Ruth X. Raiss

Proteoglycan metabolism and viability of articular cartilage explants as modulated by the frequency of intermittent loading

OBJECTIVE This study was designed to systematically determine whether and to what extent the frequency of intermittent loading modulates the biosynthesis and release of proteoglycans (PGs), and to assess chondrocyte viability within mature bovine articular cartilage explants exposed to different loading patterns. METHODS Cultured full-thickness cartilage explants from the weight-bearing area of healthy bovine fetlock joints were exposed to intermittently applied, uniaxial cyclic loads by introducing a sinusoidal waveform of 0.1, 0.5 or 1.0Hz, frequency and a peak stress of 0.5MPa for a period of 6 days. The cyclic loads were applied for 5, 10 or 20s followed by a period of unloading lasting 10, 100 or 1000s. The incorporation of radiolabeled sulfate into glycosaminoglycans (GAGs) during the final 18h, the content of GAGs and DNA, the deformation of loaded explants as well as the viability of chondrocytes within the different zones of explants were determined. RESULTS PG synthesis and loss of endogenous PGs were non-linearly and independently regulated by the frequency of the chosen intermittent load, whereas the release of newly synthesized PGs remained unaffected. The viability of chondrocytes within the superficial zone decreased drastically under intermittent loading in a manner independent of the frequency applied. CONCLUSIONS Our results confirm the hypothesis that the frequency of intermittent loading is an important mechanical factor controlling the metabolic activities of chondrocytes. They also implicate that an initially healthy cartilage explant can be mechanically manipulated to generate an in vitro model of degenerative, osteoarthritic-like cartilage.

Intermittent cyclic loading of cartilage explants modulates fibronectin metabolism

OBJECTIVE The aim of this study was to evaluate systematically the effect of tissue load, its amplitude, time of intermittence and duration of loading on the biosynthesis and release of fibronectin by intermittently loaded mature bovine articular cartilage explants. METHODS Cyclic compressive pressure was introduced using a sinusoidal waveform of 0.5 Hz-frequency with a peak stress of 0.1, 0.5 or 1.0 MPa for a period of 10 s followed by an unloaded period lasting 10, 100 or 1000 s. Fibronectin and total proteins were radiolabeled with 10 microCi/ml [3H]-phenylalanine during the final 18 h of the 1, 3 or 6 day experiments. The content of endogenous fibronectin was determined using enzyme-linked immunosorbant assay (ELISA), whereas the viability of explants was measured using sections of cartilage explants stained with fluorescein diacetate and propidium iodide. The deformation of loaded explants was determined using a load-displacement transducer system. RESULTS The mechanical factor time of intermittence significantly altered the synthesis and release of fibronectin by cartilage explants, whereas the tested range of load magnitudes, as well as the duration of loading, seemed to be of subordinate importance. Loading affected the viability of the superficial zone in the cartilage, whereas the chondrocytes of the intermediate and deep zone remained viable. The compression of loaded explants was dependent on the magnitude of stress, as well as on the duration of unloading between each loading cycle. Synthesis of fibronectin, the retention of newly synthesized fibronectin within the extracellular matrix, and the portion of newly synthesized proteins that were fibronectin was significantly increased in cartilage explants which were cyclically compressed with 0.5 MPa for 10 s followed by a period of unloading lasting 100 s. CONCLUSIONS Previous studies reporting that cartilage explants of human and animal osteoarthritic joints synthesize and retain elevated amounts of fibronectin imply that in our experiments mechanical stimuli can induce a fibronectin metabolism in vitro which mimics some of the osteoarthritic characteristics.

