Decreased FoxO1 expression contributes to facet joint osteoarthritis pathogenesis by impairing chondrocyte migration and extracellular matrix synthesis.

Facet joint osteoarthritis (FJOA), a condition commonly observed in individuals of middle to old age, has been relatively under-researched compared to other subtypes of osteoarthritis (OA). This study investigated the role of transcription factor FoxO1 in FJOA using a Col2a1-creERT knock-in mouse model. It was found that FoxO1 deletion led to severe osteoarthritic changes, indicating that FoxO1 played a critical role in cartilage homeostasis. Transcriptome sequencing was performed on degenerated cartilage from FoxO1-deleted mice. This process identified differentially expressed genes (DEGs), offering insights into the molecular mechanisms underlying FJOA. Bioinformatics analysis, including Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA) and protein-protein interaction (PPI) network analysis, identified Itgb3, Itga1, Itga6, Itga7, Itga8, Itga10, Col1a1, and Il6, as potential key contributors to FJOA after FoxO1 deletion. Importantly, overexpression of Itgb3 and inhibition of Il6 counteracted FoxO1 knockdown-induced impairments in chondrocyte migration and extracellular matrix synthesis, respectively. This study discovered FoxO1 as a key regulator of the pathogenesis of FJOA, helped unravel the complex molecular mechanisms underlying FJOA, and contributed to the development of promising therapeutic avenues toward FJOA.

MiR-337-3p confers protective effect on facet joint osteoarthritis by targeting SKP2 to inhibit DUSP1 ubiquitination and inactivate MAPK pathway.

OBJECTIVE: To probe the performance of miR-337-3p on the facet joint osteoarthritis (FJOA) and its underlying mechanism. METHODS: qRT-PCR and Western blot were utilized to analyze the levels of miR-337-3p and DUSP1 in the synovial tissues from 36 FJOA patients and 10 healthy controls. The human synovial fibroblasts of FJOA were isolated and cultured followed by cell transfection. Then, cells were exposed to 10 ng/mL of IL-1ß to induce inflammatory response of synovial fibroblasts. The alternation on cell biological function in cell models was determined. The binding of miR-337-3p and SKP2 was predicted by StarBase, TargetScan, DIANA-microT and miRmap, and further verified by RIP assay and dual-luciferase reporter assay. Co-IP experiment and ubiquitination assay were used to display the binding of SKP2 and DUSP1 as well as the ubiquitination and degradation of DUSP1. After that, the FJOA rat model was established and miR-337-3p mimic or negative control was given to rats by tail vein injection. The pathological changes of synovial tissues, synovitis score, and inflammation level in rats were assessed. RESULTS: The low expressions of miR-337-3p and DUSP1 were noticed in the synovial tissues of FJOA patients and in IL-1ß-induced synovial fibroblasts, and highly expressed p-p38 MAPK was noticed. Upregulation of miR-337-3p/DUSP1 or downregulation of SKP2 inhibited IL-1ß-induced proliferation and inflammatory response of synovial fibroblasts. SKP2 was the target gene of miR-337-3p, and SKP2 induced the ubiquitination and degradation of DUSP1. MiR-337-3p exerted a protective effect on FJOA rats by alleviating damage of rat synovial tissues, promoting cell apoptosis and repressing inflammatory response. CONCLUSION: MiR-337-3p plays a protective role in FJOA by negatively targeting SKP2 to suppress DUSP1 ubiquitination and inactivate the p38 MAPK pathway.

Localization of Nerve Growth Factor Expression to Structurally Damaged Cartilaginous Tissues in Human Lumbar Facet Joint Osteoarthritis.

