Farrerol suppresses osteoclast differentiation and postmenopausal osteoporosis by inhibiting the nuclear factor kappa B signaling pathway.

Excessive bone resorption caused by upregulated osteoclast activity is a key factor in osteoporosis pathogenesis. Farrerol is a typical natural flavanone and exhibits various pharmacological actions. However, the role and mechanism of action of farrerol in osteoclast differentiation regulation remain unclear. This study aimed to evaluate the effects and mechanism of farrerol on the inhibition of osteoclastogenesis. Tartrate-resistant acid phosphatase staining, F-actin staining, and the pit formation assay were performed to examine the differentiation and functions of osteoclasts in vitro. The expression of proteins associated with the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways was analyzed by western blotting. Dual X-ray absorptiometry, microcomputed tomography, and histopathological and immunohistochemical analyses were performed to determine the therapeutic effect of farrerol in vivo bone loss prevention. The effects of farrerol on osteoblastic bone formation were assessed using alkaline phosphatase, alizarin red S staining, and calcein-alizarin red S double labeling. Farrerol inhibited osteoclastogenesis and bone resorption in osteoclasts by suppressing nuclear factor kappa B signaling rather than mitogen-activated protein kinase signaling in vitro. Farrerol protected mice against ovariectomy-induced bone loss by inhibiting osteoclast-mediated bone resorption, instead of promoting osteoblast-mediated bone formation in vivo. The findings of the current study revealed that farrerol is a potential therapeutic agent for osteoporosis.

Ferroptosis in Osteocytes as a Target for Protection Against Postmenopausal Osteoporosis.

Ferroptosis is a necrotic form of iron-dependent regulatory cell death. Estrogen withdrawal can interfere with iron metabolism, which is responsible for the pathogenesis of postmenopausal osteoporosis (PMOP). Here, it is demonstrated that estrogen withdrawal induces iron accumulation in the skeleton and the ferroptosis of osteocytes, leading to reduced bone mineral density. Furthermore, the facilitatory effect of ferroptosis of osteocytes is verified in the occurrence and development of postmenopausal osteoporosis is associated with over activated osteoclastogenesis using a direct osteocyte/osteoclast coculture system and glutathione peroxidase 4 (GPX4) knockout ovariectomized mice. In addition, the nuclear factor erythroid derived 2-related factor-2 (Nrf2) signaling pathway is confirmed to be a crucial factor in the ferroptosis of osteocytic cells. Nrf2 regulates the expression of nuclear factor kappa-B ligand (RANKL) by regulating the DNA methylation level of the RANKL promoter mediated by DNA methyltransferase 3a (Dnmt3a), which is as an important mechanism in osteocytic ferroptosis-mediated osteoclastogenesis. Taken together, this data suggests that osteocytic ferroptosis is involved in PMOP and can be targeted to tune bone homeostasis.

IL-1ß contributes to the secretion of sclerostin by osteocytes and targeting sclerostin promotes spinal fusion at early stages.

BACKGROUND: Despite extensive research, there is still a need for safe and effective agents to promote spinal fusion. Interleukin (IL)-1ß is an important factor which influences the bone repair and remodelling. The purpose of our study was to determine the effect of IL-1ß on sclerostin in osteocytes and to explore whether inhibiting the secretion of sclerostin from osteocytes can promote spinal fusion at early stages. METHODS: Small-interfering RNA was used to suppress the secretion of sclerostin in Ocy454 cells. MC3T3-E1 cells were cocultured with Ocy454 cells. Osteogenic differentiation and mineralisation of MC3T3-E1 cells were evaluated in vitro. SOST knock-out rat generated using the CRISPR-Cas9 system and rat spinal fusion model was used in vivo. The degree of spinal fusion was assessed by manual palpation, radiographic analysis and histological analysis at 2 and 4 weeks. RESULTS: We found that IL-1ß level had a positive association with sclerostin level in vivo. IL-1ß promoted the expression and secretion of sclerostin in Ocy454 cells in vitro. Inhibition of IL-1ß-induced secretion of sclerostin from Ocy454 cells could promote the osteogenic differentiation and mineralisation of cocultured MC3T3-E1 cells in vitro. The extent of spinal graft fusion was greater in SOST-knockout rats than in wild-type rats at 2 and 4 weeks. CONCLUSIONS: The results demonstrate that IL-1ß contributes to a rise in the level of sclerostin at early stages of bone healing. Suppressing sclerostin may be an important therapeutic target capable of promoting spinal fusion at early stages.

