MiR-124 regulates osteoblast differentiation through GSK-3ß in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a spastic and spinal joint disease with the characteristic of pathological ossification. Bioinformatics analysis demonstrated that there is a complementary binding site between microRNA-124 (miR-124) and the 3'-UTR of glycogen synthase kinase-3ß (GSK-3ß) mRNA. We aimed to investigate the role of miR-124 in regulating GSK-3ß expression, Wnt/ß-catenin pathway activity, and osteoblast differentiation of spinal ligament fibroblasts. PATIENTS AND METHODS: The ligament tissues of AS and the femoral neck fracture patients were collected. MiR-124 and GSK-3ß mRNA expressions were detected by using quantitative Real-time PCR (qRT-PCR). GSK-3ß and ß-catenin protein expressions were detected by using Western blot. Ligament fibroblasts were isolated and induced to differentiate into osteoblasts. Alizarin red S staining (ARS) was used to identify osteoblast differentiation. Expressions of miR-124, GSK-3ß, ß-catenin, Osterix, and runt-related transcription factor 2 (RUNX2) were detected during differentiation. The cells were divided into two groups as agomiR-normal control (NC) transfection group and agomir miR-124 transfection group. Alkaline phosphatase (ALP) activity and Alizarin Red S staining were detected. RESULTS: MiR-124 and ß-catenin expressions in the ligament of AS patients increased, while GSK-3ß level reduced compared with control. MiR-124, ß-catenin, Osterix, and RUNX2 expressions gradually elevated, whereas GSK-3ß level gradually declined following increased osteoblasts differentiation. Antagomir miR-124 transfection significantly up-regulated the expression of GSK-3ß in osteoblast differentiation, significantly decreased the expression of ß-catenin, Osterix, and RUNX2, and significantly inhibited osteoblast differentiation. CONCLUSIONS: MiR-124 decreased and GSK-3ß elevated in AS ligament tissue. Down-regulation of miR-124 expression enhanced GSK-3ß expression, weakened Wnt/ß-catenin pathway activity, and inhibited the differentiation of ligament fibroblasts into osteoblasts.

Modulation of MMP-2 and MMP-9 through connected pathways and growth factors is critical for extracellular matrix balance of intra-articular ligaments.

The healing mechanism of cruciate ligaments is not well elucidated. Crosstalk between adjacent tissues in the knee joint plays an important role in wound healing and tissue regeneration, but the gelatinase modulation in posterior cruciate ligament fibroblasts (PCLfs) and synovial cells (SCs) based on co-culture is still elusive. The present study sought to systematically elucidate the gelatinase modulation in both PCLfs and SCs based on in vitro co-culture and in a rabbit PCL-injury model in vivo. It was found that injured PCLfs and SCs can secrete high gelatinases after co-culture. Cytokines promote greater gelatinase secretion by both injured PCLfs and SCs in the form of monomers and dimers. Pathway inhibitors can reduce injury-induced gelatinase activities, but the presence of cytokines restores the higher activity. Inhibitor cocktails can reduce gelatinase expression to a normal level even in the presence of cytokines. Growth factors promote wound healing of the injured PCL by enhancing cell migration, proliferation and collagen synthesis, but also upregulate gelatinases. Modified inhibitor cocktails containing growth factor can also reduce gelatinase expression to a normal level. This gelatinase modulation was also verified in a rabbit PCL injury model in vivo. Together, the results aids understanding of the mechanism of gelatinase modulation in injured PCL ligament post-crosstalk with synovium and infers that the gelatinases could be a potential as a therapeutic target for acute ligament injury. Copyright © 2016 John Wiley & Sons, Ltd.

Nitric oxide and metalloproteinases in canine articular ligaments: a comparison between the cranial cruciate, the medial genual collateral and the femoral head ligament.

