor-mediated endocytosis. The restrained LDL-Rs in the LDL/LDL-R complex were degraded in lysosomes.

Fluorescent activatable gadofullerene nanoprobes as NIR-MR dual-modal in vivo imaging contrast agent.

Dual mode imaging technology is widely developed to achieve the early-stage precision cancer diagnosis. Here we designed a dual-modal magnetic resonance/near infrared fluorescence optical imaging contrast agent (GdF-SS-NIR783) with the fluorescence activatable and safer gadofullerene. The nanoprobes were fabricated by conjugating the gadofullerene derivatives with a NIR fluorescence imaging agent (NIR783) via the disulfide bond. The obtained nanoprobes showed no fluorescence (OFF), but the fluorescence turned on when incubated within reduction environment such as GSH solution. The clear fluorescence signal in tumor site was observed obviously after their intratumor injection. The nanoprobes also revealed efficient MRI contrast enhancement both in vitro and in vivo. And they showed good biocompatibility and did not demonstrate any tissue toxicity in vivo. This work gave the new possibility in designing more efficient and safer nanoprobes for future medical diagnoses.

Small size fullerenol nanoparticles suppress lung metastasis of breast cancer cell by disrupting actin dynamics.

BACKGROUND: Tumor metastasis is the primary cause of mortality in cancer patients. Migratory breast cancer cells in lymphatic and blood vessels seek new sites and form metastatic colonies in the lung and bone, and then these cancer cells often wreak considerable havoc. With advances in nanotechnology, nanomaterials and nanotechnologies are widely applied in tumor therapy. In this paper, small size fullerenol nanoparticles, which are separated by isoelectric focusing electrophoresis (IFE) for discrepancy of isoelectric point (pI), are used in the study of tumor metastasis. RESULTS: In this study, the commendable inhibition of tumor metastasis was uncovered by intravenous injection of purified fullerenol fraction with special surface charge and functional groups, which was separated by IFE for discrepancy of pI. By investigating the actin dynamics in several cancer cell lines, we found these small size fullerenol nanoparticles disturbed actin dynamics. Young's modulus detection and cell migration assays revealed that fullerenol lowered stiffness and restrained migration of breast cancer cells. Filopodia, the main supporting structures of actin bundles, are important for cell motility and adhesion. Scanning electron microscopy showed that fullerenol reduced the number and length of filopodia. Simultaneously, the inhibition of integrin to form clusters on filopodias, which was likely induced by reorganizing of actin cytoskeleton, impacted cancer cell adhesion and motility. CONCLUSIONS: With intravenous injection of these fullerenol nanoparticles, tumor metastasis is well inhibited in vivo. The underlying mechanism most likely to be attributed to the effect of fullerenol nanoparticles on disturbing actin dynamics. With the disordered actin fiber, cell function is varied, including decreased cell stiffness, reduced filopodia formation, and inactivated integrin.

Effects of Fullerenol Nanoparticles on Rat Oocyte Meiosis Resumption.

The excellent biocompatibility and biological effects of fullerenol and its derivatives make their biomedical application promising. The potential effects of fullerenol in mammals have been extensively studied, but little is known about its effects on female reproduction. Using canonical oocyte-granulosa cell complexes (OGCs) in vitro maturation culture model, we investigated the effect of fullerenol on the first oocyte meiotic resumption. In the surrounding granulosa cells, fullerenol nanoparticles occluded the extracellular domain of the epidermal growth factor receptor (EGFR) to reduce EGFR-ligand binding and subsequent extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation, which involved the regulation of connexin 43 (CX43) expression and internalization. Downregulation of CX43 expression and the retraction of transzonal projections (TZPs) interrupted the gap junction channel and TZPs based mass transportation. This effect decreased cyclic adenosine monophosphate (cAMP) levels in the oocyte and thereby accelerated rat oocyte meiosis resumption. Moreover, perinuclear distribution of CX43 and EGFR was observed in granulosa cells, which could further exacerbate the effects. Fullerenol nanoparticles interfered with the strict process of oocyte meiosis resumption, which likely reduced the oocyte quality.

Quantitative Analysis of Multiple Proteins of Different Invasive Tumor Cell Lines at the Same Single-Cell Level.

Tumor cell invasion is pivotal to the development, metastasis, and prognosis of tumors. It is reported that the invasive ability of tumor cells is mainly dependent on the expression levels of membrane type-1 matrix metalloproteinase (MT1-MMP) and integrin αV ß3 proteins on cell membranes. To precisely distinguish between tumor cells with different invasive abilities, it is important to establish a highly sensitive and precise quantification method to differentiate the expression levels of MT1-MMP and integrin αV ß3 in the same single tumor cell at the same time. Herein, two functional peptides to construct red-emissive Au26 clusters and green-emissive Ag12 clusters are reported. Moreover, the Au26 clusters and Ag12 clusters have the ability to specifically target MT1-MMP and integrin αV ß3 , respectively, in the same single cell at the same time. By utilizing the fluorescent properties and metallic compositions of metal clusters, the MT1-MMP and integrin αV ß3 levels of the more invasive SiHa cells or the less invasive HeLa cells are simultaneously and quantitatively differentiated via laser ablation inductively coupled plasma mass spectrometry. This method of quantitatively detecting multiple invasive proteins on the same cell is of great value for accurately diagnosing aggressive tumors and monitoring the invasiveness of these tumors.

Extracorporeal Shock Wave Rebuilt Subchondral Bone In Vivo and Activated Wnt5a/Ca2+ Signaling In Vitro.

BACKGROUND: This study aimed to identify the optimal extracorporeal shock wave (ESW) intensity and to investigate its effect on subchondral bone rebuilt in vivo and Wnt5a/Ca2+ signaling in vitro using an osteoarthritis (OA) rat model and bone marrow mesenchymal stem cells (BMMSCs), respectively. METHODS: OA rats treated with (OA + ESW group) or without (OA group) ESW (n = 12/group) were compared with healthy controls (control group, n = 12). Gait patterns and subchondral trabecular bone changes were measured. Western blot and quantitative real-time polymerase chain reaction detected protein expression and gene transcription, respectively. RESULTS: The gait disturbances of OA + ESW group were significantly improved compared with the OA group at 6th and 8th weeks. The micro-CT analysis indicated that the BMD, BSV/BV, BV/TV, Tr.S, and Tr.Th are significantly different between OA group and OA + ESW group. Expression of Wnt5a was increased rapidly after ESW treatment at 0.6 bar and peaked after 30 min. CONCLUSIONS: ESW were positive for bone remodeling in joint tibial condyle subchondral bone of OA rat. ESW prevented histological changes in OA and prevented gait disturbance associated with OA progression. Optimal intensity of ESW induced changes in BMMSCs via activation of the Wnt5a/Ca2+ signaling pathway.