Exosomes derived from bone marrow mesenchymal stem cells pretreated with decellularized extracellular matrix enhance the alleviation of osteoarthritis through miR-3473b/phosphatase and tensin homolog axis.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative articular disease for which there is no effective treatment. Progress has been made in mesenchymal stem cell (MSC)-based therapy in OA, and the efficacy has been demonstrated to be a result of paracrine exosomes from MSCs. Decellularized extracellular matrix (dECM) provides an optimum microenvironment for the expansion of MSCs. In the present study, we aimed to investigate whether exosomes isolated from bone marrow mesenchymal stem cells (BMSCs) with dECM pretreatment (dECM-BMSC-Exos) enhance the amelioration of OA. METHODS: Exosomes from BMSCs with or without dECM pretreatment were isolated. We measured and compared the effect of the BMSC-Exo and dECM-BMSC-Exo on interleukin (IL)-1ß-induced chondrocytes by analyzing proliferation, anabolism and catabolism, migration and apoptosis in vitro. The in vivo experiment was performed by articular injection of exosomes into DMM mice, followed by histological evaluation of cartilage. MicroRNA sequencing of exosomes was performed on BMSC-Exo and dECM-BMSC-Exo to investigate the underlying mechanism. The function of miR-3473b was validated by rescue studies in vitro and in vivo using antagomir-3473b. RESULTS: IL-1ß-treated chondrocytes treated with dECM-BMSC-Exos showed enhanced proliferation, anabolism, migration and anti-apoptosis properties compared to BMSC-Exos. DMM mice injected with dECM-BMSC-Exo showed better cartilage regeneration than those injected with BMSC-Exo. Interestingly, miR-3473b was significantly elevated in dECM-BMSC-Exos and was found to mediate the protective effect in chondrocytes by targeting phosphatase and tensin homolog (PTEN), which activated the PTEN/AKT signaling pathway. CONCLUSIONS: dECM-BMSC-Exo can enhance the alleviation of osteoarthritis via promoting migration, improving anabolism and inhibiting apoptosis of chondrocytes by upregulating miR-3473b, which targets PTEN.

Berbamine inhibits RANKL- and M-CSF-mediated osteoclastogenesis and alleviates ovariectomy-induced bone loss.

Osteoporosis is a common public health problem characterized by decreased bone mass, increased bone brittleness and damage to the bone microstructure. Excessive bone resorption by osteoclasts is the main target of the currently used drugs or treatment for osteoporosis. Effective antiresorptive drugs without side effects following long-term administration have become a major focus of anti-osteoporotic drugs. In the present study, we investigated the effect of berbamine, a small molecule natural product from Berberis amurensis Rupr, a traditional Chinese medicine, on RANKL-induced osteoclast differentiation in vitro and ovariectomy-induced bone loss in vivo. The results demonstrated that berbamine at a safe and effective dose inhibited osteoclastogenesis and bone resorption function in vitro by suppressing the nuclear factor-κB signaling pathway. In addition, berbamine protected against osteoporosis by inhibiting osteoclastogenesis and bone resorption function without affecting osteogenesis in the ovariectomy mouse model. These findings revealed that berbamine has a protective role against osteoporosis and may represent a novel promising treatment strategy for osteoporosis.

Immune checkpoint inhibitors in osteosarcoma: A hopeful and challenging future.

