MiR-99a alleviates apoptosis and extracellular matrix degradation in experimentally induced spine osteoarthritis by targeting FZD8.

BACKGROUND: Our previous study identified miR-99a as a negative regulator of early chondrogenic differentiation. However, the functional role of miR-99a in the pathogenesis of osteoarthritis (OA) remains unclear. METHODS: We examined the levels of miR-99a and Frizzled 8 (FZD8) expression in tissue specimens. Human SW1353 chondrosarcoma cells were stimulated with IL-6 and TNF-α to construct an in vitro OA environment. A luciferase reporter assay was performed to analyze the relationship between miR-99a and FZD8. CCK-8 assays, flow cytometry, and ELISA assays were used to assess cell viability, apoptosis, and inflammatory molecule expression, respectively. Percutaneous intra-spinal injections of papain mixed solution were performed to create an OA Sprague-Dawley rat model. Alcian Blue staining, Safranin O Fast Green staining, and Toluidine Blue O staining were performed to detect the degrees of cartilage injury. RESULTS: MiR-99a expression was downregulated in the severe spine OA patients when compared with the mild spine OA patients, and was also decreased in the experimentally induced in vitro OA environment when compared with the control environment. Functionally, overexpression of miR-99a significantly suppressed cell apoptosis and extracellular matrix degradation stimulated by IL-6 and TNF-α. FZD8 was identified as a target gene of miR-99a. Furthermore, the suppressive effects of miR-99a on cell injury induced by IL-6 and TNF-α were reversed by FZD8 overexpression. Moreover, the levels of miR-99a expression were also reduced in the induced OA model rats, and miR-99a agomir injection relieved the cartilage damage. At the molecular level, miR-99a overexpression downregulated the levels of MMP13, ß-catenin, Bax, and caspase-3 protein expression and upregulated the levels of COL2A1 and Bcl-2 protein expression in the in vitro OA-like chondrocyte model and also in the experimental OA model rats. CONCLUSIONS: Our data showed that miR-99a alleviated apoptosis and extracellular matrix degradation by targeting FZD8, and thereby suppressed the development and progression of experimentally induced spine osteoarthritis.

Upregulation of P2Y12 inhibits chondrocyte apoptosis in lumbar osteoarthritis through the PI3K/AKT signaling pathway.

Lumbar facet osteoarthritis (FJOA) is a major cause of severe lower back pain and disability worldwide. However, the mechanism underlying cartilage degeneration in FJOA remains unclear. The purpose of this study was to investigate the regulation and mechanism of P2Y12 on chondrocyte apoptosis in FJOA. The experimental rats were randomly divided into non-operation (n = 20) and operation groups (n = 20). In the operation group, Sodium iodoacetate (MIA, Sigma, 200 mg/mL) was injected into the right L4/5 facet process using a blunt nanoneedle 26 (WPI, Sarasota, FL, USA) under the control of an injection pump. The final injection volume was 5µL and the injection rate was 2µL/min. The facet joint was removed four weeks after surgery. After the operation, samples were stored at -80 °C until further use, whereby the right facet joints in each group were tested. Hematoxylin and eosin (HE) and iron-red solid green staining were used to observe the degeneration of articular chondrocytes in rats. Immunohistochemistry and western blotting were used to observe the expressions of P2Y12, Matrix metalloproteinase 13 (MMP13), Collagen II (COL2), and other cartilage degeneration and apoptosis-related genes. Co-localization of P2Y12-cleaved caspase-3 in the apoptosis model was detected by dual-standard immunofluorescence staining. Apoptosis was also detected by flow cytometry and TUNEL assay.P2Y12 is highly expressed in OA cartilage tissue, and inhibits IL-1ß -induced chondrocyte apoptosis through PI3K/AKT signaling pathway, thus playing a certain protective role on cartilage.

Knockdown of TRAF6 inhibits chondrocytes apoptosis and inflammation by suppressing the NF-κB pathway in lumbar facet joint osteoarthritis.

