Piezo1-mediated mechanotransduction promotes entheseal pathological new bone formation in ankylosing spondylitis.

OBJECTIVE: The aim of this study was to identify the role of Piezo1-mediated mechanotransduction in entheseal pathological new bone formation and to explore the underlying molecular mechanism. METHODS: Spinal ligament tissues were collected from 14 patients with ankylosing spondylitis (AS) and 14 non-AS controls and bulk RNA sequencing was conducted. Collagen antibody-induced arthritis models were established to observe pathological new bone formation. Pharmacological inhibition and genetic ablation of Piezo1 was performed in animal models to identify the essential role of Piezo1. Entheseal osteo-chondral lineage cells were collected and in vitro cell culture system was established to study the role and underlying mechanism of Piezo1 in regulation of chondrogenesis, osteogenesis and its own expression. RESULTS: Piezo1 was aberrantly upregulated in ligaments and entheseal tissues from patients with AS and animal models. Pharmaceutical and genetic inhibition of Piezo1 attenuated while activation of Piezo1 promoted pathological new bone formation. Mechanistically, activation of CaMKII (Calcium/calmodulin dependent protein kinase II) signalling was found essential for Piezo1-mediated mechanotransduction. In addition, Piezo1 was upregulated by AS-associated inflammatory cytokines. CONCLUSION: Piezo1-mediated mechanotransduction promotes entheseal pathological new bone formation through CaMKII signalling in AS.

Tenascin-C-mediated suppression of extracellular matrix adhesion force promotes entheseal new bone formation through activation of Hippo signalling in ankylosing spondylitis.

OBJECTIVES: The aim of this study was to identify the role of tenascin-C (TNC) in entheseal new bone formation and to explore the underlying molecular mechanism. METHODS: Ligament tissue samples were obtained from patients with ankylosing spondylitis (AS) during surgery. Collagen antibody-induced arthritis and DBA/1 models were established to observe entheseal new bone formation. TNC expression was determined by immunohistochemistry staining. Systemic inhibition or genetic ablation of TNC was performed in animal models. Mechanical properties of extracellular matrix (ECM) were measured by atomic force microscopy. Downstream pathway of TNC was analysed by RNA sequencing and confirmed with pharmacological modulation both in vitro and in vivo. Cellular source of TNC was analysed by single-cell RNA sequencing (scRNA-seq) and confirmed by immunofluorescence staining. RESULTS: TNC was aberrantly upregulated in ligament and entheseal tissues from patients with AS and animal models. TNC inhibition significantly suppressed entheseal new bone formation. Functional assays revealed that TNC promoted new bone formation by enhancing chondrogenic differentiation during endochondral ossification. Mechanistically, TNC suppressed the adhesion force of ECM, resulting in the activation of downstream Hippo/yes-associated protein signalling, which in turn increased the expression of chondrogenic genes. scRNA-seq and immunofluorescence staining further revealed that TNC was majorly secreted by fibroblast-specific protein-1 (FSP1)+fibroblasts in the entheseal inflammatory microenvironment. CONCLUSION: Inflammation-induced aberrant expression of TNC by FSP1+fibroblasts promotes entheseal new bone formation by suppressing ECM adhesion forces and activating Hippo signalling.

Matching correction of main and compensatory curves is critical for immediate postoperative coronal balance in correction of severe adult idiopathic scoliosis.

PURPOSE: To analyze the correlation between immediate postoperative coronal imbalance and the matching degree of the correction rates of the main curve and compensatory curves in the surgical treatment of severe adult idiopathic scoliosis. METHODS: Patients were categorized into three types based on the preoperative coronal balance status (type A = balance, type B = shifted to cave side and type C = shifted to convex side), and each type was further divided into two subgroups based on the postoperative coronal balance status (balance and imbalance). Different coronal parameters before and after operations were calculated and compared. RESULTS: The rate of postoperative CIB was highest in type C patients (53.8%) and lowest in type A patients (31.5%). To avoid postoperative CIB, the value of the postoperative CRmain should fall within the range of 1.001 × CRcomp ± 2.84% in type A patients, 1.112 × CRcomp + 3.3% ± 5.02% in type B patients and 0.907 × CRcomp - 2.5% ± 4.38% in type C patients. CONCLUSION: Mismatch between the correction rates of the main curve and compensation curves is a critical cause of immediate postoperative CIB. The relatively equal correction of the main curve and compensatory curves is essential for type A patients to achieve postoperative coronal balance, while the correction rate of the main curve should be higher than the compensatory curves in type B patients and vice versa in type C patients. Three formulas for the three different types were developed to provide helpful guidance information for surgical planning.

