Tenascin-C levels in synovial fluid are elevated after injury to the human and canine joint and correlate with markers of inflammation and matrix degradation.

OBJECTIVE: We have previously shown the capacity of tenascin-C (TN-C) to induce inflammatory mediators and matrix degradation in vitro in human articular cartilage. The objective of the present study was to follow TN-C release into knee synovial fluid after acute joint injury or in joint disease, and to correlate TN-C levels with markers of cartilage matrix degradation and inflammation. METHOD: Human knee synovial fluid samples (n = 164) were from a cross-sectional convenience cohort. Diagnostic groups were knee healthy reference, knee anterior cruciate ligament rupture, with or without concomitant meniscus lesions, isolated knee meniscus injury, acute inflammatory arthritis (AIA) and knee osteoarthritis (OA). TN-C was measured in synovial fluid samples using an enzyme-linked immunosorbent assay (ELISA) and results correlated to other cartilage markers. TN-C release was also monitored in joints of dogs that underwent knee instability surgery. RESULTS: Statistically significantly higher levels of TN-C compared to reference subjects were observed in the joint fluid of all human disease groups and in the dogs that underwent knee instability surgery. Statistically significant correlations were observed between the TN-C levels in the synovial fluid of the human patients and the levels of aggrecanase-dependent Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments and matrix metalloproteinases 1 and 3. CONCLUSIONS: We find highly elevated levels of TN-C in human knee joints after injury, AIA or OA that correlated with markers of cartilage degradation and inflammation. TN-C in synovial fluid may serve dual roles as a marker of joint damage and a stimulant of further joint degradation.

Elevated aggrecanase activity in a rat model of joint injury is attenuated by an aggrecanase specific inhibitor.

OBJECTIVE: To evaluate aggrecanase activity after traumatic knee injury in a rat model by measuring the level of aggrecanase-generated Ala-Arg-Gly-aggrecan (ARG-aggrecan) fragments in synovial fluid, and compare with ARG-aggrecan release into joint fluid following human knee injury. To evaluate the effect of small molecule inhibitors on induced aggrecanase activity in the rat model. METHOD: An enzyme-linked immunosorbent assay (ELISA) was developed to measure ARG-aggrecan levels in animal and human joint fluids. A rat model of meniscal tear (MT)-induced joint instability was used to assess ARG-aggrecan release into joint fluid and the effects of aggrecanase inhibition. Synovial fluids were also obtained from patients with acute joint injury or osteoarthritis and assayed for ARG-aggrecan. RESULTS: Joint fluids from human patients after knee injury showed significantly enhanced levels of ARG-aggrecan compared to uninjured reference subjects. Similarly, synovial fluid ARG-aggrecan levels increased following surgically-induced joint instability in the rat MT model, which was significantly attenuated by orally dosing the animals with AGG-523, an aggrecanase specific inhibitor. CONCLUSIONS: Aggrecanase-generated aggrecan fragments were rapidly released into human and rat joint fluids after injury to the knee and remained elevated over a prolonged period. Our findings in human and preclinical models strengthen the connection between aggrecanase activity in joints and knee injury and disease. The ability of a small molecule aggrecanase inhibitor to reduce the release of aggrecanase-generated aggrecan fragments into rat joints suggests that pharmacologic inhibition of aggrecanase activity in humans may be an effective treatment for slowing cartilage degradation following joint injury.

The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse.

AIM: To describe a histologic scoring system for murine osteoarthritis (OA) that can be applied universally to instability, enzymatic, transgenic and spontaneous OA models. METHODS: Scientists with expertise in assessing murine OA histopathology reviewed the merits and drawbacks of methods described in the literature. A semi-quantitative scoring system that could reasonably be employed in any basic cartilage histology laboratory was proposed. This scoring system was applied to a set of 10 images of the medial tibial plateau and femoral condyle to yield 20 scores. These images were scored twice by four experienced scorers (CL, SG, MC, TA), with a minimum time interval of 1 week between scores to obtain intra-observer variability. An additional three novice scorers (CR, CL and MM) with no previous experience evaluated the images to determine the ease of use and reproducibility across laboratories. RESULTS: The semi-quantitative scoring system was relatively easy to apply for both experienced and novice scorers and the results had low inter- and intra-scorer variability. The variation in scores across both the experienced and novice scorers was low for both tibia and femur, with the tibia always having greater consistency. CONCLUSIONS: The semi-quantitative scoring system recommended here is simple to apply and required no specialized equipment. Scoring of the tibial plateaus was highly reproducible and more consistent than that of the femur due to the much thinner femoral cartilage. This scoring system may be a useful tool for both new and experienced scorers to sensitively evaluate models and OA mechanisms, and also provide a common paradigm for comparative evaluation across the many groups performing these analyses.