Osteoarthritis due to cranial cruciate ligament (CCL) rupture or hip dysplasia is one of the most important causes of chronic lameness in dogs. This study aimed at comparing nitric oxide (NO) production by the CCL with that of the femoral head ligament (FHL) and the medial collateral ligament (MCL), and investigating the pathway of NO production and the concomitant metalloproteinase (MMP) activity in the presence or absence of an inflammatory stimulus. Ligaments of normal dogs were subjected to different stimuli, and NO and MMP activity from explant culture supernatants were compared. The results showed that in explant cultures of the canine CCL more NO was produced than in those of the other two ligaments. A higher level of NO was produced when CCLs were exposed to the inducible nitric oxide synthase (iNOS)-inducing cocktail TNF/IL-1/LPS, and NO synthesis could be inhibited by both l-NMMA, a general nitric oxide synthase (NOS) inhibitor and l-NIL, a specific iNOS inhibitor. However, a correlation between NO synthesis and iNOS expression levels as determined by immunohistochemistry was not observed. In contrast to CCL, no evidence for iNOS-dependent NO synthesis was observed for MCL and FHL. The CCL produced less MMP than MCL and FHL, and no correlation between MMP and NO could be demonstrated. MMP activity in the CCL increased significantly after 48 h of incubation with the inflammatory stimulus. The results suggest that in canine osteoarthritis NO synthesized by canine CCL plays a more important role in the pathogenesis of osteoarthritis of the stifle than that synthesized by FHL and MCL.

Quantitative cytochemical evidence for local increases in bone turnover at the acromial enthesis of the human coracoacromial ligament.

OBJECTIVE: Enthesophytic bone outgrowths are found at many ligament attachment sites, and while their incidence is associated with many pathologies, the mechanism by which they form remains controversial. We hypothesized that changes in local cell behavior, provoked by mechanical alterations within the coracoacromial ligament (CAL), lead to acromial enthesophyte formation. We investigated whether cell behavior at acromial entheses is consistent with this. METHODS: We used quantitative enzyme cytochemistry to measure glucose 6-phosphate dehydrogenase (G6PD), alkaline phosphatase (ALP; osteoblastic activity), and tartrate-resistant acid phosphatase (TRAP; osteoclastic phenotype) activities in cells of the acromial attachment into the CAL in patients with rotator cuff tears. RESULTS: (1) Resident osteoblasts on the acromion's inferior aspect express elevated activity of G6PD and ALP, indicative of increases in osteogenic potential. (2) These activities are selectively raised at the "leading edge" of acromial bone CAL enthesis. (3) In contrast, distribution of TRAP-positive cells does not exhibit a spatial correlation with enthesis architecture. We also found that cells situated close to the CAL attachment into the acromion exhibited elevated levels of G6PD and ALP activity, but intriguingly, also showed higher TRAP activity than neighboring cells distant from entheses. CONCLUSION: These results suggest that the acromion in these patients undergoes bone accretion at the inferior attachment of the CAL, and that enthesial ligament cells close to the bone express characteristics consistent with enthesophyte formation at the leading edge of this bony spur's extension into the ligament.

Ligament grafts become more susceptible to creep within days after surgery: evidence for early enzymatic degradation of a ligament graft in a rabbit model.

Clinical evidence suggests that some ligament grafts stretch after surgery. Our purpose in this study was to quantify early postoperative creep behavior of ligament autografts in an animal model, and to explore potential mechanisms of that behavior. 38 New Zealand white rabbits underwent a unilateral, fresh, anatomic medial collateral ligament (MCL) autograft procedure and were killed immediately (time-zero), at 2 days, 3 weeks, or 8 weeks after surgery (n = 7-11 in each group). We compared the creep behavior of the autografts to normal MCLs (n = 8). An additional 7 MCL specimens were incubated for 2 days in a low concentration collagenase solution and then similarly creep-tested. All grafts were slower to recover their original length after creep than either normal ligaments or time-zero controls. These grafts started to become more vulnerable to elongation in cyclic and static creep tests within 2 days of surgery, compared to time-zero controls. This vulnerability to creep increased over the next 3 weeks, and was maintained at 8 weeks of healing. 2-day collagenase-soaked MCL specimens had the same creep strains as the 2-day autografts. These results suggest that even fresh anatomic ligament autografts become vulnerable to creep within a few days after surgery by mechanisms that may involve degradative enzymes such as collagenase.

Collagenases from periarticular ligaments and tendon: enzyme levels during the development of joint contracture.

Our laboratories have previously demonstrated that normal rabbit periarticular ligaments, anterior cruciate ligaments (ACL), medial collateral ligaments (MCL) and patellar tendon (PT) secrete collagenase. In this current study we examined these connective tissues following an immobilization period of 4 weeks. In the ligaments producing collagenase, activity was expressed only in the control, not in the immobilized joint. Control and experimental patellar tendon samples produce collagenolytic activity, suggesting that the expression of enzyme is less affected in tendons as compared to ligaments. Characterization of these collagenases was carried out using an antiserum directed against rabbit synovial collagenase. We demonstrated that ligament (ACL) and tendon (PT) collagenases cross react with this antibody in a double immunodiffusion assay. Protein blots of PT, ACL and MCL collagenases identified one major species (Mr = 45,000) and a minor species (Mr = 50,000) of immunoreactive proteins in all three connective tissues. Differences between control and experimental enzyme levels appear to be due to less collagenase protein being produced by immobilized ligaments.