Correlation study of radiographic characteristics and operative difficulty in lateral-anterior lumbar interbody fusion (LaLIF) at the L4-5 level: a novel classification for case selection.

PURPOSE: To analyze correlations between the realistic surgical difficulty of LaLIF and anatomic characteristics in radiographic images, in order to develop a simple classification to provide guiding information for case selection and evaluate the potential risks of the technique. METHODS: Ninety-six consecutive cases who underwent LaLIF surgeries at the L4-5 level with MR T2-weighted images were analyzed. A novel classification based on the anatomic relationships among the disk, great vessels, and psoas muscle was used for grouping. Clinical outcomes and realistic surgical difficulty parameters were recorded, and comparisons were made among different types of classifications. RESULTS: Of the 96 analyzed cases, the time of surgical exposure was significantly longer for type C than for type B, and both of these were longer than that of type A. The VAS and ODI were significantly improved at a 1-year follow-up. There was no statistically significant difference among the three types. Type C had the highest incidence of complications, while Type A had the lowest. Analyses of another 304 MRI cases obtained in outpatient clinics showed that the distribution of the three types among these cases was consistent with that of the surgical cohort. CONCLUSION: Our novel and simple classification provides useful information for case selection. Type A provided the best indication and is most appropriate for a beginner in this technique. Type C includes the most challenging situations, which may have a high incidence of complications and require sophisticated surgical skills to achieve satisfactory outcomes and avoid approach-related complications.

Wnt4 signaling mediates protective effects of melatonin on new bone formation in an inflammatory environment.

Growing evidence shows that the inhibitory effect of inflammatory cytokines on new bone formation by osteogenic precursor cells is a critical cause of net bone-density reduction. Melatonin has been proven to be a potential therapeutic candidate for osteoporosis. However, whether it is capable of antagonizing the suppressing effect of inflammatory cytokines on osteogenic precursor cells is so far elusive. In this study, using the cell culture system of human bone marrow stromal cells and MC3T3-E1 preosteoblasts, we recorded the following vital observations that provided insights of melatonin-induced bone formation: 1) melatonin induced bone formation in both normal and inflammatory conditions; 2) Wnt4 was essential for melatonin-induced bone formation in inflammatory stimulation; 3) melatonin- and Wnt4-induced bone formation occurred via activation of ß-catenin and p38-JNK MAPK pathways by interaction with a distinct frizzled LDL receptor-related protein complex; 4) melatonin suppressed the inhibitory effect of NF-κB on osteogenesis in a Wnt4-dependent manner; and 5) melatonin induced Wnt4 expression through the ERK1/2-Pax2-Egr1 pathway. In summary, we showed a novel mechanism of melatonin-induced bone formation in an inflammatory environment. Melatonin-induced Wnt4 expression is essential for its osteoinductive effect and the inhibitory effect of NF-κB on bone formation. Our novel findings may provide useful information for its potential translational application.-Li, X., Li, Z., Wang, J., Li, Z., Cui, H., Dai, G., Chen, S., Zhang, M., Zheng, Z., Zhan, Z., Liu, H. Wnt4 signaling mediates protective effects of melatonin on new bone formation in an inflammatory environment.

Physiochemically and Genetically Engineered Bacteria: Instructive Design Principles and Diverse Applications.

