Investigation of Anterior Cruciate Ligament Fibroblast Biomarkers for Cell Characterization by Immunohistochemistry

Approximately 200,000 anterior cruciate ligament (ACL) injuries occur in the United States per year. This ligament is also the most commonly injured ligament of the knee joint. Surgical reconstruction using autografts from the patient is the main method of treatment to date; however, cadaver tendon allografts may also be used. A less invasive and more promising method for repair is ligament engineering. Growth factors, a proper matrix, and suitable cells, are a few of the necessary components of a ligament construct. A common protocol to engineer a ligament is to use ACL fibroblasts and to seed them into a matrix or scaffold that allows the creation of a similar tissue. However, to date no successful construct has been engineered. In order for an engineered construct to be successful and resemble a human ACL, an understanding of the cellular components of the ACL fibroblasts is necessary. The existence and role of the cellular components in the human ACL allows for the ligament to maintain its mechanical properties. Therefore, it is necessary to investigate every aspect of this tissue, including using reliable biomarkers to properly characterize human ACL fibroblasts. In this study, we will identify a combination of biomarkers specific to these fibroblasts using the method of immunohistochemistry. The central hypothesis of this study is that the biomarkers of interest, vimentin, fibronectin, alpha-smooth muscle actin(α-SMA), Fibroblast Growth Factor Receptor 1 (FGFR1), and collagen III will be a suitable and reliable combination of markers to characterize anterior cruciate ligament (ACL) fibroblasts because 100% of the cells are expected to stain positive for these biomarkers by the method of immunohistochemistry. However, our results indicate that none of the biomarkers were detected in cells at a rate of 100%. Fibronectin and collagen III abundance in the ECM made it difficult to determine specific production by an isolated fibroblast. However, both biomarkers were detected in abundance by fluorescence intensity. FGFR1 and vimentin stained positive in over 50% of cells, but less than 95%. Alpha-SMA was more variable due to two of the ligaments having 0% of cells stain positive for this biomarker. However, samples that did show expression of alpha-SMA that stained over 46% of cells.