Purpose: Nerve Growth Factor (NGF) is a pivotal mediator of chronic pain and plays a role in bone remodelling. Through its high affinity receptor TrkA, NGF induces substance P (SP) as key downstream mediator of pain and local inflammation. Here we analysed NGF, TrkA and SP tissue distribution in facet joint osteoarthritis (FJOA), a major cause of chronic low back pain. Methods: FJOA specimens (n=19) were harvested from patients undergoing intervertebral fusion surgery. Radiologic grading of FJOA and spinal stenosis, followed by immunohistochemistry for NGF, TrkA and SP on consecutive tissue sections, was performed in ten specimens. Explant cultures (n=9) were used to assess secretion of NGF, IL-6, and SP by FJOA osteochondral tissues under basal and inflammatory conditions. Results: NGF was predominantly expressed in damaged cartilaginous tissues (80%), occasionally in bone marrow (20%), but not in osteochondral vascular channels. NGF area fraction in cartilage was not associated with the extent of proteoglycan loss or radiologic FJOA severity. Consecutive sections showed that NGF and SP expression was localized at structurally damaged cartilage, in absence of TrkA expression. SP and TrkA were expressed in subchondral bone marrow in both presence and absence of NGF. Low level NGF, but not SP secretion, was detected in four out of eighteen FJOA explants under both basal or inflammatory conditions (n=2 each). Conclusion: NGF is associated with SP expression and structural cartilage damage in osteoarthritic facet joints, but not with radiologic disease severity. NGF tissue distribution in FJOA differs from predominant subchondral bone expression reported for knee OA.

BAG3 protects chondrocytes against lumbar facet joint osteoarthritis by regulating autophagy and apoptosis.

Bcl2-associated athanogene3 (BAG3) protein, mainly induced by stressful stimuli, has been confirmed to participate in apoptosis and autophagy. In recent studies, BAG3 has gradually become a key molecule in tumors. However, the role of BAG3 in the progression of lumbar facet joint osteoarthritis (FJOA) and whether it can regulate chondrocyte apoptosis and autophagy are still unknown. In both human and FJOA rat models, we observed an upregulation of BAG3 and apoptosis and autophagy-related proteins compared with healthy tissues. Then, we established the chondrocytes injury model in vitro by using IL-1ß to stimulate human SW1353 cells. Western blot analysis data showed significant expression of BAG3, apoptosis, and autophagy-related proteins in SW1353 cells. Finally, by knocking down and overexpressing BAG3, we discovered possible anti-apoptotic and autophagy-promoted effects of BAG3 in FJOA through various experimental methods. This study demonstrated that BAG3 actively participates in regulating chondrocyte apoptosis and autophagy in FJOA and may be a highly interesting target for pharmacological interventions.

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.

Toll-like receptor involvement in adolescent scoliotic facet joint degeneration.

Facet joint osteoarthritis is prevalent in young patients with adolescent idiopathic scoliosis (AIS) and might contribute to back pain. Toll-like receptors (TLR) have been linked to cartilaginous tissue degeneration but their involvement in facet joint osteoarthritis in AIS patients is still unknown. We compared baseline gene expression levels of TLRs -1, -2, -4, and -6 in scoliotic and non-scoliotic chondrocytes and found higher expression levels in scoliotic chondrocytes with significantly higher TLR2 levels. Furthermore, TLR expression correlated strongly and significantly with inflammatory and catabolic markers in scoliotic but not in non-scoliotic chondrocytes. TLR activation with a synthetic TLR2/6 agonist resulted in a robust induction and release of pro-inflammatory and catabolic factors which exacerbated proteoglycan loss in scoliotic but not in non-scoliotic cartilage. We also detected a higher abundance of alarmins including S100A8/9 and biglycan in scoliotic cartilage. Finally, the small-molecule antagonists Sparstolonin B and o-Vanillin reduced catabolism following induction with naturally occurring alarmins and the synthetic TLR2/6 agonist. The high baseline expression, robust responsiveness and strong and significant correlation with proteases and pro-inflammatory cytokines suggest that TLRs are key regulators of facet joint degeneration in AIS. Blocking their activity could therefore potentially modify disease progression.

Interaction between C/EBPß and RUNX2 promotes apoptosis of chondrocytes during human lumbar facet joint degeneration.