With the comprehensive understanding of microorganisms and the rapid advances of physiochemical engineering and bioengineering technologies, scientists are advancing rationally-engineered bacteria as emerging drugs for treating various diseases in clinical disease management. Engineered bacteria specifically refer to advanced physiochemical or genetic technologies in combination with cutting edge nanotechnology or physical technologies, which have been validated to play significant roles in lysing tumors, regulating immunity, influencing the metabolic pathways, etc. However, there has no specific reviews that concurrently cover physiochemically- and genetically-engineered bacteria and their derivatives yet, let alone their distinctive design principles and various functions and applications. Herein, the applications of physiochemically and genetically-engineered bacteria, and classify and discuss significant breakthroughs with an emphasis on their specific design principles and engineering methods objective to different specific uses and diseases beyond cancer is described. The combined strategies for developing in vivo biotherapeutic agents based on these physiochemically- and genetically-engineered bacteria or bacterial derivatives, and elucidated how they repress cancer and other diseases is also underlined. Additionally, the challenges faced by clinical translation and the future development directions are discussed. This review is expected to provide an overall impression on physiochemically- and genetically-engineered bacteria and enlighten more researchers.

Boosting Oxygen Reduction at Pt(111)|Proton Exchange Ionomer Interfaces through Tuning the Microenvironment Water Activity.

A proton exchange ionomer is one of the most important components in membrane electrode assemblies (MEAs) of polymer electrolyte membrane fuel cells (PEMFCs). It acts as both a proton conductor and a binder for nanocatalysts and carbon supports. The structure and the wetting conditions of the MEAs have a great impact on the microenvironment at the three-phase interphases in the MEAs, which can significantly influence the electrode kinetics such as the oxygen reduction reaction (ORR) at the cathode. Herein, by using the Pt(111)|X ionomer interface as a model system (X = Nafion, Aciplex, D72), we find that higher drying temperature lowers the onset potential for sulfonate adsorption and reduces apparent ORR current, while the current wave for OHad formation drops and shifts positively. Surprisingly, the intrinsic ORR activity is higher after properly correcting the blocking effect of Pt active sites by sulfonate adsorption and the poly(tetrafluoroethylene) (PTFE) skeleton. These results are well explained by the reduced water activity at the interfaces induced by the ionomer/PTFE, according to the mixed potential effect. Implications for how to prepare MEAs with improved ORR activity are provided.

Cyclosporin-A reduced the cytotoxicity of propranolol in HUVECs via p38 MAPK signaling.

ABSTRACT: Propranolol (PROP) is a nonselective ß-adrenergic receptor antagonist used to treat hypertension and cardiac arrhythmias. Oral administration of PROP has recently emerged as a new treatment modality for hemangiomas. However, the side effects of PROP at the cellular level have not been adequately described.The present study investigates and highlights the mechanisms of coupling of the drugs cyclosporin-A (CyA) and PROP on cell proliferation and the occurrence of apoptosis. It also relays the antioxidant effect of PROP on human umbilical vein endothelial cells (HUVECs).HUVECs were treated with CyA and PROP. At 24 hours after treatment, the levels of reactive oxygen species (ROS), cell proliferation, and apoptosis were determined using the ROS kit, MTT assay, and Annexin V staining. In addition, the related proteins of phospho-p38 mitogen-activated protein kinase were determined by western blotting. Subsequently, HUVECs pretreated with CyA or PROP were treated with the p38 inhibitor (SB203580). Finally, the ROS level, cell proliferation, and apoptosis were measured again in both active HUVECs and HUVECs, in which the p38 proteins were inhibited.The combination of CyA and PROP reversed the effect of CyA on cell viability, reduced the ROS level and the cell apoptosis induced by PROP. Moreover, inhibition of p38 protein catalase activity immediately stopped the effect of CyA-propranolol in HUVECs.The effect of the CyA-propranolol combination on HUVECs is associated with the p38 pathway changes, which is proven to be a potential chemotherapeutic agent that minimizes the side effects of PROP in hemangioma therapy.

