| The knee joint of animals and humans represents a complex system involving both biological and biomechanical homeostatic mechanisms. Tissue responsiveness to microenvironmental changes, external loads, trauma and injury is controlled by structural and cellular elements within the tissue. The principle objective of this study was to understand the molecular biology of knee joint connective tissues, with particular emphasis on the expression of mRNA levels for various molecules involved in the maintenance of structural and functional integrity, and homeostasis.;The data presented will be discussed in terms of the various physiological states on expression and regulation of a critical subset of mRNAs in knee joint connective tissues that include the medial collateral ligament, anterior cruciate ligament, synovium, articular cartilage, and meniscus. The physiological models analyzed in this study consist of: (i) medial collateral scar/wound healing, (ii) medial collateral ligament and anterior cruciate ligament maturation and the influence of load deprivation, (iii) anterior cruciate ligament transection/osteoarthritic development, and (iv) ovariohysterectomy and the influence of sex steroid hormones in knee joint connective tissue homeostasis.;In the first series of experiments, using a medial collateral ligament wound healing model, a subset of growth factors and growth factor receptors were found to be differentially expressed during the healing phases of the medial collateral ligament compared to controls. The second set of experiments was designed to assess the effects of biomechanical deprivation (i.e immobilization) on ligament maturation. Based on the analysis of molecules involved in matrix synthesis, degradation and remodeling, as well as potential regulatory molecules (eg. growth factors), immobilization of the MCL and ACL appeared to shift the balance in favor of a catabolic environment within the tissue. This altered cellular balance in activity is the likely mechanism for the observed failure of immobilized ligaments to mature. A specific matrix remodeling enzyme, matrix metalloproteinase-13, was demonstrated to be significantly elevated in immobilized ligaments compared to controls. The third series of experiments analysed expression of matrix metalloproteinase-13 at both the mRNA and protein level in normal and healing medial collateral ligament as well as articular cartilage and meniscus using a rabbit anterior cruciate ligament transection (ie. Osteoarthritis) model. Results of these studies demonstrated tissue specific elevations in matrix metalloproteinase-13 levels. A final series of experiments were designed based on observations that an inverse correlation existed between expression of matrix metalloproteinase-13 and the steroid receptor, estrogen receptor alpha, at the mRNA level. Preliminary experiments demonstrated the presence of estrogen receptor alpha at the mRNA level in rabbit knee joint connective tissues. Cotransfection experiments using a luciferase promoter construct containing the proximal 667 base pairs of the rabbit matrix metalloproteinase-13 promoter and the human estrogen receptor alpha expression vector demonstrated that estrogen receptor alpha may regulate matrix metalloproteinase-13 expression at the promoter level. The data presented in this dissertation demonstrates that knee joint connective tissue homeostasis is complex and involves contributions from a diverse family of genes in a tissue specific manner. |
