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ACL rupture occurs in hundreds of thousands active adolescents and young adults each year. Despite current treatment, post-traumatic osteoarthritis following these injuries is commonplace within a decade of injury in these young patients. Thus, there is widespread clinical and scientific interest in improving patient outcomes and preventing osteoarthritis. The current emphasis on the removal of the torn ACL and subsequent replacement with a tendon graft (ACL reconstruction) stems from adherence to a long held and widely accepted doctrine that the ACL has only a limited healing response and therefore cannot heal or regenerate with suture repair. Recent work has shown that the premature loss of the provisional scaffold in the wound site after ACL rupture with or without repair prevents healing. Additional studies have detailed findings after placement of a substitute provisional scaffold in the wound site of the ACL injury to initiate healing of the ruptured ligament after primary repair. This technique, called enhanced primary repair, has significant potential advantages over current ACL reconstruction techniques, including the preservation of the complex attachment sites and innervation of these structures, thus retaining much of the biomechanical and proprioceptive function of these tissues. This manuscript summarizes the recent in vitro and in vivo studies in the area of enhancing ACL healing using biologic supplementation. Subsequent work in this area may lead to the development of a novel approach to treatment of this important injury.
Ruptures of the anterior cruciate ligament of the knee affect over 175,000 patients every year, including an estimated 38,000 high school students1. These injuries are devastating, not only at the time of the acute injury, but also as patients who sustain an ACL tear have a 78% risk of radiographic osteoarthritis within only 14 yrs following their injury (Figure 2), whether they undergo surgical reconstruction or not 2. For a high school or college student with an ACL tear, that is a striking and troubling statistic.
The anterior cruciate ligament has long been thought to have poor capacity for healing, even with suture repair. Rates of failure of healing (non-union) and structural laxity of the ACL, even with surgical repair, range from 40 to 100% 3–5. This is in contrast to other ligaments, such as the medial collateral ligament (MCL), where successful healing is essentially universally achievable with only six weeks of brace treatment. The lack of functional healing seen in the ACL after suture repair has been previously attributed to the “hostile” environment of synovial fluid6; 7, to alterations in the cellular metabolism after injury8; 9, and to intrinsic cell deficiencies10–16. This has led to abandonment of suture repair, and almost universal adoption of ACL reconstruction for treatment. In ACL reconstruction, the torn ACL tissue is removed from the knee surgically and replaced with a tendon graft harvested either from the medial hamstrings, the middle third of the patellar tendon, or allograft tissue.
However, although ACL reconstruction is an excellent operation for restoring the sagittal plane stability of the knee, significant problems remain. In the short term, ACL reconstruction requires harvesting of other tissues from the knee, a procedure with its own associated morbidities. Allograft tissue carries the risk of disease transmission and delayed biologic incorporation, and also presents issues related to cost and availability. It also removes the native ACL tissue and the sensory nerve fibers (and thus neuromuscular function) of the ligament. Lastly, it replaces a complex, fan-shaped bundle of 17 different ligament fascicles with a single or double bundle of tendon fibers. The point-to-point graft is unable to restore the normal rotational kinematics of the knee. Some of these deficiencies may contribute to the premature degeneration of the joint which occurs after ACL injury, even with ACL reconstruction 2. Perhaps the most concerning problem, however, is the fact that even with ACL reconstruction, patients have a high risk for premature post-injury osteoarthritis. Thus, even with our gold standard of treatment for ACL injury as many as 78% of patients will have radiographic signs of arthritis at only 14 years after surgery2.
The histologic response to injury in the human ACL has been found to be significantly different from that previously reported in the medial collateral ligament (MCL). One study examined human ACL tissue before and after rupture17; 18 and compared the cellular responses in the ACL with those previously reported for the MCL6; 19. Using histology and immunohistochemistry techniques, it was found that like MCL cells, the ACL cells within the ligament proliferate and the ligament revascularizes after rupture6; 18; 19( FIGURE 3). Collagen production was also noted to continue within the ligament up to one year out from injury20. Cells in both the intact and ruptured ACL were found to migrate easily into a simulated wound site21; 22.
However, the provisional scaffold which reconnected the torn collagen fascicles in the wound site of the MCL was missing in the ACL, where the two ends of the ruptured ligament were simply churning around in synovial fluid and never able to reconnect 18. Thus, it appeared that while the cells and vascularity of the ACL were capable of mounting a histologic healing response, there was no structural evidence of a filling in at the wound site in the ACL (FIGURE 4).