CXCL12/CXCR4-Rac1-mediated migration of osteogenic precursor cells contributes to pathological new bone formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a chronic inflammatory disease characterized by inflammatory back pain and spinal ankylosis due to pathological new bone formation. Here, we identified CXCL12 as a critical contributor to pathological new bone formation through recruitment of osteogenic precursor cells (OPCs). CXCL12 was found highly expressed in the regions that would potentially develop pathological new bone. OPCs were recruited to the regions where CXCL12 was up-regulated. Inhibition of CXCL12/CXCR4 axis with AMD3100 or conditional knockout of CXCR4 attenuated OPCs migration and subsequent pathological new bone formation in animal models of AS. By contrast, a genetically engineered animal model with CXCL12 overexpression developed a joint ankylosis phenotype. Furthermore, Rac1 was found essential for OPCs migration and pathological new bone formation. These findings ravel the novel role of CXCL12 in AS and indicate a potential strategy for targeting the CXCL12/CXCR4-Rac1 axis to prevent progression of axial skeleton ankylosis.

BRD4 promotes heterotopic ossification through upregulation of LncRNA MANCR.

AIMS: Acquired heterotopic ossification (HO) is a debilitating disease characterized by abnormal extraskeletal bone formation within soft-tissues after injury. The exact pathogenesis of HO remains unknown. It was reported that BRD4 may contribute to osteoblastic differentiation. The current study aims to determine the role of BRD4 in the pathogenesis of HO and whether it could be a potential target for HO therapy. METHODS: Achilles tendon puncture (ATP) mouse model was performed on ten-week-old male C57BL/6J mice. One week after ATP procedure, the mice were given different treatments (e.g. JQ1, shMancr). Achilles tendon samples were collected five weeks after treatment for RNA-seq and real-time quantitative polymerase chain reaction (RT-qPCR) analysis; the legs were removed for micro-CT imaging and subsequent histology. Human bone marrow mesenchymal stem cells (hBMSCs) were isolated and purified bone marrow collected during surgeries by using density gradient centrifugation. After a series of interventions such as knockdown or overexpressing BRD4, Alizarin red staining, RT-qPCR, and Western Blot (Runx2, alkaline phosphatase (ALP), Osx) were performed on hBMSCs. RESULTS: Overexpression of BRD4 enhanced while inhibition of Brd4 suppressed the osteogenic differentiation of hBMSCs in vitro. Overexpression of Brd4 increased the expression of mitotically associated long non-coding RNA (Mancr). Downregulation of Mancr suppressed the osteoinductive effect of BRD4. In vivo, inhibition of BRD4 by JQ1 significantly attenuated pathological bone formation in the ATP model (p = 0.001). CONCLUSION: BRD4 was found to be upregulated in HO and Brd4-Mancr-Runx2 signalling was involved in the modulation of new bone formation in HO. Cite this article: Bone Joint Res 2021;10(10):668-676.

Visfatin promotes intervertebral disc degeneration by inducing IL-6 expression through the ERK/JNK/p38 signalling pathways.

Visfatin reportedly induces the expression of proinflammatory cytokines. Severe grades of intervertebral disc disease (IVDD) exhibit higher expression of visfatin than mild ones. However, the direct relationship between visfatin and IVDD remains to be elucidated. This study aimed to clarify whether stimulation of visfatin in IVDD is mediated by IL-6. To investigate the role of visfatin in IVDD, a rat model of anterior disc puncture was established by injecting visfatin or PBS using a 27-gauge needle. Results revealed an obvious aggravation of the histological morphology of IVDD in the visfatin group. On treating human NP cellswith visfatin, the levels of collagenII and aggrecan decreased and those of matrix metallopeptidase 3 and IL-6 gradually increased. A rapid increase in ERK, JNK, and p38 phosphorylation was also noted after visfatin treatment. Compared to those treated with visfatin alone, NP cells pretreated with ERK1/2, JNK, and p38 inhibitors or siRNA targeting p38, ERK, and JNK exhibited a significant suppression of IL-6. Our data represent the first evidence that visfatin promotes IL-6 expression in NP cells via the JNK/ERK/p38-MAPK signalling pathways. Further, our findings suggest epidural fat and visfatin as potential therapeutic targets for controlling IVDD-associated inflammation.

Painful intervertebral disc degeneration and inflammation: from laboratory evidence to clinical interventions.

