In this study, we found histologic changes in the insertion site after transection and enhanced repair of the ACL, and that these changes were dependent on the degree of return of mechanical strength in the repairs. In immature and adolescent animals treated with bioenhnaced repair, reasonable return of mechanical strength was seen in both groups.18
Accordingly, the initial changes of fibroblast proliferation and loss of collagen alignment in the fibrous zone, as well as osteoclastic resorption of the fibrocartilage layers, were followed by increased collagen alignment in the fibrous zone and partial restoration of the fibrocartilage zones. However, in adult animals and adolescent animals which had ACL transection only and which healed with poor mechanical strength,18
the changes over time were more gradual, with the fibrous zone becoming relatively acellular with steadily increasing disorganization of the fibrocartilage zones. The changes in these animals were more consistent with degeneration of the insertion site.
Ligaments and tendons do not consistently regenerate the mineralized and unmineralized fibrocartilage layers of the native insertion site after injury or reconstruction.9,10,11,21,22,23
From the results of the present study, it appears that fibrocartilage layers are not completely reformed by 15 weeks after bioenhanced repair of the ACL. By 15 weeks, while there is a reappearance of chondrocytes arranged in a columnar arrangement perpendicular to bone in layers, the organization of these layers is not as structured as in a native insertion site. Although fibroblastic proliferation and osteoclastic presence are mediators in this process, it is unclear whether they augment or hinder the development of the fibrocartilage layer in primary healing of the ACL.
The disorganization of the columnar organization within the fibrocartilage layers, as well as the apoptotic appearance of the chondrocytes and the disappearance of distinct lacunae, as observed in the insertion site of adult animals and non-treated adolescent animals at 15 weeks, are changes similar to those seen in insertion sites after rupture of the ACL. Prior studies have shown increasing chondrocyte apoptosis and decreasing thickness of both the nonmineralized and mineralized fibrocartilage layers in the ACL insertion site after substance resection by 6 weeks.15,16,17
Our study suggests that the insertion site of non-healing ligaments, even with bioenhanced repair, demonstrates degenerative characteristics similar to non-treated ruptured ligament insertion sites. Conversely, the insertion site of healing ligaments, as seen in the immature and adolescent animals, do not demonstrate these degenerative changes.
Prior models on insertion site healing have investigated changes after injury of the MCL,14,24
flexor tendons of the hand,22,23
the rotator cuff tendons,25,26
and during healing of an ACL graft in a bone tunnel,9,10,11
and collectively, these studies show that ligament or tendon-to-bone healing occurs initially by fibroblastic proliferation at the bone-ligament junction. In the present study, fibroblastic proliferation also was found to occur at the insertion site during bioenhanced repair of the ACL after transection, as demonstrated by the highly cellular fibroblastic layer which develops between 2 and 4 weeks in the fibrous zone of immature and adolescent, but not adult animals. Similar to prior investigations,,9,10,11,14,21,24
this early fibroblastic proliferation is characterized by highly disorganized collagen alignment in the fibrous layer of the insertion site. The collagen layer in the fibrous zone undergoes progressive organization, and by 15 weeks a relatively large proportion of collagen is again oriented perpendicular to the insertion site in a densely packed arrangement, although it is still hypercellular relative to a normal insertion site.
Studies on rotator cuff healing25,26
and ACL reconstruction27,28
demonstrate that the process of fibroblastic proliferation at the insertion site is regulated by inflammatory mediators and growth factors which direct the differential expression and remodeling of collagen within the fibrous and fibrocartilage zones. These studies show that the ability to form fibrocartilage during insertion site healing may be impaired by a reactive inflammatory response which directs fibroblasts to initially form reparative scar, rather than regenerative tissue. In the present study, although there is a predominance of fibroblastic proliferation, the inflammatory response appears to be minimal at the insertion site of the healing ACL. Thus, it is therefore unclear to what extent the fibroblastic response is reparative or regenerative during ACL healing with bioenhanced repair. The intra-articular environment, the presence of platelet-derived growth factors and the biomechanical loading in our model may affect the fibroblastic response in a manner that is different than prior models. Future studies are planned to determine the temporal expression of collagen subtypes to determine if the fibrous layer resembles scar or regenerative collagen and fibrocartilage.