Collagenase production by rabbit ligaments and tendon.

Three periarticular connective tissues from normal rabbits were examined for collagenolytic activity. Enzyme activity was secreted by cultures of anterior cruciate ligament (ACL), medial collateral ligament (MCL) and patellar tendon (PT). A lag period of six days or more was often observed prior to the detection of active collagenase. We attributed this to the presence of an excess of inhibitor in the early days of culture. We quantitated the amount of enzyme and inhibitor produced in 13 days. The levels of collagenase in the ACL and MCL were comparable. The PT, however, consistently secreted more enzyme than the two periarticular (ACL and MCL) ligaments. The reaction products were analyzed for all three collagenases and compared to those generated by the rabbit skin enzyme. We observed the characteristic TCA and TCB collagen fragments for MCL and PT enzymes. Collagen cleavage by the ACL cultures resulted in a product with a molecular weight intermediate between the alpha 2 chain and the TCA piece. These data suggest that quantitative and qualitative differences exist in the ability of these similar connective tissues to degrade collagen.

Effects of the hormone relaxin on the metabolism of the glycosaminoglycans in the mouse symphysis pubis.

The influence of the peptide hormone relaxin on the glycosaminoglycan (GAG) metabolism was investigated in the pubic ligament of the symphysis pubis and in serum of the virgin mouse. Fresh weight DNA and GAG content per 1 ligament is significantly increased, the level of water soluble protein is not affected. A shift in the electrophoretic GAG pattern by an increasing amount of hyaluronic acid and a decreasing amount of chondroitin sulfate and dermatan sulfate can be observed. Concerning GAG-splitting enzymes (N-acetylglucosaminidase, arylsulfatase, beta-glucuronidase) the N-acetylglucosaminidase reveals a significant increase of its activity in the interpubic ligament and in the serum. The data demonstrate that relaxin treatment induces some changes in the GAG metabolism.

Enzymatic adaptation in ligaments during immobilization.

Ligaments are a composite of fibroblasts and collagen in a proteoglycan matrix. Seventy-five percent of the organic solid is collagen and 23% is proteoglycan. Fibroblasts are responsible for the overall composition of the ligament, that is the synthesis and the degradation of macromolecular components. Like muscle and bone, ligaments are dynamic, undergoing hypertrophy with exercise and atrophy with immobilization. This paper reviews the structure and composition of ligaments and discusses the cellular events responsible for atrophy of ligaments with immobilization. As an experimental model, one knee of New Zealand White rabbits was immobilized with a pin. After 2, 4, and 8 weeks of immobility, the medial collateral ligaments were isolated and enzyme analysis was performed. Gross and microscopic changes were apparent after 2 weeks. As for enzyme changes, lactic dehydrogenase and malic dehydrogenase decreased in activity. The lysosomal hydrolases responsible for glycosaminoglycan degradation increased in activity, suggesting that enzymatic adaptations mediate the physical and chemical changes in the ligament. The cells switch from an anabolic synthetic state to a catabolic, degradative state during immobility. It would seem from the biochemical viewpoint that, whenever possible, cast-bracing and functional splints may be preferable to rigid plasters in many sports-related ligamentous injuries.

Synovitis after surgical division of the anterior cruciate ligament of the dog.

The synovial reaction to the surgical division of an anterior cruciate ligament in inbred beagle dogs, was studied. The left ligaments of 16 animals were divided by a 'blind' technique: the right stifle joints were also explored under aseptic conditions but the cruciate ligaments were not cut. Operation was followed by the formation of a blood-stained sterile effusion and by acute synovitis, sustained on the left, transient on the right. The effusion gradually diminished, becoming serous, but haemosiderin remained within subsynoviocytic macrophages for at least 84 days and lymphocytic foci and synovial cell hyperplasia persisted for similar times. It is suggested that interpretation of the cartilage changes in this cruciate ligament division model of osteoarthrosis should take account of the extent and duration of the post-operative inflammation.