Low back pain (LBP), as a leading cause of disability, is a common musculoskeletal disorder that results in major social and economic burdens. Recent research has identified inflammation and related signaling pathways as important factors in the onset and progression of disc degeneration, a significant contributor to LBP. Inflammatory mediators also play an indispensable role in discogenic LBP. The suppression of LBP is a primary goal of clinical practice but has not received enough attention in disc research studies. Here, an overview of the advances in inflammation-related pain in disc degeneration is provided, with a discussion on the role of inflammation in IVD degeneration and pain induction. Puncture models, mechanical models, and spontaneous models as the main animal models to study painful disc degeneration are discussed, and the underlying signaling pathways are summarized. Furthermore, potential drug candidates, either under laboratory investigation or undergoing clinical trials, to suppress discogenic LBP by eliminating inflammation are explored. We hope to attract more research interest to address inflammation and pain in IDD and contribute to promoting more translational research.

Growth differentiation factor-6 attenuates inflammatory and pain-related factors and degenerated disc-induced pain behaviors in rat model.

Previous studies have indicated that growth differentiation factor 6 (GDF6) is a potential candidate for intervertebral disc (IVD) degeneration (IDD) treatment. Here, we investigated the effect of GDF6 on IDD by examining changes in disc structure and the expression of inflammatory and pain-related factors. A rat posterior disc puncture model of single segments and three consecutive segments was constructed, and GDF6 or phosphate-buffered solution was administered via intradiscal injection 1 or 2 weeks after surgery. Magnetic resonance imaging showed a clear degeneration signal in the punctured disc, which was inhibited by GDF6. Histological staining revealed that GDF6 did not significantly improve the structure of IVDs in rats 8 weeks after puncture surgery, but it had an inhibitory effect on expression of the tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-1ß in the IVD. Furthermore, GDF6 was found to protect the morphology and structure of the IVD 32 weeks after surgery. Mechanical and thermal hyperalgesia tests suggested that GDF6 injection can significantly improve mechanical and thermal-stimulated pain behavior in rats and inhibit the expression of inflammatory factors TNF-α and IL-1ß and the pain factor calcitonin gene-related peptide in the dorsal root ganglion. A rat protein array test indicated that GDF6 could reduce the expression of cytokines IL-6, intercellular cell adhesion molecule-1, matrix metalloproteinase-13, IL-1ß, and TNF-α and increase the expression of tissue inhibitor of metalloproteinases 1, Transforming growth factor-beta 2, IL-10, and resistin in a TNF-α-induced IDD cell model. Thus, our study demonstrates that GDF6 can improve the structure of the IVD, inhibit the expression of inflammatory and pain-related factors, and improve pain behavior in rats. Clinical Significance: To establish further preclinical research and clinical trials, comprehensive data are needed to validate the regenerative properties of GDF6. Ideally, a regenerative agent should also be able to relieve discogenic pain, achieving the best clinical outcomes.

Role of the miR-133a-5p/FBXO6 axis in the regulation of intervertebral disc degeneration.