The sulfation pattern of chondroitin sulfate from articular cartilage explants in response to mechanical loading

Chondrocytes within articular cartilage experience complete unloading between loading cycles thereby utilizing mechanical signals to regulate their own anabolic and catabolic activities. Structural alterations of proteoglycans (PGs) during aging and the development of osteoarthritis (OA) have been reported; whether these can be attributed to altered load or compression is largely unknown. We report here on experiments in which the effect of intermittent loading on the fine structure of newly synthesized chondroitin sulfate (CS) in bovine articular cartilage explants was examined. Tissues were subjected for 6 days to cyclic compressive pressure using a sinusoidal waveform of 0.1, 0.5 or 1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5, 10 or 20 s, followed by an unloading period lasting 10, 100 or 1000 s. During the final 18 h of the culture, cartilage explants were radiolabeled with 50 microCi/ml D-6-[3H]glucosamine, and newly synthesized as well as endogenous CS chains were isolated after proteinase solubilization of the tissue. CS chains were depolymerized with chondroitinase ABC and ACII, and the 3H-digestion products were quantified after fractionation by high-performance anion-exchange chromatography using a CarboPac PA1 column. Intermittently applied cyclic mechanical loading did not affect the proportion of 4- and 6-sulfated disaccharide repeats, but caused a significant decrease in the abundance of the 4,6-disulfated nonreducing terminal galNAc residues. In addition, loading induced elongation of CS chains. Taken together, these data provide evidence for the first time that long-term in vitro loading results in marked and reproducible changes in the fine structure of newly synthesized CS, and that accumulation of such chains may in turn modify the physicochemical and biological response of articular cartilage. Moreover, data presented here suggest that in vitro dynamic compression of cartilage tissue can induce some of the same alterations in CS sulfation that have previously been shown to occur during the development of degenerative joint diseases such as OA.

Fibronectin metabolism of cartilage explants in response to the frequency of intermittent loading

Chondrocytes within articular cartilage experience complete unloading between loading cycles and in so doing utilize mechanical signals to regulate their own metabolic activities. A strongly elevated fibronectin content is an early feature in osteoarthritis and appears to be related to increases in both the synthesis and retention of this glycoprotein. The objectives of this study were to investigate systematically whether the frequency of intermittently applied cyclic mechanical loading of cartilage explants alters the biosynthesis and retention of fibronectin, and to assess whether it is possible to induce in vitro osteoarthritic‐like changes of this metabolic parameter by mechanical means over a period of 6 days. Cartilage plugs consisting of viability‐checked chondrocytes were exposed to sinusoidal cyclic compressive pressure alterations of 0.1, 0.5 or 1.0 Hz frequency with a peak stress of 0.5 MPa for a period of 5, 10 or 20 s, followed by an unloading period of 10, 100 or 1000 s, and compared to unloaded reference plugs from the same joint and topographic origin. The incorporation of radioactive precursor into fibronectin during the last 18 h, the content of fibronectin, and the viability of chondrocytes were determined. Our data revealed that (a) the fibronectin synthesis was selectively, but non‐linearly affected by the frequency of intermittent loads applied (as defined by the frequency of the applied force, the duration of the loading cycle and the duration of the force‐free period between each loading cycle), and that (b) the retention of endogenous fibronectin and proteins within loaded cartilage explants is strongly elevated. These data support our hypothesis that the mechanical factor “frequency of intermittent loading” seems to be the crucial mechanical parameter controlling the metabolism of chondrocytes. The effect of the frequency of intermittent loading cannot be described by a simple statistical correlation, so that no specific predictions are possible. However, our results imply that distinct loading protocols have been established that can induce alterations of the fibronectin metabolism similar to those observed in human and animal osteoarthritis.