The pathophysiological changes in cartilage are a crucial feature of lumbar facet joint (LFJ) degeneration and arthritis. However, the molecular mechanism of human LFJ degeneration remains largely defined. This study aimed to examine the changes in chondrocytes at different stages of degenerative LFJ using hematoxylin and eosin and Safranin O staining. The significant loss of chondrocytes in grades 2 and 3 of LFJs was observed. The expression levels of CCAAT enhancer binding protein ß (C/EBPß), Runt-related transcription factor 2 (RUNX2), and matrix metalloproteinase 13 (MMP13) also increased with the aggravation of degeneration (4.89, 5.77, and 6.3 times by Western blot). In vitro, chondrocytes scraped from the LFJs during surgery were stimulated by interleukin (IL)-1ß to establish the injury model. The association of C/EBPß and RUNX2 with active caspase-3 on chondrocytes was analyzed. The high expression level of C/EBPß, RUNX2, and MMP13 was consistent with that of caspase-3, which reached a peak after 36 h of stimulation. Immunofluorescence suggested that C/EBPß, RUNX2, and MMP13 co-labeled with active caspase-3. Moreover, immunoprecipitation data prompted that C/EBPß was able to interact with RUNX2. The knockdown of C/EBPß significantly decreased the expression levels of MMP13 and active caspase-3 (2.48 and 2.89 times as detected by Western blot analysis) and inhibited chondrocyte apoptosis, which was further demonstrated using flow cytometry. Taken together, the findings of this study uncovered that C/EBPß could interact with RUNX2 to induce chondrocyte apoptosis in human LFJ degeneration by regulating the expression of MMP13.

Isolation and characterization of stem cells from differentially degenerated human lumbar zygapophyseal articular cartilage.

The present study aimed to verify the presence of stem cells with multilineage differentiation potential in human lumbar zygapophyseal articular cartilage (LZAC) and to compare the chondrogenic potential of cells obtained from differentially degenerated articular cartilage samples. Surgically obtained human lumbar zygapophyseal joint tissues were classified into the normal, mildly degenerated and severely degenerated groups, according to their pathological characteristics. Primary chondrocytes from these groups were cultured, and stem cells were selected using a monoclonal cell culture method. Differences in stem cell morphology between the three groups were observed using inverted microscopy and phalloidin staining. In addition, stem cell chondrogenic potential was determined through induced differentiation and cellular staining. Gene and protein expression levels of the chondrogenic­specific markers aggrecan, collagen type­II and SRY­related high­mobility­group box 9 were determined using reverse transcription­quantitative polymerase chain reaction and western blotting. The clonogenic ability of stem cells in the three groups was determined using a clonogenic assay. It was revealed that stem cells with multilineage differentiation potential were isolated from all three cartilage groups; however, the cells obtained from severely degenerated articular cartilage resulted in severe fibrosis, whilst those obtained from mildly degenerated articular cartilage possessed stronger chondrogenic and clonogenic abilities. Taken together, stem cells with multilineage differentiation potential and clonal properties were identified in human LZAC, and these characteristics were more prominent in mildly degenerated as compared with severely degenerated articular cartilage.

Up-regulation of TRAF2 inhibits chondrocytes apoptosis in lumbar facet joint osteoarthritis.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) has been demonstrated that it plays a significant role in cell death receptor signal transduction. The purpose of this study was to investigate the expression of TRAF2 and its possible role in FJOA. We observed an up-regulation of TRAF2 in FJOA by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) compared to normal tissues. In vitro, we used TNF-α to stimulate Human SW1353 chondrosarcoma cells to establish the chondrocytes injury model. Western blot analysis revealed significant expression of TRAF2 and cleaved caspase-3/8 in SW1353 cells. Co-localization of TRAF2/cleaved caspase-3/8 was detected in the cells injury model by double-labeling immunofluorescent staining. We demonstrated a possible anti-apoptotic effect of TRAF2 in chondrocyte apoptosis in FJOA by knockdown of its expression with siRNA. Moreover, TRAF2 knockdown was demonstrated to enhance TNF-α-induced apoptosis by flow cytometry assay. In conclusion, our results show that the up-regulation of TRAF2 may play an important role in the inhibition of chondrocyte apoptosis of FJOA.

