Arthrofibrosis Following Total Knee Arthroplasty: Insight into Molecular Mechanisms|
Date: May 18, 2007
at 4:00 PM
Location: Matheson Hall, Room: 109
Marla Steinbeck, M.T.(ASCP), Ph.D.
Department of Orthopaedic Surgery
Thomas Jefferson University
Following TKA 3-4% of patients develop idiopathic stiffness (arthrofibrosis) and undergo a second surgery to alleviate their discomfort and to improve joint mobility. Understanding the molecular mechanism of arthrofibrosis may allow implementation of novel therapeutic modalities to address this undesirable complication. Our hypothesis is that reactive oxygen and nitrogen species (RONS) are responsible for the aggressive fibroblast proliferation and ECM deposition observed in arthrofibrosis. Tissues from the periarticular region of 10 patients undergoing TKA for arthrofibrosis were analyzed using various molecular techniques including; histology, histomorphometry, immunohistochemistry and micro-CT. Additionally, cell viability was analyzed by the TUNEL assay. Three distinct morphological regions were observed within the collected tissue; a fibrotic region, a fibrocartilage/calcified region and an area of increased vascularization. The fibrotic region was characterized by elevated numbers of fibroblasts, disorganized matrix, increased proteoglycan deposition, low vascularization and few TUNEL positive cells. Adjacent to the fibrotic regions were areas of fibrocartilage/calcification. The cells in this region showed the characteristic chondrocyte phenotype (rounded cells surrounded by clear lacuna and embedded in a proteoglycan rich matrix), no blood vessels and few TUNEL positive cells. Within this area were calcified regions where, trabecular-like bone formation was found. The third region, separate from the other two, was highly vascularized and contained the highest number of TUNEL positive cells.
To evaluate inflammation based on the presence of immune cells, tissue sections were stained with Giemsa (inflammatory cells), Toluidine blue (mast cells), CD68 (macrophage) and elastase (neutrophils). Mast cells and macrophages were observed in the fibrous and vascular regions. No neutrophils were detected, which is consistent with the absence of tissue infection in these patients. To determine the presence of an RONS source, myeloperoxidase (MPO) immunohistochemistry, which stains neutrophils and some macrophages, was performed. Macrophages showed intense staining for this enzyme. To assess RONS matrix modification and intracellular production, tissue sections were evaluated by immunohistochemistry for the presence of nitrotyrosine. Nitrosylation was observed in regions containing inflammatory cells and chondrocytes. Taken together, all of the patient tissues showed evidence of metaplasia, inflammatory cell infiltration, increased MPO expression and protein nitrosylation. The presence of RONS and MPO suggests they may play a role in mediating the fibrotic nature of this condition and that anti-oxidant therapy may alleviate at least part of the fibrotic scar tissue formation.
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Dr. Marla Steinbeck has been active in the areas of inflammation, reactive oxygen chemistry and osteoclast biology where her work has focused on evaluating their involvement in the development of osteoarthritis, idiopathic arthrofibrosis and wear debris-mediated osteolysis following joint replacement. Currently, she is an Assistant Professor in the Department of Orthopaedic Surgery at Thomas Jefferson University. Before joining Thomas Jefferson University, she was an Instructor in the Department of Pathology at Harvard Medical School. She has successfully developed an assay to measure singlet oxygen production by inflammatory cells and has determined the involvement of myeloperoxidase in the generation of this highly reactive oxidant. She has shown that osteoclasts express the enzyme NADPH-oxidase and that the generation of reactive oxygen species is involved in both the differentiation of osteoclasts and in their function as bone resorptive cells. Her most recent studies show the involvement of myeloperoxidase in early osteoarthritis. Her current work is in developing biomarker assays for osteoarthritis and idiopathic arthrofibrosis diagnostics and therapeutics using genomic and proteomic approaches to understand the molecular mechanisms of disease. Her laboratory is heavily invested in the area of biomarker development. This effort includes the use of microarray analysis, RT-PCR, liquid chromatography, mass spectrometry and immunoassays for protein detection/measurement.
Matheson Hall is located at 32nd and Market Streets.