Gene profiling of the rat medial collateral ligament during early healing using microarray analysis

Ligament heals in a synchronized and complex series of events. The remodeling process may last months or years. Experimental evidence suggests the damaged ligament does not recover its normal functional properties. Specific mechanisms to prevent scar formation and to regenerate the original mechanical function remain elusive but likely involve regulation of creeping substitution. Creeping substitution creates a larger hypercellular, hypervascular, and disorganized granulation tissue mass that results in an inefficient and nonregenerative wound healing process for the ligament. Control of creeping substitution may limit the extent of this tissue compromise and reduce the time necessary for healing. The objective of this study is to better understand the mechanism behind scar formation by identifying the extracellular matrix factors and other unique genes of interest differentially expressed during rat ligament healing via microarray. For this study, rat medial collateral ligaments were either surgically transected or left intact. Ligaments were collected at day 3 or 7 postinjury and used for microarray, quantitative PCR, and/or immunohistochemistry. Results were compared with the normal intact ligament. We demonstrate that early ligament healing is characterized by the modulation of several inflammatory and extracellular matrix factors during the first week of injury. Specifically, a number of matrix metalloproteinases and collagens are differentially and significantly expressed during early ligament healing. Additionally, we demonstrate the modulation of three novel genes, periostin, collagen-triple helix repeat containing-1, and serine protease 35 in our ligament healing model. Together, control of granulation tissue creeping substitution and subsequent downstream scar formation is likely to involve these factors. ligament healing involves a complex, coordinated series of events that results in a ligament that is more scar-like in character than the native tissue. The repair process may extend from months to years. Experimental evidence suggests the injured ligament incompletely recovers its original mechanical properties (25, 28). In an effort to develop an efficient repair process, researchers have tested a number of treatments including tissue engineering approaches, nonsteroidal anti-inflammatory drugs, and ultrasonic or electrical stimulation (28, 35). All show promise, but an incomplete understanding of the healing process makes optimal treatment regimes elusive. With tissue engineering and regenerative medicine, it is essential to understand the normal healing process in a ligament, thereby providing a basis to formulate and evaluate innovative new treatments. Previous work from our laboratory (8) established a highly active cellular and vascular scenario within the first week after ligament injury. Specifically, macrophage infiltration crested between days 3–5 while blood vessel accumulation peaked between days 7–11 postinjury (Fig. 1). Creeping substitution of damaged tissue into normal uninjured ligament, resulting in the expansion of the original wound size, was also evident during this time (8). The reduction of creeping substitution during inflammation and early proliferation may limit downstream scar formation, but the genetic players remain largely undefined.