Osteosarcoma (OS), the most common malignant tumor in the musculoskeletal system, mainly occurs in adolescents. OS results in high mortality and disability rates due to a fatal metastatic tendency and subsequent iatrogenic damage caused by surgery, radiotherapy and chemotherapy. Recently, immunotherapies have resulted in promising prognoses with reduced side effects compared with traditional therapies. Immune checkpoint inhibitors (ICIs), which are a representative immunotherapy for OS, enhance the antitumor effects of immune cells. ICIs have shown satisfactory outcomes in other kinds of malignant tumors, especially hemopoietic tumors. However, there is still a high percentage of failures or severe side effects associated with the use of ICIs to treat OS, leading to far worse outcomes. To reveal the underlying mechanisms of drug resistance and side effects, recent studies elucidated several possible reasons, including the activation of other inhibitory immune cells, low immune cell infiltration in the tumor microenvironment, different immune properties of OS subtypes, and the involvement of osteogenesis and osteolysis. According to these mechanisms, researchers have developed new methods to overcome the shortcomings of ICIs. This review summarizes the recent breakthroughs in the use of ICIs to treat OS. Although numerous issues have not been solved yet, ICIs are still the most promising treatment options to cure OS in the long run.

Magnetically Guided Intracartilaginous Delivery of Kartogenin Improves Stem Cell-Targeted Degenerative Arthritis Therapy.

Background: Degenerative joint disease or osteoarthritis (OA) is a leading cause of disability worldwide. Intra-articular injection is the mainstay nonsurgical treatment for OA. However, dense cartilage and a lack of vasculature often limit the ability of drugs to reach cell or tissue targets at the concentrations necessary to elicit the desired biological response. Kartogenin (KGN), a small molecular compound, possesses a strong capacity to promote chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the rapid clearance of KGN from the intra-articular cavity limits its feasibility. Materials and Methods: We constructed a magnetically guided biodegradable nanocarrier system (MNP) which enabled intracartilaginous delivery of KGN to promote chondrogenic differentiation by MSCs embedded within the articular matrix. Moreover, in preclinical models of OA, KGN-loaded MNPs exhibited increased tissue penetration and retention within the joint matrix under external magnetic guidance. Results: Histological examination showed that compared with KGN alone, KGN-loaded MNPs enhanced chondrogenic differentiation and improved the structural integrity of both articular cartilage and subchondral bone. Conclusion: This study demonstrates a practical method for intracartilaginous delivery using engineered nanocarriers, thus providing a new strategy to improve the efficacy of molecular therapeutic agents in the treatment of OA.

Omaveloxolone inhibits IL-1ß-induced chondrocyte apoptosis through the Nrf2/ARE and NF-κB signalling pathways in vitro and attenuates osteoarthritis in vivo.

Osteoarthritis (OA) is a common degenerative joint disease. Effective drugs that can halt or decelerate osteoarthritis progression are still lacking. Omaveloxolone is a semisynthetic oleanane triterpenoid exerting antioxidative and anti-inflammatory effects. The present study aims to determine whether omaveloxolone has a therapeutic effect on OA. Chondrocytes were treated with interleukin (IL)-1ß to establish an OA cell model in vitro. Indicators of cell viability, oxidative stress, inflammation, cell apoptosis and extracellular matrix (ECM) degradation were investigated. Proteins related to the Nuclear factor erythroid derived-2-related factor 2 (Nrf2)/antioxidant response element (ARE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signalling pathways were assessed using Western blotting. A destabilized medial meniscus surgery-induced OA rat model was used in vivo. Gait analysis, microcomputed tomography analysis, and histopathological and immunohistochemical analyses were performed to determine the therapeutic effect of omaveloxolone on attenuating osteoarthritis in vivo. The results showed that omaveloxolone exerts antioxidative, anti-inflammatory, antiapoptotic and anti-ECM degradation effects via activation of the Nrf2/ARE signalling pathway and inhibition of the NF-κB signalling pathway in chondrocytes in vitro and attenuates OA progression in vivo, suggesting that omaveloxolone may be a potential therapeutic agent for OA.

Cartilage targeting therapy with reactive oxygen species-responsive nanocarrier for osteoarthritis.

