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Tendon-to-bone healing occurs by formation of a fibrous, scar tissue interface rather than regeneration of a normal insertion. Because inflammatory cells such as macrophages lead to formation of fibrous scar tissue, we hypothesized immobilization would allow resolution of acute inflammation and result in improved tendon-bone healing. We reconstructed the ACL of 60 Sprague-Dawley rats using a tendon autograft. An external fixation device was used to immobilize the surgically treated knee in 30 rats. We evaluated tendon-bone interface width, collagen fiber continuity, and new osteoid formation histologically. Immunohistochemistry was used to localize ED1+ and ED2+ macrophages at the tendon-bone interface at 2 and 4 weeks. Biomechanical testing was performed at 4 weeks. Interface width was smaller and collagen fiber continuity was greater in the immobilized group. Immobilized animals exhibited fewer ED1+ macrophages at the healing interface at 2 and 4 weeks. In contrast, there were more ED2+ macrophages at the interface in the immobilized group at 2 weeks. Failure load and stiffness were similar between groups at 4 weeks. The data suggest early immobilization diminishes macrophage accumulation and may allow improved tendon-bone integration.

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-beta) 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-beta 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-beta production.

Background: Clinical studies have demonstrated a high rate of incomplete healing of rotator cuff tendon repair. Since healing of such a repair is dependent on bone ingrowth into the repaired tendon, we hypothesized that osteoinductive growth factors would improve rotator cuff tendon-healing., Methods: Seventy-two skeletally mature sheep underwent detachment of the infraspinatus tendon followed by immediate repair. The animals received one of three treatments at the tendon-bone interface: (1) an osteoinductive bone protein extract on a Type-I collagen sponge carrier, (2) the collagen sponge carrier alone, and (3) no implant. The animals were killed at six and twelve weeks, and the repaired rotator cuff was evaluated with use of magnetic resonance imaging, plain radiographs, histologic analysis, and biomechanical testing., Results: A gap consistently formed between the end of the repaired tendon and bone in this model, with reparative scar tissue and new bone spanning the gap. Magnetic resonance imaging showed that the volume of newly formed bone (p < 0.05) and soft tissue (p < 0.05) in the tendon-bone gap were greater in the growth factor-treated animals compared with the collagen sponge control group at both time-points. Histologic analysis showed a fibrovascular tissue in the interface between tendon and bone, with a more robust fibrocartilage zone between the bone and the tendon in the growth factor-treated animals. The repairs that were treated with the osteoinductive growth factors had significantly greater failure loads at six weeks and twelve weeks (p < 0.05); however, when the data were normalized by tissue volume, there were no differences between the groups, suggesting that the treatment with growth factor results in the formation of poor-quality scar tissue rather than true tissue regeneration. The repairs that were treated with the collagen sponge carrier alone had significantly greater stiffness than the growth factor-treated group at twelve weeks (p = 0.005)., Conclusions: This model tests the effects of growth factors on scar tissue formation in a gap between tendon and bone. The administration of osteoinductive growth factors resulted in greater formation of new bone, fibrocartilage, and soft tissue, with a concomitant increase in tendon attachment strength but less stiffness than repairs treated with the collagen sponge carrier alone.

Background: Healing of a tendon graft in a bone tunnel depends on bone ingrowth into the interface between tendon and bone. Excessive osteoclastic activity may contribute to bone resorption, tunnel widening, and impaired healing. We hypothesized that inhibition of osteoclastic activity by osteoprotegerin (OPG) would increase bone formation around a tendon graft in anterior cruciate ligament reconstruction in a rabbit model, while increased osteoclastic activity due to the application of receptor activator of nuclear factor-kappa B ligand (RANKL) would impair bone ingrowth., Methods: Sixty skeletally mature, male New Zealand White rabbits underwent bilateral anterior cruciate ligament reconstruction. OPG (100 microg per tunnel) or RANKL (10 microg per tunnel) was delivered to the tendon-bone interface with use of a synthetic calcium phosphate carrier vehicle. Twenty animals were killed at two, four, and eight weeks after surgery. Two rabbits from each group were prepared for histological evaluation, and the other rabbits were used for biomechanical testing., Results: A significantly greater amount of bone surrounded the tendon at the healing tendon-bone interface in the OPG-treated limbs compared with the controls and the RANKL-treated limbs at all time-points (p < 0.05). There were significantly fewer osteoclasts in the OPG-treated limbs compared with the controls and the RANKL-treated limbs (p < 0.05). The average tunnel area in the OPG group was significantly smaller than that in the RANKL group (p = 0.003 at two weeks and p = 0.004 at four weeks). The femur-anterior cruciate ligament-tibia complex of the OPG-treated limbs had significantly increased stiffness compared with RANKL-treated limbs at eight weeks (p = 0.04)., Conclusions: Osteoprotegerin significantly improves bone formation around the grafted tendon and improves the stiffness at the healing tendon-bone junction in a rabbit model.