Tumor necrosis factor receptor-associated factor 6 (TRAF6), a regulator of NF-κB signaling, has been discovered recently to be probably related to osteoarthritis, while the function of TRAF6 in lumbar facet joint osteoarthritis(FJOA)still remains unknown. The aim of this study was to probe the specific function of TRAF6 in chondrocytes and its connection with the pathophysiology of FJOA. We found upregulation of TRAF6 in FJOA cartilage by western blot analysis. In vitro, we stimulated immortalized human chondrocytes by LPS to establish the cells apoptosis model. Western blot analysis demonstrated that levels of TRAF6 and cleaved caspase-3/8 in the chondrocyte injury model increased significantly. Knockdown of TRAF6 suppressed the expression of matrix metallopeptidase-13 (MMP-13) and interleukin-6 (IL-6) induced by LPS, and alleviated cell apoptosis. Meanwhile, western blot and immunofluorescent staining demonstrated that IκBα degradation and p65 nuclear transportation were also inhibited, revealing that knockdown of TRAF6 suppressed activation of the NF-κB pathway in LPS-induced chondrocytes apoptosis model. Collectively, our findings suggest that TRAF6 plays a crucial role in FJOA development by regulating NF-κB signaling pathway. Knockdown of TRAF6 may supply a potential therapeutic strategy for FJOA.

The Use of Allogenic Stromal Vascular Fraction (SVF) Cells in Degenerative Joint Disease of the Spine in Dogs.

BACKGROUND/AIM: Stem cells are widely used in regenerative medicine and in clinical practice for the treatment of damaged nerve tissue, myocytes, tendons, and ligaments. The aim of the study was to monitor VEGF levels after the administration of allogenic cellular material (SVF) in the course of treatment of dogs suffering from degenerative joint disease in the spinal region. MATERIALS AND METHODS: The study was conducted on 10 dogs of both genders, aged between 6 and 13 years in which allogenic stromal vascular fraction of stem cells (SVF) was administered intravenously. The control group was composed of 10 clinically healthy dogs. Before treatment and after 2- and 8-week intervals blood samples were obtained from the study group dogs in order to determine VEGF levels via immunoenzymatic test. RESULTS: in a few days after the therapy, alleviation of pain symptoms and reduction of lameness were noticed. The VEGF level in 2 weeks after the therapy was significantly elevated (median: 38.77 pg/ml), while in 8 weeks a decrease was observed (median: 18.37 pg/ml). Conlusion: Administration of allogenic stem cells has a positive influence on elevation of the VEGF levels in the blood serum of affected animals as well as their regeneration capacity.

Expression of adiponectin in the subchondral bone of lumbar facet joints with different degrees of degeneration.

BACKGROUND: Osteoarthritis research has been most commonly performed in the setting of the articular cartilage of the knee. To the best of our knowledge, no studies have evaluated the role of adiponectin in osteoarthritis of the lumbar facet joint (FJOA). Therefore, in this study, we explored whether adiponectin was expressed in the lumbar facet joints and evaluated the role of adiponectin in FJOA. METHODS: We enrolled patients who underwent lumbar computed tomography (CT) and magnetic resonance imaging (MRI) at the Orthopedic Department of the First Affiliated Hospital of Nanchang from May 2015 to June 2016. Lumbar facet joints were obtained from 135 patients at the time of lumbar fusion surgery and divided into three groups according to the Weishaupt grade. Cytokine levels in the subchondral bones were evaluated by enzyme-linked immunosorbent assays (ELISAs), and adiponectin levels were determined by immunohistochemistry, western blotting, and quantitative polymerase chain reaction (qPCR). RESULTS: By ELISA, adiponectin levels were examined in the subchondral bone for lumbar facet joint, and adiponectin was found to be negatively correlated with BMI in 52 patients (p < 0.001, r = -0.861). By immunohistochemistry analysis, adiponectin was found to be expressed in the subchondral bone of the lumbar facet, whereas the cartilage area was negative for adiponectin expression. Immunostaining intensity and area was related to the degeneration of the lumbar facet joint, and, in our research, considerably decreased staining intensity and area were observed in more severely degenerated lumbar facet joints. Furthermore, the expression of adiponectin was also reduced in degenerated lumbar facet joints, and the level of decline corresponded to degeneration detected by western blotting and qPCR analysis (n = 27, p < 0.0001). CONCLUSIONS: Adiponectin expression was observed in the subchondral bone of the lumbar facet joint and decreased as the degree of degeneration increased. Thus, the results of this study provide new insights into the relationship between adiponectin and osteoarthritis.