Histopathology atlas of animal model systems - overview of guiding principles.

Animal model systems represent an important adjunct and surrogate for studies of osteoarthritis (OA) in humans. They provide a means to study OA pathophysiology as well as aid in the development of therapeutic agents and biological markers for diagnosing and prognosing the disease. Thus, it is of great importance for the OA scientific community, both in academic as well as industrial research, to standardize scoring systems for evaluating the OA disease process and to make results between different studies comparable. The task of the histopathology initiative of OARSI was to achieve a consensus of scoring systems for the most important species used in OA animal model research (dog, guinea pig, horse, mouse, rabbit, rat, and sheep/goat), which are presented in the various chapters in this special volume of Osteoarthritis & Cartilage together with extra chapters on basic methodology (histochemistry, statistics, morphometry), the specific terminology and a general discussion of animal models in OA research. Standardized definitions are suggested for basic but essential terms such as "grading" and "staging" in order to promote their consistent use and thereby promote improved understanding and data interpretation across all model systems. Thus, this introductory chapter presents an overview of the guiding principles for assessment of important OA animal model systems. Use of such systems, independently or in conjunction with other systems in parallel, should facilitate comparability of results across animal model studies.

Inhibition of osteoclasts prevents cartilage loss and pain in a rat model of degenerative joint disease.

OBJECTIVE: To investigate the relationship between efficacy of a bisphosphonate, pain and extent of joint damage in the monosodium iodoacetate (MIA) model of painful degenerative joint disease. METHODS: Zoledronate treatment was initiated prior to and at various times following model induction, including late time points representing advanced disease. Radiographic and histological structural parameters were correlated with pain as measured by weight bearing. RESULTS: Intraarticular (IA) MIA resulted in a progressive loss of bone mineral density (BMD) and chondrocytes, thinning of cartilage, loss of proteoglycan, resorption of calcified cartilage and subchondral bone, as well as pain. This was completely prevented by pre-emptive chronic zoledronate treatment with joint sections being histologically indistinguishable from saline-injected controls. When initiation of treatment was delayed efficacy was reduced. In animals with advanced joint degeneration, treatment partially restored BMD and had a significant, but limited, effect on pain. We confirmed these radiographic and behavioral findings in the medial meniscal tear model. To understand the mechanism-of-action of zoledronate we investigated an early time point 4 days post-model induction when chondrocytes were histologically viable, with minor loss of proteoglycan and generalized synovitis. Osteoclast-mediated resorption of the calcified cartilage was observed and was prevented by two doses of zoledronate. CONCLUSION: Subchondral bone remodeling plays an important role in nociception and the pathobiology of the MIA model with osteoclasts being implicated in both bone and cartilage resorption. Inhibition of osteoclastic activity when initiated early leads to improved efficacy.

ADAMTS5-/- mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and subchondral bone changes.