OBJECTIVE: Low back pain is a leading cause of disabilities worldwide, and intervertebral disc degeneration (IVDD)-related disorders have been recognised as one of the main contributors. Nevertheless, the underlying mechanism has not yet been fully understood. The aim of this study was to investigate the role of the miR-133a-5p/FBXO6 axis in the regulation of IVDD. METHODS: RT-qPCR, WB and IHC were performed to assess the expression of FBXO6 in human IVD tissues. Nucleus pulposus (NP) cells were treated with IL-1ß to induce IVDD cellular model. Silence of FBXO6 was achieved using specific siRNAs. CCK-8 assay, flow cytometry, TUNEL assay, RT-qPCR and WB were used to evaluate the role and mechanism of FBXO6 in the process of IVDD. Online tools, GSE datasets and RT-qPCR were used to search the candidate miRNAs targeting FBXO6. The direct binding sites between FBXO6 and miR-133a-5p were further verified by a dual luciferase assay. RT-qPCR, WB and rescue experiments were conducted to identify the regulatory function of miR-133a-5p on the expression of aggrecan, collagen Ⅱ, MMP3, ADAMTS5, IL-6 and COX2. In addition, the role of the NF-κB pathway in regulating miR-133a-5p was studied using lentiviral shRNA, WB and RT-qPCR. RESULTS: Results showed that FBXO6 mainly expressed in the NP tissue of IVD and the expression of FBXO6 decreased with the process of IVDD as well as under IL-1ß stimulation. The silence of FBXO6 led to the decreased expression of aggrecan and collagen Ⅱ and the increased expression of MMP3, ADAMTS5, IL-6 and COX2, which further induced the degeneration of NP cells. The bioinformatic analysis showed that miR-133a-5p was the candidate miRNA targeting FBXO6. miR-133a-5p was upregulated in IVDD tissues and significantly inhibited the expression of FBXO6. The inhibition of miR-133a-5p ameliorated the acceleration of IVDD induced by the silence of FBXO6 in vitro. Moreover, it was demonstrated that IL-1ß regulated the expression of the miR-133a-5p/FBXO6 axis via the NF-κB pathway in NP cells. CONCLUSION: miR-133a-5p was upregulated by IL-1ß to aggravate intervertebral disc degeneration via sponging FBXO6. Inhibiting miR-133a-5p expression or rescuing FBXO6 expression may be promising strategies for the treatment of IVDD. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study suggests that the miR-133a-5p/FBXO6 axis could regulate NP cells proliferation, apoptosis, synthesis and degradation of extracellular matrix, which provides a promising therapeutic target and strategy for the treatment of IVDD.

Aberrant upregulation of CaSR promotes pathological new bone formation in ankylosing spondylitis.

Pathological new bone formation is a typical pathological feature in ankylosing spondylitis (AS), and the underlying molecular mechanism remains elusive. Previous studies have shown that the calcium-sensing receptor (CaSR) is critical for osteogenic differentiation while also being highly involved in many inflammatory diseases. However, whether it plays a role in pathological new bone formation of AS has not been reported. Here, we report the first piece of evidence that expression of CaSR is aberrantly upregulated in entheseal tissues collected from AS patients and animal models with different hypothetical types of pathogenesis. Systemic inhibition of CaSR reduced the incidence of pathological new bone formation and the severity of the ankylosing phenotype in animal models. Activation of PLCγ signalling by CaSR promoted bone formation both in vitro and in vivo. In addition, various inflammatory cytokines induced upregulation of CaSR through NF-κB/p65 and JAK/Stat3 pathways in osteoblasts. These novel findings suggest that inflammation-induced aberrant upregulation of CaSR and activation of CaSR-PLCγ signalling in osteoblasts act as mediators of inflammation, affecting pathological new bone formation in AS.

Melatonin alleviates intervertebral disc degeneration by disrupting the IL-1ß/NF-κB-NLRP3 inflammasome positive feedback loop.

The inflammatory response is induced by the overexpression of inflammatory cytokines, mainly interleukin (IL)-1ß, and is one of the main causes of intervertebral disc degeneration (IVDD). NLR pyrin domain containing 3 (NLRP3) inflammasome activation is an important source of IL-1ß. As an anti-inflammatory neuroendocrine hormone, melatonin plays various roles in different pathophysiological conditions. However, its roles in IVDD are still not well understood and require more examination. First, we demonstrated that melatonin delayed the progression of IVDD and relieved IVDD-related low back pain in a rat needle puncture IVDD model; moreover, NLRP3 inflammasome activation (NLRP3, p20, and IL-1ß levels) was significantly upregulated in severely degenerated human discs and a rat IVDD model. Subsequently, an IL-1ß/NF-κB-NLRP3 inflammasome activation positive feedback loop was found in nucleus pulposus (NP) cells that were treated with IL-1ß. In these cells, expression of NLRP3 and p20 was significantly increased, NF-κB signaling was involved in this regulation, and mitochondrial reactive oxygen species (mtROS) production increased. Furthermore, we found that melatonin disrupted the IL-1ß/NF-κB-NLRP3 inflammasome activation positive feedback loop in vitro and in vivo. Melatonin treatment decreased NLRP3, p20, and IL-1ß levels by inhibiting NF-κB signaling and downregulating mtROS production. Finally, we showed that melatonin mediated the disruption of the positive feedback loop of IL-1ß in vivo. In this study, we showed for the first time that IL-1ß promotes its own expression by upregulating NLRP3 inflammasome activation. Furthermore, melatonin disrupts the IL-1ß positive feedback loop and may be a potential therapeutic agent for IVDD.