Targeting cartilage is a promising strategy for the treatment of osteoarthritis, and various delivery vehicles were developed to assist the therapeutic agents into cartilage. However, the underlying biomechanisms and potential bioactivities remain oversimplified. Inspired by oxidative stress in the pathogenesis of osteoarthritis, we firstly testified the antioxidant capacity of a synthetic small molecule compound, oltipraz (OL), to the chondrocytes treated by IL-1ß. Then a functional reactive oxygen species (ROS) responsive nanocarrier, mesoporous silica nanoparticles (MSN) modified with methoxy polyethylene glycol-thioketal, was constructed. In vitro biomolecular results showed that compared with OL alone, MSN-OL could significantly activate Nrf2/HO-1 signaling pathway, which exhibited better ROS-scavenging proficiency and greater anti-apoptotic ability to protect mitochondrial membrane potential of chondrocytes. Further bioinformatics analysis revealed that MSN-OL suppressed clusters of genes associated with extracellular matrix organization, cell apoptosis and cellular response to oxidative stress. Animal experiments further confirmed the great cartilage-protecting ability of MSN-OL through upregulating the expression of Nrf2/HO-1 signaling pathway without obvious toxicity. In summary, this study provided a delivery system through ROS-responsive regulation of the therapeutic agents into chondrocytes of the cartilage, and confirmed the exact biological mechanisms of this innovative strategy.

Iron overload-induced ferroptosis of osteoblasts inhibits osteogenesis and promotes osteoporosis: An in vitro and in vivo study.

Growing evidence indicates that iron overload is an independent risk factor for osteoporosis. However, the mechanisms are not fully understood. The purpose of our study was to determine whether iron overload could lead to ferroptosis in osteoblasts and to explore whether ferroptosis of osteoblasts is involved in iron overload-induced osteoporosis in vitro and in vivo. Ferric ammonium citrate was used to mimic iron overload conditions, while deferoxamine and ferrostatin-1 were used to inhibit ferroptosis of MC3T3-E1 cells in vitro. The ferroptosis, osteogenic differentiation and mineralization of MC3T3-E1 cells were assessed in vitro. A mouse iron overload model was established using iron dextran. Immunohistochemical analysis was performed to determine ferroptosis of osteoblasts in vivo. Enzyme-linked immunosorbent assays and calcein-alizarin red S labelling were used to assess new bone formation. Dual x-ray absorptiometry, micro-computed tomography and histopathological analysis were conducted to evaluate osteoporosis. The results showed that iron overload reduced cell viability, superoxide dismutase and glutathione levels, increased reactive oxygen species generation, lipid peroxidation, malondialdehyde levels and ferroptosis-related protein expression, and induced ultrastructural changes in mitochondria. Iron overload could also inhibit osteogenic differentiation and mineralization in vitro. Inhibiting ferroptosis reversed the changes described above. Iron overload inhibited osteogenesis, promoted the ferroptosis of osteoblasts and induced osteoporosis in vivo, which could also be improved by deferoxamine and ferrostatin-1. These results demonstrate that ferroptosis of osteoblasts plays a crucial role in iron overload-induced osteoporosis. Maintaining iron homeostasis and targeting ferroptosis of osteoblasts might be potential measures of treating or preventing iron overload-induced osteoporosis.

Acellular nerve grafts supplemented with induced pluripotent stem cell-derived exosomes promote peripheral nerve reconstruction and motor function recovery.

Peripheral nerve injury is a great challenge in clinical work due to the restricted repair gap and weak regrowth ability. Herein, we selected induced pluripotent stem cells (iPSCs) derived exosomes to supplement acellular nerve grafts (ANGs) with the aim of restoring long-distance peripheral nerve defects. Human fibroblasts were reprogrammed into iPSCs through non-integrating transduction of Oct3/4, Sox2, Klf4, and c-Myc. The obtained iPSCs had highly active alkaline phosphatase expression and expressed Oct4, SSEA4, Nanog, Sox2, which also differentiated into all three germ layers in vivo and differentiated into mature peripheral neurons and Schwann cells (SCs) in vitro. After isolation and biological characteristics of iPSCs-derived exosomes, we found that numerous PKH26-labeled exosomes were internalized inside SCs through endocytotic pathway and exhibited a proliferative effect on SCs that were involved in the process of axonal regeneration and remyelination. After that, we prepared ANGs via optimized chemical extracted process to bridge 15 mm long-distance peripheral nerve gaps in rats. Owing to the promotion of iPSCs-derived exosomes, satisfactory regenerative outcomes were achieved including gait behavior analysis, electrophysiological assessment, and morphological analysis of regenerated nerves. Especially, motor function was restored with comparable to those achieved with nerve autografts and there were no significant differences in the fiber diameter and area of reinnervated muscle fibers. Taken together, our combined use of iPSCs-derived exosomes with ANGs demonstrates good promise to restore long-distance peripheral nerve defects, and thus represents a cell-free strategy for future clinical applications.