115 CARTILAGE BIOMARKERS AFTER MENISCECTOMY

Purpose: Study of 846 as anabolic marker and C2C as catabolic marker of post-traumatic osteoarthritis (OA) in the rabbit model of anterior cruciate ligament transection (ACLT). Methods: Transection of the anterior cruciate ligament was performed in adult male New Zealand white rabbits (n = 32). An open arthrotomy surgery of only the right knees was used. Left knees remained untreated and served as intraindividual control knees. At time 0 before surgery and at 2, 4, 8 and 12 weeks before sacrifice, synovial fluid was aspirated from both, right and left knee joints. Use of all animals had been permitted by both, the University’s ethics committee and the government ethics committee (Regierungsprasidium Darmstadt, Germany). Macroscopic grading was performed of all right and left knee joints. 9 areas per each medial and lateral tibia, respectively medial and lateral femur and the patella were evaluated. Histologic grading was performed with a grading system accounting for proteoglycan content, matrix structure, cellularity, and osteophyte formation. 846 levels and C2C levels were measured in the synovial fluid lavages with immunoassays. The distributions of synovial fluid levels of 846 and C2C were statistically analysed (Kolmogorov-Smirnov-test). Differences between left and right knees were then statistically analysed by student’s t-test. Results: All rabbits had a normal postoperative course after ACLT. At 2 weeks after ACLT, macroscopically visible lesions were only detected at the medial femoral site (p< 0.05). With time, lesion size increased and lesions appeared at more joint sites. At 12 weeks after ACLT, medial and lateral tibiae as well as both femoral condyles showed statistically significant lesions. Histologic scores showed early OA after ACLT, when compared with the unoperated contralateral side. 846 levels in the synovial fluid lavages of the operated knee joints were increased at all timepoints (p< 0.05) when compared to the non-operated contralateral knees. The highest levels were measured from 2 to 8 weeks with a decrease at 12 weeks. The C2C levels at 4 and 8 weeks were higher than at 2 weeks after ACLT (p< 0.05). Conclusions: To our knowledge, this is the first report of 846 and C2C studied in the same rabbit synovial fluid samples. Both markers have been reported as potential markers of metabolic processes in post-traumatic OA, probably reflecting degrading, respectively rebuilding cartilage processes. Biologically, they might be markers of the ensuing macroscopic and microscopic changes. While the reparative response, as possibly indicated by 846, is not sufficient to stop the destructive process of OA, possibly indicated by C2C, the exact meaning of these marker levels has to be characterised in more detail.

114 CARTILAGE BIOMARKERS AFTER ACLT

Purpose: Study of 846 as anabolic marker and C2C as catabolic marker of post-traumatic osteoarthritis (OA) in the rabbit model of anterior cruciate ligament transection (ACLT). Methods: Transection of the anterior cruciate ligament was performed in adult male New Zealand white rabbits (n = 32). An open arthrotomy surgery of only the right knees was used. Left knees remained untreated and served as intraindividual control knees. At time 0 before surgery and at 2, 4, 8 and 12 weeks before sacrifice, synovial fluid was aspirated from both, right and left knee joints. Use of all animals had been permitted by both, the University’s ethics committee and the government ethics committee (Regierungsprasidium Darmstadt, Germany). Macroscopic grading was performed of all right and left knee joints. 9 areas per each medial and lateral tibia, respectively medial and lateral femur and the patella were evaluated. Histologic grading was performed with a grading system accounting for proteoglycan content, matrix structure, cellularity, and osteophyte formation. 846 levels and C2C levels were measured in the synovial fluid lavages with immunoassays. The distributions of synovial fluid levels of 846 and C2C were statistically analysed (Kolmogorov-Smirnov-test). Differences between left and right knees were then statistically analysed by student’s t-test. Results: All rabbits had a normal postoperative course after ACLT. At 2 weeks after ACLT, macroscopically visible lesions were only detected at the medial femoral site (p< 0.05). With time, lesion size increased and lesions appeared at more joint sites. At 12 weeks after ACLT, medial and lateral tibiae as well as both femoral condyles showed statistically significant lesions. Histologic scores showed early OA after ACLT, when compared with the unoperated contralateral side. 846 levels in the synovial fluid lavages of the operated knee joints were increased at all timepoints (p< 0.05) when compared to the non-operated contralateral knees. The highest levels were measured from 2 to 8 weeks with a decrease at 12 weeks. The C2C levels at 4 and 8 weeks were higher than at 2 weeks after ACLT (p< 0.05). Conclusions: To our knowledge, this is the first report of 846 and C2C studied in the same rabbit synovial fluid samples. Both markers have been reported as potential markers of metabolic processes in post-traumatic OA, probably reflecting degrading, respectively rebuilding cartilage processes. Biologically, they might be markers of the ensuing macroscopic and microscopic changes. While the reparative response, as possibly indicated by 846, is not sufficient to stop the destructive process of OA, possibly indicated by C2C, the exact meaning of these marker levels has to be characterised in more detail.