Characterization of subchondral bone histopathology of facet joint osteoarthritis in lumbar spinal stenosis.

Facet joint osteoarthritis may be a cause of low back pain in degenerative spine diseases including lumbar spinal stenosis. Subchondral bone is regarded as a potential therapeutic target for osteoarthritis treatment. The goal of this study was to characterize subchondral bone histopathology in osteoarthritic facet joints from lumbar spinal stenosis patients. Fifteen patients with degenerative spinal stenosis scheduled for transforaminal lumbar interbody fusion surgery were recruited for this study. Osteoarthritis severity was graded on T1- and T2-weighted MRI images using Weishaupt scoring system. Dissected osteoarthritic facet joints were subjected to histological and immunohistochemistry analyses to study relative abundance of osteoblast, osteoclasts, and macrophages using van Gieson's, tartrate-resistant acid phosphatase and CD68-antibody staining, respectively. Presence of nerve fibers was evaluated by PGP9.5-antibody staining. Differential bone histopathology, independent from radiological osteoarthritis grade, was observed in facet joints. Extensive de novo bone formation was found in subchondral bone tissues of eight of fifteen specimens. Regions of bone formation showed high abundance of blood vessels and CD68-positive macrophages, but were devoid of multinucleated osteoclasts. Additional pathological changes in subchondral marrow spaces, including inflammatory infiltration and enhanced osteoclast activity, were characterized by macrophage-rich tissues. PGP9.5-positive nerve fibers were detected near arterioles, but not in regions displaying bone pathology. Individual histopathological parameters did not associate with clinical features or radiological osteoarthritis severity. Subchondral bone histopathology of facet joint osteoarthritis in lumbar spinal stenosis is characterized by marrow infiltration by macrophage-rich tissues and enhanced de novo bone formation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1475-1480, 2016.

Granulation Tissue Eroding the Subchondral Bone Also Promotes New Bone Formation in Ankylosing Spondylitis.

OBJECTIVE: We previously suggested that fibroblast-rich granulation tissue eroding the subchondral bone is instrumental in the joint remodeling that occurs in ankylosing spondylitis (AS). The purpose of this study was to determine if this granulation tissue also carries bone-forming capabilities, which we approached by searching for bone-forming cells (hypertrophic chondrocytes, osteoblasts) in its vicinity. We also assessed adipogenic tissue transformation, which has been suggested to be an intermediate feature in AS bone formation based on imaging studies. METHODS: The facet joints of AS patients, osteoarthritis (OA) patients, and autopsy subjects (controls) were screened for subchondral granulation tissue. We searched for hypertrophic chondrocytes by assessing RUNX-2, type X collagen, and matrix metalloproteinase 13 (MMP-13) expression, for osteoblasts by analyzing RUNX-2, CD56, and type I collagen expression, as well as for signs of new bone formation. Adipocytes and lipid accumulation were assessed in Safranin O-stained sections. RESULTS: In the joints of AS and OA patients, RUNX-2-positive cells were found to be lining the granulation tissue. These cells coexpressed type I collagen but lacked type X collagen and MMP-13 expression, confirming their osteoblastic nature. In 91% of AS joints and in 20% of OA joints (P < 0.05), we observed foci of new bone formation at contact zones between the granulation tissue and the cartilage. Joints containing bony spots showed greater replacement of the adjacent bone marrow by granulation tissue than did joints without bone formation (P < 0.05). The granulation tissue often contained adipocytes and lipid accumulations. Replacement of the subchondral bone marrow by fat tissue was also frequently found but was not associated with new bone formation. CONCLUSION: The subchondral granulation tissue carries osteoblasts, which promote new bone formation, leading to intraarticular ankylosis of the facet joints in AS.