In vitro and in vivo effects of hyperglycemia and diabetes mellitus on nucleus pulposus cell senescence.

Diabetes mellitus contributes to intervertebral disc degeneration. Nucleus pulposus cell senescence plays an important role in intervertebral disc degeneration. However, the effects of hyperglycemia on human nucleus pulposus cells and the underlying process remains poorly understood. In the current study, we evaluated the effects of high glucose levels on human nucleus pulposus cell senescence in vitro and the effects of hyperglycemia on rat nucleus pulposus aging in vivo. Human nucleus pulposus cells were cultured in high-glucose medium (200 mM glucose) for 48 h. Senescence-associated ß-galactosidase staining, western blot analysis, and enzyme-linked immunosorbent assays were performed to evaluate human nucleus pulposus cell senescence. Flow cytometry and enzyme-linked immunosorbent assays were used to evaluate reactive oxygen species and advanced glycation end-product levels. Transcriptome sequencing followed by bioinformatics analysis was used to understand the abnormal biological processes of nucleus pulposus cells cultured in high-glucose medium. Diabetes mellitus rat models were established and histopathological and immunohistochemical analysis was conducted to examine nucleus pulposus tissue senescence in vivo. Exposure to a high glucose concentration promoted human nucleus pulposus cell senescence and increased the senescence-related secretion phenotype in human nucleus pulposus cells in vitro and in rat nucleus pulposus tissue in vivo. Bioinformatics analysis showed that hub genes were involved in nucleus pulposus cell cycle activities and cell senescence. The results suggest that appropriate blood glucose control may be key to preventing intervertebral disc degeneration in diabetic patients.

Accumulation of LDL/ox-LDL in the necrotic region participates in osteonecrosis of the femoral head: a pathological and in vitro study.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a common but intractable disease that appears to involve lipid metabolic disorders. Although numerous studies have demonstrated that high blood levels of low-density lipoprotein (LDL) are closely associated with ONFH, there is limited evidence to explain the pathological role of LDL. Pathological and in vitro studies were performed to investigate the role of disordered metabolism of LDL and oxidized LDL (ox-LDL) in the femoral head in the pathology of ONFH. METHODS: Nineteen femoral head specimens from patients with ONFH were obtained for immunohistochemistry analysis. Murine long-bone osteocyte Y4 cells were used to study the effects of LDL/ox-LDL on cell viability, apoptosis, and metabolism process of LDL/ox-LDL in osteocytes in normoxic and hypoxic environments. RESULTS: In the pathological specimens, marked accumulation of LDL/ox-LDL was observed in osteocytes/lacunae of necrotic regions compared with healthy regions. In vitro studies showed that ox-LDL, rather than LDL, reduced the viability and enhanced apoptosis of osteocytes. Pathological sections indicated that the accumulation of ox-LDL was significantly associated with impaired blood supply. Exposure to a hypoxic environment appeared to be a key factor leading to LDL/ox-LDL accumulation by enhancing internalisation and oxidation of LDL in osteocytes. CONCLUSIONS: The accumulation of LDL/ox-LDL in the necrotic region may contribute to the pathology of ONFH. These findings could provide new insights into the prevention and treatment of ONFH.

Bardoxolone-Methyl Prevents Oxidative Stress-Mediated Apoptosis and Extracellular Matrix Degradation in vitro and Alleviates Osteoarthritis in vivo.