Altered Wnt and NF-κB Signaling in Facet Joint Osteoarthritis: Insights from RNA Deep Sequencing.

Facet joint osteoarthritis is common lumbar osteoarthritis characterized by facet joint cartilage degeneration. However, the molecular basis of facet joint osteoarthritis remains largely undetermined. In the current study, we collected facet joint tissue samples from 10 control patients and 48 patients with facet joint osteoarthritis (20 patients with moderate degeneration and 28 with severe degeneration). The control patients underwent internal fixation of the lumbar spine due to vertebral fracture. RNA deep sequencing was performed, and Bioinformatic tools were applied. Among top 30 enriched signaling pathways, we focused on two inflammation-related signaling pathways, Wnt and NF-κB signaling pathways. Subsequently, using the quantitative RT-PCR analysis, we confirmed that in Wnt signaling pathway, the mRNA levels of Dickkopf WNT Signaling Pathway Inhibitor 2 (DKK2), Sex-determining Region Y-box 17 (SOX17), MYC, Cyclin D1, Calcium/Calmodulin Dependent Protein Kinase II Alpha (CAMK2A), and Wnt Family Member 11 and 5 were increased in facet joint osteoarthritis, while the mRNA levels of WNT Inhibitory Factor 1, Casein Kinase 1 Alpha 1, Transcription Factor 7/Lymphoid Enhancer Binding Factor 1 (TCF7/LEF1), and VANGL Planar Cell Polarity Protein 2 were decreased. In NF-κB signaling pathway, the mRNA levels of C-C Motif Chemokine Ligand 4 (CCL4) and C-C Motif Chemokine Ligand 4 Like 2 (CCL4L2) were increased, while the mRNA levels of BCL2 Related Protein A1 were decreased. These results suggest that Wnt and NF-κB signaling may be altered in the process of facet joint cartilage degeneration. The present study will expand our understanding of the molecular bases underlying facet joint osteoarthritis.

Transcriptional information revealed differentially expressed circular RNAs in facet joint osteoarthritis.

Circular RNAs are newly identified RNAs that regulate gene expression and execute various biological activities. Facet joint osteoarthritis (FJOA) is a severe facet joint cartilage degeneration disease that induces chronic low back pain to a larger population. The molecular mechanisms of FJOA, especially the presence and changes of circular RNAs in FJOA have not been investigated. In the current study, we analyzed RNA deep sequencing outcomes from healthy control group and FJOA group, identified and annotated circular RNAs, and determined the expressions of circular RNAs. Differentially expressed circular RNAs in the FJOA group were then screened and the host genes of these differentially expressed circular RNAs were functional analyzed by Gene Ontology (GO), Kyoto Enrichment of Genes and Genomes (KEGG), and protein-protein interaction network analysis. Seven circRNAs were arbitrarily selected to confirm the trustworthiness of these profiles by quantitative qRT-PCR. The potential binding sites of microRNAs (miRNAs) and identified circular RNAs were also predicted. Our current study provided valuable bioinformation regarding circular RNAs in FJOA and expanded our knowledge of FJOA.

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.

Intra-articular nerve growth factor regulates development, but not maintenance, of injury-induced facet joint pain & spinal neuronal hypersensitivity.

