Matrix‐degrading enzyme synthesis by cells isolated from the canine cranial cruciate ligament
Authors: Anderson V.M. , Vaughan‐Thomas A. , Innes J.F. 
Published: September 2009

Introduction  Cruciate ligament disease or injury is common in humans and dogs. There is some evidence to indicate that both its incidence and the degree of tissue pathology may vary between breeds and in relation to body weight, age, gender and body condition. In order to investigate a direct role of endocrine influences on pathology, we have attempted to establish a model system using isolated canine cranial cruciate ligament (CCL) cells in culture. In this preliminary study, we isolated CCL cells and characterized the matrix metalloproteinase (MMP) activities expressed by these cells in culture. MMPs contribute to the extracellular matrix turnover of connective tissue, and an imbalance between matrix synthesis and degradation leads to changes in the matrix, which may compromise ligament integrity and strength.

Materials and methods  CCLs were collected from animals following euthanasia for reasons other than joint disease. Ligament cells were isolated using collagenase digestion and maintained in culture in DMEM/F12 containing 10%(v/v) fetal bovine serum. Early passage number cells were incubated in medium containing reduced serum concentrations [1% (v/v)] in order to determine levels of MMP production.

Gelatinase activities (MMP‐2 and MMP‐9) in the media of cultured cells were determined using gelatin zymography. The identification of MMP was confirmed by using EDTA to inhibit gelatinolytic activity.

In order to determine whether the cells express collagenase activities, a fluorogenic substrate assay was used in which cell‐conditioned medium was treated with APMA prior to incubation with the substrate (MCA‐Pro‐Cha‐Gly‐Nva‐His‐Ala‐Dpa‐NH2).

Hormones (relaxin, leptin, growth hormone and oestrogen) were added at near physiological serum levels, where known, to 12‐well plates after cells were 100% confluent, and medium samples were analysed 72 h after treatment.

Results  Pro‐MMP‐2 was the predominant gelatinase activity expressed by canine cruciate ligament cells in culture. The active forms of both MMP‐2 and MMP‐9 were observed at very low levels or not at all, differences being observed between animals. The volumes of medium samples analysed were adjusted such that the levels of MMP detected were within the range of activities that yielded a linear densitometric response curve. Our preliminary results show no change in levels of pro and active forms of MMP‐2 and MMP‐9 in cell culture after treatment with the hormones, oestradiol, relaxin, growth hormone and leptin. Collagenase activity was detected in the ligament cell‐conditioned medium using the fluorogenic substrate, and the volume of medium used for assay was adjusted to lie within the standard curve. Our preliminary data suggest that hormonal treatment may modulate the production of collagenase activity. However, these findings require confirmation.

Discussion  These results show that pro‐MMP‐2 is the predominant gelatinase activity synthesized by cultured ligament cells. Previous studies have shown that this is also the predominant gelatinase activity present in extracts of canine cruciate tissue. We have also shown that CCL cells express a collagenase activity in culture. Therefore, we intend to use the CCL cell‐culture system as a model to investigate the effects of systemic and local endocrine influences on the matrix degradative cascades. This model will allow us to obtain information on the roles of hormones in pathological processes prior to ligament rupture in vivo.