PURPOSE: Oxidative stress-induced chondrocyte apoptosis and extracellular matrix (ECM) degradation plays an important role in the progression of osteoarthritis (OA). Bardoxolone methyl (BM), a semisynthetic triterpenoid, exerts strong effect against oxidative stress. The purpose of the present study was to determine the effectiveness of bardoxolone-methyl (BM) in preventing oxidative stress-induced chondrocyte apoptosis and extracellular ECM degradation in vitro and the role of alleviating OA progression in vivo. METHODS: Oxidative damage was induced by 25 mM tert-butyl hydroperoxide (TBHP) for 24 h in rat chondrocytes. 0.025 and 0.05 µM bardoxolone-methyl (BM) were used in vitro treatment. Ex-vivo cartilage explant model was established to evaluate the effect of BM on oxidative stress-induced ECM degradation. The mouse OA model was induced by surgical destabilization of the medial meniscus. RESULTS: In vitro, 0.025 and 0.05 µM BM reduced TBHP-induced excessive ROS generation, improved cell viability, increased malondialdehyde level and decreased superoxide dismutase level. 0.025 and 0.05 µM BM prevented TBHP-induced mitochondrial damage and apoptosis in chondrocytes BM activated heme oxygenase-1 (HO-1)/NADPH quinone oxidoreductase 1 (NOQ1) signaling pathway through targeting nuclear factor erythroid derived-2-related factor 2 (Nrf2). Additionally, BM treatment enhanced the expression levels of aggrecan and collagen II and inhibited the expression levels of matrix metalloproteinase 9 (MMP 9), MMP 13, Bax and cleaved-caspase-3. BM increased proteoglycan staining area and IOD value in ex vivo cultured experiment cartilage explants and improved the OARSI score, stands, max contact mean intensity, print area and duty cycle in mouse OA model. CONCLUSION: BM prevented oxidative stress-induced chondrocyte apoptosis and ECM degradation in vitro and alleviated OA in vivo, suggesting that BM serves as an effective drug for treatment with OA.

Engeletin Protects Against TNF-α-Induced Apoptosis and Reactive Oxygen Species Generation in Chondrocytes and Alleviates Osteoarthritis in vivo.

PURPOSE: Osteoarthritis (OA) is a progressive disease characterized by pain and impaired joint functions. Engeletin is a natural compound with anti-inflammatory and antioxidant effects on other diseases, but the effect of engeletin on OA has not been evaluated. This study aimed to elucidate the protective effect of engeletin on cartilage and the underlying mechanisms. METHODS: Chondrocytes were isolated from rat knee cartilage, and TNF-α was used to simulate OA in vitro. After treatment with engeletin, the expression of extracellular matrix (ECM) components (collagen II and aggrecan) and matrix catabolic enzymes (MMP9 and MMP3) was determined by Western blotting and qPCR. Chondrocyte apoptosis was evaluated using Annexin V-FITC/PI and flow cytometry. Apoptosis-related proteins (Bax, Bcl-2, and cleaved caspase-3) were evaluated by Western blotting. The mitochondrial membrane potential of chondrocytes was measured with JC-1, and intracellular reactive oxygen species (ROS) levels were determined with DCFH-DA. Changes in signaling pathways (Nrf2, NF-κB and MAPK) were evaluated by Western blotting. In vivo, anterior cruciate ligament transection (ACLT) was used to induce the rat OA model, and engeletin was administered intraarticularly. The therapeutic effect of engeletin was analyzed by histopathological analysis. RESULTS: Pretreatment with engeletin alleviated TNF-α-induced inhibition of ECM components (collagen II and aggrecan) and upregulation of matrix catabolic enzymes (MMP9 and MMP3). Engeletin ameliorated chondrocyte apoptosis by inhibiting Bax expression and upregulating Bcl-2 expression. Engeletin maintained the mitochondrial membrane potential of chondrocytes and scavenged intracellular ROS by activating the Nrf2 pathway. The NF-κB and MAPK pathways were inhibited by treatment with engeletin. In vivo, ACLT-induced knee OA in rats was alleviated by intraarticular injection of engeletin. CONCLUSION: Engeletin ameliorated OA in vitro and in vivo. It may be a potential therapeutic drug for OA.

Nanostructured Coating of Non-Crystalline Tantalum Pentoxide on Polyetheretherketone Enhances RBMS Cells/HGE Cells Adhesion.

