Scar formation of ligaments after rupture and ligament grafts after reconstruction remains a great challenge. Ligament healing involves a complex, coordinated series of events that form a neo-ligament, which is more disorganized and flbrotic in character than the native tissue. The repair process may extend from months to years, and the injured ligament never fully recovers its original mechanical properties. With little intrinsic healing potential, ruptures of the anterior cruciate ligament (ACL) are usually reconstructed. The "healed" tissues, however, do not regenerate native tissues or recapitulate their mechanical function. ACL grafts often lengthen (incidents range from 40-100%) and their strength can drop by ~50% after remodeling. Reconstructed knees are often less stable and fail to restore normal joint kinematics. These deficiencies are likely the cause of premature joint degeneration and osteoarthritis.

Our overall goal is to improve ligament and ligament graft healing, making them more regenerative. The first 2 studies characterized ligament healing in a spatial and temporal manner over 28 days. The experiments demonstrated creeping substitution and the potential role of the immune system to control the repair and/or regenerative process. From these studies, macrophages were identified as a potentially significant player during healing. Macrophages paralleled creeping substitution, were abundant within the healing ligament, and potentially played a destructive role via matrix phagocytosis.

The role of macrophages during early ligament healing was then evaluated using liposome-encapsulated clodronate to inhibit phagocytosing macrophages. Clodronate attenuated the early infiltration of macrophages, resulting in delayed structural and functional healing. Macrophage re-infiltration into the wound resulted in continued ligament healing. These results suggested that early inhibition of phagocytosing macrophages is detrimental to ligament healing.

The final experiment evaluated the effects of interleukin-4 on ligament healing. lnterleukin-4 (IL-4) is reported to stimulate the Th2 lymphocyte/M2 macrophage pathway, reducing inflammation and stimulating remodeling. IL-4 dose- and time-dependently stimulated early ligament regeneration but was unable to maintain the response during later healing.

In summary, this work demonstrated the association between the immune cells and ligament healing, indicating a potential for obtaining a more regenerative response by modulating the immune response in a time, dose, and spatial manner.