Influences of Tumor Necrosis Factor-α on Lysyl Oxidases and Matrix Metalloproteinases of Injured Anterior Cruciate Ligament and Medial Collateral Ligament Fibroblasts.

The anterior cruciate ligament (ACL) fails to heal after injury, even after a primary surgical attempt. In contrast, the medial collateral ligament (MCL) can heal relatively well and restore the full joint function. The difference in intrinsic properties of these ligament cells can be due to their different responses to their local factors. TNF-α is considered to be an important chemical mediator in the wound healing of the ligaments. However, TNF-α-induced expression of lysyl oxidases (LOXs) and matrix metalloproteinases (MMPs) after injury is poorly understood. In this study, we use equi-biaxial stretch chamber to realize 12% stretch, which could mimic the injury to the ACL and MCL fibroblasts in vitro, and aim to determine the intrinsic differences between injured ACL and MCL by characterizing the differential expressions of LOXs and MMPs in response to TNF-α. The methods included Semiquantitative PCR, quantitative real-time PCR, Western blot, and zymography. We found that the mRNAs of LOXs had temporal increases in injured ACL and MCL. Moreover, the increases were higher in injured MCL than those in injured ACL (up to 1.77 ± 0.13-fold in LOX, 1.73 ± 0.21-fold in LOXL-1, 2.23 ± 0.27-fold in LOXL-2, 1.95 ± 0.11-fold in LOXL-3, 1.97 ± 0.28-fold in LOXL-4). On the other hand, the expressions of MMPs in injured ACL were much more prominent than those in injured MCL fibroblasts (up to 2.63 ± 0.20-fold in MMP-1, 3.73 ± 0.18-fold MMP-2, 1.58 ± 0.11-fold MMP-3, 4.23 ± 0.31-fold MMP-12). Similar results were observed at the protein level. The differential expression of LOXs and MMPs between the injured ACL and MCL fibroblasts in this study may help explain the healing abilities of the two different ligaments.

Differential MMP-2 activity of ligament cells under mechanical stretch injury: an in vitro study on human ACL and MCL fibroblasts.

Recent studies have revealed that following injuries, ligament tissues such as anterior cruciate ligaments (ACL), release large amounts of matrix metalloproteinases (MMPs). These enzymes have a devastating effect on the healing process of the injured ligaments. Although these enzymes are produced following ligament injuries, because of different healing capacities seen between the medial collateral ligament (MCL) and ACL, we were curious to find if the MMP activity was expressed and modulated differently in these tissues. For this purpose ACL and MCL fibroblasts were seeded on equi-biaxial stretch chambers and were stretched in different levels. The stretched cells were assayed using Zymography, Western Blot and global MMP activity assays. The results showed that within 72 h after injurious stretch, production of 72 kD pro-MMP-2 increased in both ACL and MCL. However, the ACL fibroblasts generated significantly more pro-MMP-2 than the MCL fibroblasts. Furthermore we found in ACL pro-MMP-2 was converted more into active form. With 4-aminophenyl mercuric acetate (APMA) treatment, large amounts of pro-MMP-2 were converted into active form in both. This indicates that there is no significant difference between ACL and MCL fibroblasts in post-translational modification of MMP-2. The fluorescent MMP activity assays revealed that the MMP family activities were higher in the injured ACL fibroblasts than the MCL. Since the MMPs are critically involved in extracellular matrix (ECM) turnover, these findings may explain one of the reasons why the injured ACL hardly repairs. The higher levels of active MMP-2 seen in the ACL injuries may disrupt the delicate balance of ECM remodeling process. These results suggest that the generation and modulation of MMP-2 may be directly involved in the different responses seen in ACL and MCL injuries.

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells.

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.

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.

Compressive compared with tensile loading of medial collateral ligament scar in vitro uniquely influences mRNA levels for aggrecan, collagen type II, and collagenase.

To test the hypothesis that loading conditions can be used to engineer early ligament scar behaviors, we used an in vitro system to examine the effect that cyclic hydrostatic compression and cyclic tension applied to 6-week rabbit medial collateral ligament scars had on mRNA levels for matrix molecules, collagenase, and the proto-oncogenes c-fos and c-jun. Our specific hypothesis was that tensile stress would promote more normal mRNA expression in ligament whereas compression would lead to higher levels of mRNA for cartilage-like molecules. Femur (injured medial collateral ligament)-tibia complexes were subjected to a hydrostatic pressure of 1 MPa or a tensile stress of 1 MPa of 0.5 Hz for 1 minute followed by 14 minutes of rest. On the basis of a preliminary optimization experiment, this 15-minute testing cycle was repeated for 4 hours. Semiquantitative reverse transcription-polymerase chain reaction analysis was performed for mechanically treated medial collateral ligament scars with use of rabbit specific primer sets for types I, II, and III collagen, decorin, biglycan, fibromodulin, versican, aggrecan, collagenase, c-fos, c-jun, and a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Cyclic hydrostatic compression resulted in a statistically significant increase in mRNA levels of type-II collagen (171% of nonloaded values) and aggrecan (313% of nonloaded values) but statistically significant decreases in collagenase mRNA levels (35% of nonloaded values). Cyclic tension also resulted in a statistically significant decrease in collagenase mRNA levels (66% of nonloaded values) and an increase in aggrecan mRNA levels (458% of nonloaded values) but no significant change in the mRNA levels for the other molecules. The results show that it is possible to alter mRNA levels for a subset of genes in scar tissue by supplying unique mechanical stimuli in vitro and thus that further investigation of scar engineering for potential reimplantation appears feasible.

Matrix metalloproteinase-13 expression in rabbit knee joint connective tissues: influence of maturation and response to injury.

The hypothesis of the present work was that expression of matrix metalloproteinase-13 (MMP-13, collagenase-3) would be induced during conditions involving important matrix remodeling such as ligament maturation, scar healing and joint instability. Therefore, MMP-13 expression in the medial collateral ligament (MCL) during the variable situations of tissue maturation and healing was assessed. MMP-13 expression in three intra-articular connective tissues of the knee (i.e. articular cartilage, menisci and synovium) following the transection of the anterior cruciate ligament of the knee was evaluated at 3 and 8 weeks post-injury. MMP-13 mRNA (semi-quantitative RT-PCR) and protein (immunohistochemistry and Western blotting) were detected in all of the tissues studied. Significantly higher MCL mRNA levels for MMP-13 were detected during the early phases of tissue maturation (i.e. 29 days in utero and 2-month-old rabbits) compared to later phases (5- and 12-month-old rabbits). This pattern of expression was recapitulated following MCL injury, with very high levels of expression in scar tissue at 3 weeks post-injury and then a decline to levels not significantly different from control values by 14 weeks. Elevated mRNA levels correlated with increased protein levels for MMP-13 in both menisci and synovium following the transection of the anterior cruciate ligament and during medial collateral ligament healing. These results indicate that MMP-13 expression is regulated by a number of variables and that high levels of expression occur in situations when connective tissue remodeling is very active.