PURPOSE: As a dental material, polyetheretherketone (PEEK) is bioinert that does not induce cellular response and bone/gingival tissues regeneration. This study was to develop bioactive coating on PEEK and investigate the effects of coating on cellular response. MATERIALS AND METHODS: Tantalum pentoxide (TP) coating was fabricated on PEEK surface by vacuum evaporation and responses of rat bone marrow mesenchymal stem (RBMS) cells/human gingival epithelial (HGE) were studied. RESULTS: A dense coating (around 400 nm in thickness) of TP was closely combined with PEEK (PKTP). Moreover, the coating was non-crystalline TP, which contained many small humps (around 10 nm in size), exhibiting a nanostructured surface. In addition, the roughness, hydrophilicity, surface energy, and protein adsorption of PKTP were remarkably higher than that of PEEK. Furthermore, the responses (adhesion, proliferation, and osteogenic gene expression) of RBMS cells, and responses (adhesion and proliferation) of HGE cells to PKTP were remarkably improved in comparison with PEEK. It could be suggested that the nanostructured coating of TP on PEEK played crucial roles in inducing the responses of RBMS/HGE cells. CONCLUSION: PKTP with elevated surface performances and outstanding cytocompatibility might have enormous potential for dental implant application.

Induction of notochordal differentiation of bone marrow mesenchymal­derived stem cells via the stimulation of notochordal cell­rich nucleus pulposus tissue.

The degeneration of intervertebral disc (IVD) tissue, initiated following the disappearance of notochordal cells (NCs), is characterized by the decreased number of nucleus pulposus (NP) cells (NPCs) and extracellular matrix. Transplanting proper cells into the IVD may sustain cell numbers, resulting in the synthesis of new matrix; this represents a minimally invasive regenerative therapy. However, the lack of cells with a correct phenotype severely hampers the development of regenerative therapy. The present study aimed to investigate whether porcine NC­rich NP tissue stimulates bone marrow­derived mesenchymal stem cell (BM­MSC) differentiation toward NC­like cells, which possess promising regenerative ability, for the treatment of disc degeneration diseases. BM­MSCs were successfully isolated from porcine femurs and tibiae, which expressed CD90 and CD105 markers and did not express CD45. Differentiation induction experiments revealed that the isolated cells had osteogenic and adipogenic differentiation potential. When co­cultured with NC­rich NP tissue, the BM­MSCs successfully differentiated into NC­like cells. Cell morphological analysis revealed that the cells exhibited an altered morphology, from a shuttle­like to a circular one, and the expression of NC marker genes, including brachyury, keratin­8, and keratin­18, was enhanced, and the cells exhibited the ability to generate aggrecan and collagen II. Taken together, the findings of the present study demonstrated that the primarily isolated and cultured BM­MSCs may be stimulated to differentiate into NC­like cells by porcine NC­rich NP explants, potentially providing an ideal cell source for regenerative therapies for disc degeneration diseases.

Acacetin Alleviates Inflammation and Matrix Degradation in Nucleus Pulposus Cells and Ameliorates Intervertebral Disc Degeneration in vivo.

PURPOSE: Intervertebral disc degeneration (IDD) is one of the most prevalent musculoskeletal disorders. The nucleus pulposus is the major component of the intervertebral disc, and nucleus pulposus cells (NPCs) play a significant role in the normal functioning of the intervertebral disc. Reactive oxygen species (ROS) generation, inflammation and extracellular matrix degradation in NPCs contribute to the degeneration of intervertebral discs. Acacetin is a drug that exerts antioxidant and anti-inflammatory effects on many types of cells. However, whether acacetin can relieve the degeneration of NPCs remains unknown. METHODS: NPCs were extracted from rat intervertebral discs. The NPCs were treated with tert-butyl peroxide (TBHP) to simulate a high-ROS environment, and acacetin was subsequently added. The contents of ROS, inflammatory mediators (COX-2, iNOS) and extracellular matrix components (aggrecan, collagen II, MMP13, MMP9, MMP3) were measured. Components of related signaling pathways (Nrf2, MAPK) were also evaluated. To determine the effect of acacetin in vivo, we simulated disc degeneration via needle puncture. Acacetin was then applied intraperitoneally, and the degenerative status was evaluated using MRI and histopathological analysis. RESULTS: In vitro, acacetin alleviated TBHP-induced ROS generation and upregulated the expression of antioxidant proteins, including HO-1, NQO1, and SOD. In addition, acacetin relieved the TBHP-induced generation of inflammatory mediators (COX-2, iNOS) and degradation of the extracellular matrix (aggrecan, collagen II, MMP13, MMP9, and MMP3). Acacetin exerted its effect by activating the Nrf2 pathway and inhibiting p38, JNK and ERK1/2 phosphorylation. In vivo, acacetin ameliorated puncture-induced disc degeneration in a rat tail model, which was evaluated using MRI and histopathological analysis. CONCLUSION: Acacetin alleviated IDD in vitro and in vivo and may have the potential to be developed as an effective treatment for IDD.