Background: Motion between a tendon graft and bone tunnel may impair graft incorporation and lead to tunnel widening., Hypothesis: Healing of a tendon graft in a bone tunnel is inhibited by graft-tunnel motion., Study Design: Controlled laboratory study., Methods: Anterior cruciate ligament reconstruction was performed in 5 cadaveric rabbit limbs, and 3-dimensional graft-tunnel motion was measured using micro-computed tomography. The authors then performed bilateral anterior cruciate ligament reconstruction in 15 rabbits and used histomorphometry to compare tendon-to-bone healing between the tunnel aperture, midtunnel, and tunnel exit and between the anterior and posterior aspects of the tunnel., Results: Graft-tunnel motion was greatest at the tunnel apertures and least at the tunnel exit in cadaveric testing. Healing of the graft was slowest at the tunnel apertures. Tendon-bone interface width was greater at the aperture than at the tunnel exit for the femoral tunnel (P = .04). There was an inverse correlation between time zero graft-tunnel motion and healing in the femoral tunnel (P = .005). There was closer apposition of new bone to the tendon graft in the posterior half of the interface (P < .05). Osteoclasts were found at the tunnel apertures., Conclusion: Although graft-tunnel motion was only measured in cadaveric animals, results suggest that healing may be affected by the local mechanical environment, as graft healing in the femoral tunnel was inversely proportional to the magnitude of graft-tunnel motion., Clinical Relevance: Graft-tunnel motion may impair early graft incorporation and may lead to osteoclast-mediated bone resorption, contributing to tunnel widening. Early, aggressive postoperative rehabilitation may have detrimental effects on graft-to-bone healing.

Background: Nonsteroidal anti-inflammatory drugs are commonly prescribed after rotator cuff repair. These agents can impair bone formation, but no studies have evaluated their impact on tendon-to-bone healing., Hypothesis: Traditional nonselective nonsteroidal anti-inflammatory drugs and cyclooxygenase-2-specific nonsteroidal anti-inflammatory drugs interfere with tendon-to-bone healing., Study Design: Controlled laboratory study., Methods: One hundred eighty Sprague-Dawley rats underwent acute rotator cuff repairs. Postoperatively, 60 rats received 14 days of celecoxib, a cyclooxygenase-2-specific nonsteroidal anti-inflammatory drug; 60 received indomethacin, a traditional nonselective nonsteroidal anti-inflammatory drug; and 60 received standard rat chow. Animals were sacrificed at 2, 4, and 8 weeks and evaluated by gross inspection, biomechanical testing, histologic analysis, and polarized light microscopy to quantify collagen formation and maturation., Results: Five tendons completely failed to heal (4 celecoxib, 1 indomethacin). There were significantly lower failure loads in the celecoxib and indomethacin groups compared with the control groups at 2, 4, and 8 weeks (P < .001), with no significant difference between nonsteroidal anti-inflammatory drug groups. There were significant differences in collagen organization and maturation between the controls and both nonsteroidal anti-inflammatory drug groups at 4 and 8 weeks (P < .001). Controls demonstrated progressively increasing collagen organization during the course of the study (P < .001), whereas the nonsteroidal anti-inflammatory drug groups did not., Conclusion: Traditional and cyclooxygenase-2-specific nonsteroidal anti-inflammatory drugs significantly inhibited tendon-to-bone healing. This inhibition appears linked to cyclooxygenase-2., Clinical Relevance: If the results of this study are verified in a larger animal model, the common practice of administering non-steroidal anti-inflammatory drugs after rotator cuff repair should be reconsidered.

A scar tissue interface forms rather than a normal ligament insertion site following attachment of a tendon graft to bone. The specific cell types that initiate the process of tendon-to-bone healing are unknown. We hypothesized that inflammatory cell accumulation following tendon-to-bone repair results in this scar interface. We used a rodent model to examine the temporal and spatial pattern of accumulation of hematopoietic lineage cells in the early phase of tendon-to-bone healing. Thirty-six Lewis rats underwent anterior cruciate ligament (ACL) reconstruction in the left knee using a flexor digitorum longus tendon graft. Six animals were sacrificed at 4, 7, 11, 14, 21, and 28 days after surgery. Serial sections were analyzed for proliferating cells (PCNA), recruited macrophages (ED1), resident macrophages (ED2), neutrophils, T-lymphocytes (CD3), mast cells, immature progenitor cells/pericytes (expressing the NG2 cell-surface chondroitin sulfate proteoglycan), and newly-formed blood vessels (Factor VIII). Neutrophils, ED1(+) and ED2(+) macrophages accumulated sequentially in the healing tendon graft, with progressive cell ingrowth from the interface towards the inner tendon. Neutrophils and ED1(+) cells were seen in the tendon-bone interface at 4 days after surgery, while ED2(+) macrophages were not identified until 11 days. These cells progressively repopulated the tendon graft. NG2-positive progenitor cells were found along the edge of the bone tunnel in the interface, but these cells did not invade the tendon. Occasional T-lymphocytes and mast cells were seen in the tendon-bone interface. There was no proliferation of intrinsic tendon cells, indicating that the tendon does not directly contribute to healing. We hypothesize that cytokines produced by infiltrating macrophages are likely to contribute to the formation of a fibrous scar tissue interface rather than a normal insertion site.