Intermittent parathyroid hormone (1-34) application regulates cAMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells, via the cAMP/PKA signaling pathway.

The potential effects of intermittent parathyroid hormone (1-34) [PTH (1-34)] administration on bone formation have previously been investigated. A number of studies have suggested that the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway is associated with PTH-induced osteogenic differentiation. However, the precise signaling pathways and molecular mechanism by which PTH (1-34) induces the osteogenic differentiation of bone mesenchymal stromal cells (BMSCs) remain elusive. The purpose of the present study was to investigate the mechanism underlying the effect of intermittent PTH (1-34) application on the proliferation and osteogenic differentiation of BMSCs. BMSCs were randomly divided into four groups, as follows: Osteogenic medium (control group); osteogenic medium and intermittent PTH (1-34); osteogenic medium and intermittent PTH (1-34) plus the adenylyl cyclase activator forskolin; and osteogenic medium and intermittent PTH (1-34) plus the PKA inhibitor H-89. A cell proliferation assay revealed that PTH (1-34) stimulates BMSC proliferation via the cAMP/PKA pathway. Furthermore, reverse transcription-quantitative polymerase chain reaction, alkaline phosphatase activity testing and cell examination using Alizarin Red S staining demonstrated that PTH (1-34) administration promotes osteogenic differentiation and mineralization, mediated by the cAMP/PKA pathway. Crucially, the results of western blot analyses suggested that PTH (1-34) treatment and, to a greater degree, PTH (1-34) plus forskolin treatment caused an increase in phosphorylated cAMP response element binding protein (p-CREB) expression, but the effect of PTH on p-CREB expression was blocked by H-89. In conclusion, the current study demonstrated that intermittent PTH (1-34) administration regulates downstream proteins, particularly p-CREB, in the cAMP/PKA signaling pathway, to enhance the proliferation, osteogenic differentiation and mineralization of BMSCs.

Low-magnitude, high-frequency vibration promotes the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells cultured on a hydroxyapatite-coated surface: The direct role of Wnt/ß-catenin signaling pathway activation.

The positive effect of low-magnitude, high­frequency (LMHF) vibration on implant osseointegration has been demonstrated; however, the underlying cellular and molecular mechanisms remain unknown. The aim of this study was to explore the effect of LMHF vibration on the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) cultured on hydroxyapatite (HA)-coated surfaces in an in vitro model as well as to elucidate the molecular mechanism responsible for the effects of LMHF vibration on osteogenesis. LMHF vibration resulted in the increased expression of fibronectin, which was measured by immunostaining and RT-qPCR. Stimulation of BMSCs by LMHF vibration resulted in the rearrangement of the actin cytoskeleton with more prominent F-actin. Moreover, the expression of ß1 integrin, vinculin and paxillin was notably increased following LMHF stimulation. Scanning electron microscope observations revealed that there were higher cell numbers and more extracellular matrix attached to the HA-coated surface in the LMHF group. Alkaline phosphatase activity as well as the expression of osteogenic-specific genes, namely Runx2, osterix, collagen I and osteocalcin, were significantly elevated in the LMHF group. In addition, the protein expression of Wnt10B, ß-catenin, Runx2 and osterix was increased following exposure to LMHF vibration. Taken together, the findings of this study indicate that LMHF vibration promotes the adhesion and the osteogenic differentiation of BMSCs on HA-coated surfaces in vitro, and LMHF vibration may directly induce osteogenesis by activating the Wnt/ß­catenin signaling pathway. These data suggest that LMHF vibration enhances the osseointegration of bone to a HA-coated implant, and provide a scientific foundation for improving bone-implant osseointegration through the application of LMHF vibration.