OBJECTIVE: The objective of the current study is to define whether intra-articular nerve growth factor (NGF), an inflammatory mediator that contributes to osteoarthritic pain, is necessary and sufficient for the development or maintenance of injury-induced facet joint pain and its concomitant spinal neuronal hyperexcitability. METHOD: Male Holtzman rats underwent painful cervical facet joint distraction (FJD) or sham procedures. Mechanical hyperalgesia was assessed in the forepaws, and NGF expression was quantified in the C6/C7 facet joint. An anti-NGF antibody was administered intra-articularly in additional rats immediately or 1 day following facet distraction or sham procedures to block intra-articular NGF and test its contribution to initiation and/or maintenance of facet joint pain and spinal neuronal hyperexcitability. NGF was injected into the bilateral C6/C7 facet joints in separate rats to determine if NGF alone is sufficient to induce these behavioral and neuronal responses. RESULTS: NGF expression increases in the cervical facet joint in association with behavioral sensitivity after that joint's mechanical injury. Intra-articular application of anti-NGF immediately after a joint distraction prevents the development of both injury-induced pain and hyperexcitability of spinal neurons. Yet, intra-articular anti-NGF applied after pain has developed does not attenuate either behavioral or neuronal hyperexcitability. Intra-articular NGF administered to the facet in naïve rats also induces behavioral hypersensitivity and spinal neuronal hyperexcitability. CONCLUSION: Findings demonstrate that NGF in the facet joint contributes to the development of injury-induced joint pain. Localized blocking of NGF signaling in the joint may provide potential treatment for joint pain.

Cartilage in facet joints of patients with ankylosing spondylitis (AS) shows signs of cartilage degeneration rather than chondrocyte hypertrophy: implications for joint remodeling in AS.

INTRODUCTION: In ankylosing spondylitis (AS), joint remodeling leading to joint ankylosis involves cartilage fusion. Here, we analyzed whether chondrocyte hypertrophy is involved in cartilage fusion and subsequent joint remodeling in AS. METHODS: We assessed the expression of chondrocyte hypertrophy markers runt-related transcription factor 2 (Runx2), type X collagen (COL10), matrix metalloproteinase 13 (MMP13), osteocalcin and beta-catenin and the expression of positive bone morphogenic proteins (BMPs) and negative regulators (dickkopf-1 (DKK-1)), sclerostin, (wingless inhibitory factor 1 (wif-1)) of chondrocyte hypertrophy in the cartilage of facet joints from patients with AS or osteoarthritis (OA) and from autopsy controls (CO) by immunohistochemistry. Sex determining region Y (SRY)-box 9 (Sox9) and type II collagen (COL2) expression was assessed as indicators of chondrocyte integrity and function. RESULTS: The percentage of hypertrophic chondrocytes expressing Runx2, COL10, MMP13, osteocalcin or beta-catenin was significantly increased in OA but not in AS joints compared to CO joints. Frequencies of sclerostin-positive and DKK-1-positive chondrocytes were similar in AS and CO. In contrast, wif-1- but also BMP-2- and BMP-7-expressing and Sox9-expressing chondrocytes were drastically reduced in AS joints compared to CO as well as OA joints whereas the percentage of COL2-expressing chondrocytes was significantly higher in AS joints compared to CO joints. CONCLUSIONS: We found no evidence for chondrocyte hypertrophy within hyaline cartilage of AS joints even in the presence of reduced expression of the wnt inhibitor wif-1 suggesting that chondrocyte hypertrophy is not a predominant pathway involved in joint fusion and remodeling in AS. In contrast, the reduced expression of Sox9, BMP-2 and BMP-7 concomitantly with induced COL2 expression rather point to disturbed cartilage homeostasis promoting cartilage degeneration 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.

In situ analysis of interleukin-6 expression at different sites of zygapophyseal joints from patients with ankylosing spondylitis in comparison to controls.