Oltipraz Prevents High Glucose-Induced Oxidative Stress and Apoptosis in RSC96 Cells through the Nrf2/NQO1 Signalling Pathway.

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes mellitus (DM). Schwann cell (SC) apoptosis contributes to the occurrence and development of DPN. Effective drugs to prevent SC apoptosis are required to relieve and reverse peripheral nerve injury caused by DM. Oltipraz [4-methyl-5-(2-pyrazinyl)-1,2-dithiole-3-thione], an agonist of nuclear factor erythroid derived-2-related factor 2 (Nrf2), exerts strong effect against oxidative stress in animal models or clinical patients in certain diseases, including heart failure, acute kidney injury, and liver injury. The aim of the present study was to determine the effectiveness of oltipraz in preventing SC apoptosis induced by high glucose levels. RSC96 cells pretreated with oltipraz were cultured in high-glucose medium (50 mM glucose) for 24 h, and cells cultured in medium containing 5 mM glucose were used as the control. Flow cytometry was used to evaluate the degree of apoptosis. A Cell Counting Kit-8 assay was used to assess cell viability. The mitochondrial membrane potential was assessed using JC-1 staining, and reactive oxygen species (ROS) generation was measured using 20,70-dichlorodihydrofluorescein diacetate staining. In addition, the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) levels were also evaluated using the corresponding kits. Flow cytometry was subsequently used to detect apoptosis, and western blotting was used to measure the expression levels of nuclear factor erythroid derived-2-related factor 2 and NADPH quinone oxidoreductase 1. The results showed that high glucose concentration increased oxidative stress and apoptosis in RSC96 cells. Oltipraz improved cell viability and reduced apoptosis of RSC96 cells in the high glucose environment. Additionally, oltipraz exhibited a significant antioxidative effect, as shown by the decrease in MDA levels, increased SOD levels, and reduced ROS generation in RSC96 cells. The results of the present study suggest that oltipraz exhibits potential as an effective drug for treatment with DPN.

Long non­coding RNA MALAT1 promotes high glucose­induced rat cartilage endplate cell apoptosis via the p38/MAPK signalling pathway.

Diabetes mellitus (DM) contributes to intervertebral disc degeneration (IDD). The long non­coding RNA MALAT1 has been revealed to play an important role in diabetes­associated complications. However, the specific role of MALAT1 in diabetes­associated IDD has not been determined. The aim of the present study was to evaluate the roles of MALAT1 in the apoptosis of cartilage endplate (CEP) cells induced by high glucose and to explore the mechanisms underlying this effect. Rat CEP cells were cultured in high­glucose medium (25 mM glucose) for 24 or 72 h. Cells cultured in medium containing 5 mM glucose were used as a control. Flow cytometry was used to detect the degree of apoptosis. Reverse transcription­quantitative PCR was used to measure the expression of MALAT1 mRNA. In addition, CEP cells were treated with different conditions (high glucose, high glucose + MALAT1 negative control, high glucose + MALAT1 RNAi, normal control) for 72 h. Flow cytometry was subsequently used to detect apoptosis and western blotting was used to measure the expression levels of total and phosphorylated p38. The results revealed that high glucose concentration promoted apoptosis and enhanced expression of MALAT1 in CEP cells. Furthermore, MALAT1 knockout decreased the expression levels of total and phosphorylated p38 and reduced the apoptosis of rat CEP cells. The results obtained in the present study indicated that MALAT1 may serve as an important therapeutic target for curing or delaying IDD in patients with diabetes.