The role of macrophages in early healing of a tendon graft in a bone tunnel

Background: Macrophages accumulate following tendon-to-bone repair and may contribute to the formation of a scar-tissue interface rather than to the reformation of a normal insertion site. We hypothesized that macrophage depletion may lead to improved insertion site regeneration, in a form of "scar-less" healing rather than reactive scar-tissue formation. Methods: One hundred and ninety-two Sprague-Dawley rats underwent anterior cruciate ligament reconstruction with use of a flexor tendon autograft and were divided into a control group (ninety-six rats) and a liposomal clodronate-injected group (ninety-six rats). Clodronate is a bisphosphonate that selectively induces macrophage apoptosis. Animals in the liposomal clodronate group received weekly intraperitoneal injections of liposomal clodronate (1.33 mL/ 100 g of body weight). Rats were killed at serial time points from three to forty-two days. Immunostaining identified macrophages and transforming growth factor-beta (TGF-β) at the tendon-bone interface. Fibrous interface width, osteoid formation, and collagen fiber continuity were evaluated with use of histomorphometry. Serial fluorochrome labeling was used to measure mineral apposition rate. Additional rats were killed for biomechanical testing at seven, fourteen, twenty-eight, and forty-two days. Results: Liposomal clodronate significantly decreased macrophages and TGF-β accumulation at the tendon-bone interface (p < 0.05). Specimens from rats that received liposomal clodronate exhibited a significantly narrower fibrous tissue interface between tendon and bone at all time points compared with specimens from controls (p < 0.05). In specimens from the liposomal clodronate group, healing proceeded at an accelerated rate, characterized by enhanced collagen fiber continuity and a greater degree of interface remodeling between tendon and bone. There were significant increases in osteoid formation (p < 0.05) and mineral apposition rates (p < 0.05) among experimental specimens. At forty-two days, the specimens from the liposomal clodronate group had significantly greater increases than the control specimens with respect to load to failure (mean and standard deviation, 13.5 ± 4.2 N and 9.7 ± 3.9 N, respectively; p < 0.05) and stiffness (mean, 11.5 ± 5.0 N/mm and 7.5 ± 3.2 N/mm; p < 0.05). Conclusions: Macrophage depletion following anterior cruciate ligament reconstruction resulted in significantly improved morphologic and biomechanical properties at the healing tendon-bone interface, which we hypothesize are due to diminished macrophage-induced TGF-β production. Clinical Relevance: Techniques to modulate inflammation, and specifically macrophages, may improve tendon-bone healing and may enhance the structural integrity of healing tendon grafts.