OBJECTIVES: Analysis of interleukin (IL)-6 serum levels in patients with ankylosing spondylitis (AS) has indicated that IL-6 might be a pro-inflammatory cytokine involved in AS. However, two placebo-controlled trials with monoclonal antibodies directed against the IL-6 receptor have failed to demonstrate the efficacy of the monoclonal humanized anti-human IL-6 receptor antibody over placebo for the treatment of symptoms of AS. In this study we conducted an in situ analysis of IL-6 expression at different sites of inflammation in zygapophyseal joints of patients with AS in comparison to osteoarthritis autopsy controls (CO). METHOD: Our immunohistochemical analysis involved 14 patients with AS, 12 autopsy controls (CO), and 11 patients with osteoarthritis (OA). Immunohistochemistry was performed to detect IL-6+ cells at five different sites: within subchondral bone marrow, fibrous tissue replacing subchondral bone marrow, hyaline cartilage, and the subchondral bone plate, and at entheseal sites. RESULTS: Apart from changes in subchondral bone marrow, no significant differences were observed at the sites analysed when comparing AS patients and controls. A significantly lower frequency of IL-6+ cells was evident in AS patients compared to controls (p = 0.0043). In addition, AS patients tended to have even lower percentages of IL-6+ cells than controls at subchondral bone plates and entheseal sites. A significantly lower number of IL-6 expressing cells was also seen within the fibrous tissue of AS compared to OA patients (p = 0.0237). CONCLUSIONS: This in situ analysis confirms that IL-6 is not a key player in the pathogenesis of inflammatory processes in spondyloarthritides (SpA). The relevance of pro-inflammatory agents in axial SpA might be studied better in situ in bony specimens at the primary site of inflammation.

Indian Hedgehog signaling pathway members are associated with magnetic resonance imaging manifestations and pathological scores in lumbar facet joint osteoarthritis.

Indian Hedgehog (HH) has been shown to be involved in osteoarthritis (OA) in articular joints, where there is evidence that Indian HH blockade could ameliorate OA. It seems to play a prominent role in development of the intervertebral disc (IVD) and in postnatal maintenance. There is little work on IHH in the IVD. Hence the aim of the current study was to investigate the role of Indian Hedgehog in the pathology of facet joint (FJ) OA. 24 patients diagnosed with lumbar intervertebral disk herniation or degenerative spinal stenosis were included. Preoperative magnetic resonance imaging (MRI) and Osteoarthritis Research Society International (OARSI) histopathology grading system was correlated to the mRNA levels of GLI1, PTCH1, and HHIP in the FJs. The Weishaupt grading and OARSI scores showed high positive correlation (r = 0.894) (P < 0.01). MRI Weishaupt grades showed positive correlation with GLI1 (r = 0.491), PTCH1 (r = 0.444), and HHIP (r = 0.654) mRNA levels (P < 0.05 in each case). OARSI scores were also positively correlated with GLI1 (r = 0. 646), PTCH1 (r = 0. 518), and HHIP (r = 0.762) mRNA levels (P < 0.01 in each case). Cumulatively our findings indicate that Indian HH signaling is increased in OA and is perhaps a key component in OA pathogenesis and progression.

Establishment of a rat model of lumbar facet joint osteoarthritis using intraarticular injection of urinary plasminogen activator.

Lumbar facet joint (LFJ) osteoarthritis (OA) is an important etiology of low back pain. Several animal models of LFJ OA have been established using intraarticular injection of various chemicals. This study aimed to establish a rat model of LFJ OA using urinary plasminogen activator (uPA). Sprague-Dawley rats were treated with intraarticular injection in the L5-L6 facet joints with uPA (OA group, n = 40) or normal saline (vehicle group, n = 40). Mechanical and thermal hyperalgesia in the ipsilateral hind paws were evaluated using von Frey hairs and a thermoalgesia instrument, respectively. Toluidine blue staining, hematoxylin-eosin staining, and immunohistochemical examination of the LFJ was performed. Treatment with uPA induced cartilage damage, synovitis, and proliferation of synovial cells in the fact joints. The OA group showed significantly higher hyperalgesia in the hind paws in comparison with the vehicle group and normal controls (P < 0.05). Expression of IL-1ß, TNF-α, and iNOS in the LFJ cartilage in the OA group was significantly increased (P < 0.05). A rat model of LFJ OA was successfully established using intraarticular injection of uPA. This animal model is convenient and shows good resemblance to human OA pathology.