Here, osteoclastic remodeling was observed in groups where functional ACL healing was most successful. However, prior studies have reported that osteoclastic activity results in impaired healing at the tendon-bone junction in a bone tunnel. Rodeo demonstrated that stimulation of osteoclastic activity with receptor activator of NF-kappa β ligand (RANKL) impaired formation of the bone-tendon insertion site while inhibition of osteoclastic activity with osteoprotegerin (OPG) facilitated enhanced healing at the insertion site during tendon to bone healing in ACL reconstruction in a tunnel.29
Similarly, Thomopoulos prevented bone loss associated with osteoclastic activity and showed improved insertion site healing in a flexor tendon healing model in a tunnel by the administration of alendronate after insertion site injury.23
Although the results in the present study seem contradictory to these prior reports, the environment of the insertion site for ACL healing is likely different from that of a healing tendon within a bone tunnel. For example, the vascular supply of the healing ACL insertion site is not completely disrupted as it is when a free graft is placed in a bone tunnel. In addition, the healing ACL insertion site experiences mechanical stress which is predominantly tensile, where a graft in a tunnel experiences stress which has a shear component as well. Thus, the two models of tendon and ligament to bone healing may represent fairly distinct biologic and mechanical situations, and the role of osteoclasts may be different in each case. However, because this study did not test a hypothesis for the mechanism of osteoclasts in healing, future studies would be necessary to better define their role in ACL healing. For these reasons, it is as yet unclear whether or not the osteoclasts impair insertion site healing as seen in other models, or whether they are, in fact, important mediators that facilitate healing during repair of the uninjured ACL insertion site.
In addition, the insertion site was not directly injured in the present study. The histologic changes seen are therefore likely related to something other than injury, possibly loss of tensile stress across the interface. Similar histologic changes occurred after midsubstance MCL rupture without direct injury to the insertion site14
, where there was osteoclastic remodeling of the insertion site between two and six weeks after rupture. However, the increased osteoclastic activity seen in the present study is likely not distinctly associated with mechanical unloading, as the adult animals did not demonstrate the types of osteoclastic changes at the insertion site that was observed in the younger animals, despite the fact that all age groups initially had their insertion sites unloaded after transection of the ACL. If the osteoclastic changes were due to unloading alone, one would expect to see osteoclastic changes in the insertion sites of the adult animals as well.
Interestingly, treatment with collagen-platelet composite resulted in all ligaments healing in continuity. While some repairs were stronger than others at the 15 week time point, none of the repairs frankly failed or formed gaps. The lower strength in the adult animals at 15 weeks may have resulted in relative unloading of the insertion sites and contributed to the degenerative changes noted in that group.
Although the results of our study reveal that insertion site remodeling during enhanced suture repair occurs initially by fibrovascular proliferation and osteoclastic remodeling, our results do not show what occurs at intermediate time points. Although prior studies have shown osteoblastic growth to be an important mediator which succeeds fibroblastic proliferation in tendon-to-bone healing in ACL reconstruction,29, 30
our study did not show the presence of osteoblasts at early or late time points. Future studies at intermediate time points will provide a better understanding of transitional steps in this process to determine if there is an antecedent osteoblastic or chondroblastic stage which directs the remodeling during enhanced suture repair.
One potential criticism of the present study was that there were more than twice as many platelets in the 15 wk group as in the other timepoints. However, recent studies have demonstrated no difference in mechanical performance of ACL repairs with a range of 3× to 5× the systemic concentration,31
so this difference in platelet concentration on functional healing is likely less significant. Future studies of the effect of platelet concentration on histologic changes in the repair tissue would be of interest.
One limitation of our study is that it is unknown whether or not unilateral surgery and/or more protective rehabilitation of the repaired ligaments and insertion sites would have significantly increased or decreased the fibroblastic proliferation and osteoclastic activity noted here. Unfortunately, we do not have biomechanical data on the animals tested from weeks 1–4, however, a previously published paper from our group found that there was a nadir in healing ligament yield load between 6 and 9 weeks after repair,32
after which time increases in yield load occurred up to 15 weeks post-repair.
This study has shown that specific insertion site changes occur when the ACL heals biomechanically after enhanced suture repair in skeletally immature and adolescent animals. These changes include fibroblast and osteoclast proliferation, particularly at 2 to 4 weeks after ACL injury, and these changes partially reverse by 15 weeks after successful repair. Further investigation into the age-related biology of the insertion site and the surrounding tissues, including evaluation of the matrix composition, the presence of stem cells adjacent to the injury site, the molecular signals, and potential molecular manipulations of the healing process, are all clinically relevant avenues of study that could potentially lead to a revolution in the treatment of ACL injuries, particularly for young patients.