Graduate Thesis Or Dissertation
 

Role of Cartilage and Bone Matrix Regulation in Early Equine Osteochondrosis

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/6q182r720

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  • Osteochondrosis (OC) results from disruption of the normal endochondral ossification process during articular cartilage and epiphyseal bone growth. Previous studies have revealed that cartilage canals are often associated with early OC lesions, especially along the ossification front. In early OC, extracellular matrix signaling pathways are expected to be altered, as they control matrix production and degradation associated with endochondral ossification. The aim of this study was to obtain a greater understanding of the role of cellular regulatory mechanisms in the pathogenesis of early equine osteochondrosis by identifying differences in gene and protein expression in both normal and diseased cartilage of adolescent horses. The objective of the study was to define the expression of chondrocyte signaling pathways in two specific regions, the osteochondral junction and in cells surrounding the cartilage canals. The hypothesis of this study was that when evaluating gene and protein expression of chondrocytes in diseased and normal early osteochondrosis samples, it was expected to find an upregulation of matrix metalloproteinases and a decrease in extracellular matrix gene expression within the osteochondral junction and cells surrounding the cartilage canals. Samples were previously harvested from the femoropatellar joints of foals ranging in age from 1 to 6 months. Archived paraffin-embedded osteochondral samples (6 osteochondrosis affected, 8 normal controls) and cDNA from chondrocytes captured with laser capture microdissection (4 OC, 5 normal controls) were used in this project. Quantitative real-time PCR was then performed on pre-amplified samples, evaluating 18 target genes from extracellular matrix signaling pathways. The genes included Collagen types I, IIB, and X, Aggrecan, Aggrecanase-1, MMP-1, MMP-3, SOX 5, SOX 9, Jagged 2, COMP, Runx2, Gremlin 1, Ephrin B2, Lubricin (PRG4), Osteocalcin (BGLAP), bFGF, and TGF-ß3. Immunohistochemistry was performed on paraffin-embedded osteochondral samples for osteocalcin, Sox-9, and collagen type IIB using mouse monoclonal α-human antibodies and for lubricin using rabbit polyclonal α-human antibodies. Downward trends were noted in gene expression of collagen type IIB, aggrecan, and SOX-9 in chondrocytes surrounding the cartilage canals of early osteochondrosis-affected samples compared to normal samples. In chondrocytes along the osteochondral junction, there was a trend of downregulation of lubricin and collagen type IIB in osteochondrosis samples compared to normal samples. No difference in osteocalcin, lubricin, collagen type II B, or SOX9 protein expression was apparent between OC and normal cartilage samples. Osteochondrosis is a multifactorial developmental disorder with a complex system of closely regulated signaling pathways. Recent studies have explored the early pathogenesis of osteochondrosis, with particular focus on failure of vascular anastomoses at the cartilage canals, alterations in biomechanical forces around the cartilage canals, and regulation of extracellular matrix pathways. An aim of the current study is to extend this knowledge by evaluating expression of components of the extracellular matrix in both normal and osteochondrosis-affected adolescent osteochondral specimens. Results of the current study indicate that several matrix genes may be downregulated, including collagen type IIB, aggrecan, Sox-9, and lubricin in osteochondrosis. Limitations of this study include a targeted rather than comprehensive evaluation of genes expressed in the extracellular matrix. In addition, only two cell populations were studied due to having a limited time to complete the laser capture procedure prior to sample degradation. It is also important to note that gene and protein expression studies do not address longitudinal changes or activity of molecules within the region. The results of this study contribute to the overall understanding of the pathogenesis of osteochondrosis and aid in identifying the role of cartilage and bone matrix regulation in early equine osteochondrosis.
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