OBJECTIVE: Osteoarthritis (OA) is characterized by damaged articular cartilage and changes in subchondral bone. Previous work demonstrated aggrecanase-2 deficient (ADAMTS5-/-) mice to be protected from cartilage damage induced by joint instability. This study analyzed whether this protective effect on cartilage is also reflected in the subchondral bone structure. METHODS: Right knee joints from 10-week old male wild type (WT) and ADAMTS5-/- mice received transection of the medial meniscotibial ligament to induce OA, whereas left knees were left unoperated. After 8 weeks knee joints were scanned by micro-CT. The proximal tibia was selected for further analysis. Histology was performed to evaluate cartilage damage and osteoclast presence. RESULTS: ADAMTS5-/- joints had a significantly thinner subchondral plate and less epiphyseal trabecular bone compared to WT joints. Histology confirmed previous findings that ADAMTS5-/- mice have significantly less cartilage damage than WT in the instability-induced OA model. Although the subchondral bone plate became significantly thicker at the medial tibial plateau in operated joints of both genotypes, the percentage increase was significantly smaller in ADAMTS5-/- mice (WT: 20.7+/-4.7%, ADAMTS5-/-: 8.3+/-1.2% compared to the left unoperated control joint). In ADAMTS5-/- animals a significant decrease was found in both Oc.N./BS and Oc.S./BS. Finally, in WT but not in ADAMTS5-/- mice a significant correlation was found between medial subchondral bone plate thickness and cartilage damage at the medial tibial plateau. CONCLUSION: ADAMTS5-/- joints that were protected from cartilage damage showed minor changes in the subchondral bone structure, in contrast to WT mice where substantial changes were found. This finding suggests links between the process of cartilage damage and subchondral bone changes in instability-induced OA.

The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse.

OBJECTIVE: To evaluate anterior cruciate ligament transection (ACLT) and destabilization of the medial meniscus (DMM) surgical instability models of osteoarthritis (OA) in the 129/SvEv mouse knee joint. DESIGN: Micro-surgical techniques were used to perform ACLT or DMM under direct visualization. Histological scoring was performed on multiple sections to assess cartilage damage across the entire joint. RESULTS: The ACLT model gave severe OA, chondrogenesis of the joint capsule and, in some cases, severe subchondral erosion of the posterior tibial plateau. Surgical DMM was less invasive than the ACLT procedure and resulted in lesions primarily on the central weight-bearing region of the medial tibial plateau and medial femoral condyles. Lesions in the DMM model progressed from mild-to-moderate OA at 4 weeks, to moderate-to-severe OA at 8 weeks post-surgery. Destruction of the subchondral bone was never observed in the DMM model. CONCLUSIONS: ACLT is not recommended in the mouse due to the high surgical proficiency required and the development of severe OA that may involve subchondral bone erosion. The severity and location of lesions following DMM are consistent with lesions observed in aged spontaneous mouse models of OA. The DMM model has sufficient sensitivity to show disease modification, as observed with the ADAMTS-5 knock out (KO) mouse. The DMM model should be a first choice to challenge mice with gene deletions of potential targets in OA.

Osteoarthritis severity is sex dependent in a surgical mouse model.

OBJECTIVE: To investigate the role of sex hormones in cartilage degradation and progression of osteoarthritis (OA) in a murine model induced by destabilization of the medial meniscus (DMM). DESIGN: Accelerated OA development in mice was induced by transection of the menisco-tibial ligament, which anchors the medial meniscus to the tibial plateau. Intact male and female, and orchiectomized (ORX) male and ovariectomized (OVX) female mouse knee histology were compared for signs of OA following DMM. The effect of testosterone or estrogen addition in vivo was assessed in ORX males in the surgical OA model. RESULTS: OA severity was markedly higher in males than females after DMM. OVX females developed significantly more severe OA than control females. ORX males developed significantly less severe OA than control males. When ORX male mice were supplemented with exogenous dihydrotestosterone (DHT), the severity of OA was restored to the level experienced by the control male mice. Hip cartilage from mice of both sexes demonstrated similar spontaneous and interleukin-1alpha (IL-1alpha) induced proteoglycan (PG) release in vitro. DHT and 17-beta estradiol (E2) did not significantly alter the PG release pattern when supplemented to cartilage cultures of either sex. CONCLUSION: Sex hormones play a critical role in the progression of OA in the murine DMM surgical model, with males having more severe OA than females. Intact females had more OA than OVX females, indicating that ovarian hormones decrease the severity of OA in the female mice. Male hormones, such as testosterone, exacerbate OA in male mice as demonstrated by the fact that ORX mice experienced less OA than intact males, and that addition of DHT to ORX males was able to counteract the effect of castration and re-establish severe OA.

Repair of articular cartilage defects one year after treatment with recombinant human bone morphogenetic protein-2 (rhBMP-2).