Characterization of degenerative human facet joints and facet joint capsular tissues.

OBJECTIVE: Lumbar facet joint degeneration (FJD) may be an important cause of low back pain (LBP) and sciatica. The goal of this study was to characterize cellular alterations of inflammatory factor expression and neovascularization in human degenerative facet joint capsular (FJC) tissue. These alterations in FJC tissues in pain stimulation were also assessed. DESIGN: FJs were obtained from consented patients undergoing spinal reconstruction surgery and cadaveric donors with no history of back pain. Histological analyses of the FJs were performed. Cytokine antibody array and quantitative real-time polymerase chain reaction (qPCR) were used to determine the production of inflammatory cytokines, and western blotting analyses (WB) were used to assay for cartilage-degrading enzymes and pain mediators. Ex vivo rat dorsal root ganglion (DRG) co-culture with human FJC tissues was also performed. RESULTS: Increased neovascularization, inflammatory cell infiltration, and pain-related axonal-promoting factors were observed in degenerative FJCs surgically obtained from symptomatic subjects. Increased VEGF, (NGF/TrkA), and sensory neuronal distribution were also detected in degenerative FJC tissues from subjects with LBP. qPCR and WB results demonstrated highly upregulated inflammatory cytokines, pain mediators, and cartilage-degrading enzymes in degenerative FJCs. Results from ex vivo co-culture of the DRG and FJC tissue demonstrated that degenerative FJCs increased the expression of inflammatory pain molecules in the sensory neurons. CONCLUSION: Degenerative FJCs possess greatly increased inflammatory and angiogenic features, suggesting that these factors play an important role in the progression of FJD and serve as a link between joint degeneration and neurological stimulation of afferent pain fibers.

Relationship between spine osteoarthritis, bone mineral density and bone turn over markers in post menopausal women.

BACKGROUND: Several studies have observed an inverse relationship between osteoporosis and spinal osteoarthritis, the latter being considered as possibly delaying the development of osteoporosis. The aim of this study was to determine the association between individual radiographic features of spine degeneration, bone mineral density (BMD) and bone-turn over markers. METHODS: It was a cross sectional study of 277 post menopausal women. BMD of all patients was assessed at the spine and hip using dual-energy X-ray absorptiometry. Lateral spinal radiographs were evaluated for features of disc degeneration. Each vertebral level from L1/2 to L4/5 was assessed for the presence and severity of osteophytes and disc space narrowing (DSN). For Bone turn-over markers, we assessed serum osteocalcin and C-terminal cross-linking telopeptide of type I collagen (CTX). Linear regressions and partial correlation were used respectively to determine the association between each of disc degeneration features, BMD, and both CTX and osteocalcin. RESULTS: Mean age of patients was 58.7 +/- 7.7 years. Eighty four patients (31.2%) were osteoporotic and 88.44% had spine osteoarthritis. At all measured sites, there was an increase in BMD with increasing severity of disc narrowing while there was no association between severity of osteophytes and BMD. After adjustment for age and BMI, there was a significant negative correlation between CTX and DSN. However, no significant correlation was found between CTX and osteophytes and between osteocalcin and both osteophytes or DSN. CONCLUSION: In post menopausal women the severity of disc narrowing, but not osteophytes, is associated with a generalized increase in BMD and a decreased rate of bone resorption. These results are consistent with the hypothesis that osteoarthritis, through DSN, has a protective effect against bone loss, mediated by a lower rate of bone resorption. However, spine BMD is not a relevant surrogate marker for the assessment of osteoporosis in the spine in patients with osteoarthritis and debate as to the relationship between OA and OP is still open because of the contradictory data in the literature.