BACKGROUND: Damaged articular cartilage has a limited ability to repair. Operative removal of damaged cartilage and penetration into the subchondral bone to allow population of the defect with progenitor cells can result in filling of the defect with repair tissue. However, this repair tissue often degenerates over time because of its inability to withstand the mechanical forces to which it is subjected. We previously reported that recombinant human bone morphogenetic protein-2 (rhBMP-2) improves the repair of full-thickness defects of cartilage as long as six months postoperatively. We have now extended that study to examine the quality of the repair tissue at one year. METHODS: Full-thickness defects of cartilage were created in the trochlear groove of twenty-five adult New Zealand White rabbits. Eight defects were left empty, eight were filled with a collagen sponge, and nine were filled with a collagen sponge impregnated with five micrograms of rhBMP-2. The animals were killed at fifty-two weeks postoperatively, and the gross appearance of the healed defect was assessed. The repair tissue was examined histologically and was evaluated, according to a grading scale, by four individuals who were blinded with respect to the treatment. The tissue sections were immunostained with antibodies against type-I collagen, type-II collagen, aggrecan, and link protein. The residence time of the rhBMP-2 in the cartilage defect was evaluated in vivo with use of scintigraphic imaging of radiolabeled protein. RESULTS: One year after a single implantation of a collagen sponge containing five micrograms of rhBMP-2, the defects had a significantly better histological appearance than the untreated defects (those left empty or filled with a collagen sponge). The histological features that showed improvement were integration at the margin, cellular morphology, architecture within the defect, and reformation of the tidemark. The total scores were also better for the defects treated with rhBMP-2 than for the untreated defects, but in no instance was the repair tissue identical to normal articular cartilage. The thickness of the cartilage in the defects treated with rhBMP-2 was 70 percent that of the normal cartilage, an observation that was identical to that at twenty-four weeks postoperatively. Immunostaining demonstrated significantly less type-I collagen in the defects treated with rhBMP-2 than in the untreated defects. Immunostaining for other matrix components showed no difference among the treatment groups. The mean residence time of rhBMP-2 in the cartilage defects was eight days with an elimination half-life of 5.6 days. Detectable amounts of rhBMP-2 were present as long as fourteen days after implantation. CONCLUSIONS: The problems associated with operative repair of cartilage include the formation of fibrocartilage rather than normal articular cartilage and the degeneration of that repair tissue over time. Our results demonstrate that the addition of rhBMP-2 to the operative site after creation of a full-thickness defect results in an improvement in the histological appearance and composition of the extracellular matrix at one year postoperatively. If these experimental results translate directly to the clinical situation, it is possible that the addition of rhBMP-2 to existing operative treatments for the repair of cartilage may improve the repair process and may help to maintain the integrity of the repair tissue.

Spontaneous osteoarthritis in Dunkin Hartley guinea pigs: histologic, radiologic, and biochemical changes.

Dunkin Hartley guinea pigs develop spontaneous, age-related osteoarthritis (OA) of the knee and other joints. Histologic changes are observed beginning at 3 months of age. Disease severity increases with age, and at 18 months moderate to severe OA is observed. A study was undertaken to assess the morphologic and biochemical changes of 22-month-old animals, and to compare them with values in 2-month-old guinea pigs. Biochemical indices characteristic of OA, from tibial cartilage, indicated an increase in proteoglycan content from 233 +/- 2 micrograms/mg (mean +/- SEM) at 2 months of age to 365 +/- 6 micrograms/mg at 22 months. Collagen concentration in cartilage decreased from 364 +/- 2 micrograms/mg at 2 months to 223 +/- 3 micrograms/mg at 22 months. Proteoglycan fragments found in synovial fluid measured 4.6 +/- 1 micrograms/ml at 2 months and increased to 37 +/- 2 micrograms/ml at 22 months. Radiographic changes observed at 22 months included marginal osteophytes of the tibia and femur, sclerosis of the subchondral bone of the tibial plateau, femoral condyle cysts, and calcification of the collateral ligaments. Histologic evaluation revealed severe OA, with a Mankin score of 10.7 +/- 0.5 in 22-month-old animals. In contrast, 2-month-old animals had no histologic or radiographically detectable lesions. The results of the study reported here indicate that the lesions observed in this model are similar to those of human OA. Spontaneous development of OA in guinea pigs is amenable to the study of the pathogenesis of OA and to the evaluation of potential disease-modifying agents.