The observation that there was essentially no provisional scaffold formation or wound site filling between the ends of the ruptured human ACL led to additional in vivo studies comparing the ACL and MCL histologic response to injury in a mechanically stable and contained defect24. Central defects were created in extra-articular ligaments (MCL and/or patellar ligament) and an intra-articular ligament (ACL) in canine knees and the histologic response to injury evaluated at 3 days (n=3), 7 days (n=4), 21 days (n=5) and 42 days (n=5).
The findings in these studies were that MCL and patellar ligament defects exhibited far greater filling of the wound site and increased presence in the wound site of fibrinogen, fibronectin, platelet-derived growth factor-A, transforming growth factor-beta1, fibroblast growth factor and vonWillebrand’s Factor and vWF when compared to ACL defects at all time points25 (FIGURE 5). Thus, this study supported the hypothesis that there is a lack of provisional scaffold found in the intra-articular wound site of the ACL, and this loss is also associated with a decreased presence of important extracellular matrix proteins and cytokines within the wound site of the ACL.
These findings in humans17; 18; 20–22 and in the experimental large animal model24–26, led to a unique hypothesis – namely, that perhaps it was this lack of provisional scaffold between the two ends of the torn ACL that was a key mechanism behind the failure of the ACL to heal. In the MCL and other comparable tissues which heal successfully outside of joints, the very first phase of the healing response is filling of the defect with a fibrin-platelet plug which bridges the wound edges19. This plug, or scaffold, is then subsequently invaded by reparative cells which remodel the scaffold into a healing fibrovascular scar (FIGURE 4). The formation of the early provisional scaffold is the first critical step in the wound healing process.
However, inside the joint, even though bleeding occurs after ACL injury, no fibrin-platelet plug is observed to form, even at the injury site27(FIGURE 4). One possible explanation for this finding is that circulating intra-articular plasmin breaks down the fibrin plug as fast as it can form. Recent work has shown that after trauma to the joint, the production of plasmin is upregulated via the increased secretion of urokinase plasminogen activator28 (FIGURE 6). With the additional circulating plasmin, the fibrin network is quickly destabilized within the joint environment and no fibrin-platelet plug forms.
This premature loss of the fibrin-platelet plug would have the significant clinical benefit of preventing overall joint scarring and stiffness (arthrofibrosis) and thus maintenance of joint mobility after injury. However, the degradation of the fibrin-platelet plug in the overall joint also would remove it prematurely from any wound sites. As formation of the fibrin-platelet plug is the essential first step for wound healing of musculoskeletal tissue outside the joint19, we believe that the loss of this fibrin-platelet plug inside the joint is the key mechanism behind the failure of tissues in the joint (intra-articular tissues) to heal (FIGURE 7)
The hypothesis that the premature failure of the provisional scaffold as the impediment to ACL healing is an important change from previous mechanisms proposed for ACL non-union, which focused predominantly on the intrinsic cell and vascular responses11–13; 15; 29–34. Past research based on the cell-deficiency hypotheses has focused predominantly on stimulation of cells in vitro with growth factors, or on cell transplantation into wound sites (including the use of stem cells and genetically modified cells). However, if it is failure of the provisional scaffold, an additional line of inquiry into placement of a substitute scaffold itself would be a logical step (FIGURE 1).
Based on the early studies on the provisional scaffold failure in the ACL wound site in humans and animals, materials that might be useful in the ACL wound site were evaluated. The substitute scaffold would need to withstand the physical and mechanical conditions of the intra-articular environment. The mechanical boundary conditions require the scaffold be able to withstand the relatively high physiologic strains seen by the connective tissues of interest, strains which typically approach 3% during normal knee motion35. Enzymatically, the bridge would need to be resistant to degradation by circulating intra-articular inflammatory metabolites and proteolytic enzymes that are present after injury or surgery, including plasmin28, matrix metallo-proteinases36 and glycosidases37. It is also likely that biologic stimuli would be required to successfully replicate the successful wound healing environment, either in the form of a single cytokine38; 39 (sufficiently upstream in the wound healing cascade) or in a group of growth factors, including the cohort released by platelets in other wound sites.
In vitro studies have demonstrated improved cellular proliferation and migration, as well as increased collagen production rates with the addition of growth factors including PDGF16; 40, TGF-b40, and FGF29; 40. In follow-up studies of these in vitro results, healing of the ACL using growth factors in animal models has also been studied39; 41. Kobayashi et al noted improved filling and vascularity surrounding a central defect in a canine ACL model with implantation of a bFGF pellet; however, no biomechanical testing was performed in that study41. In a study of MCL transection in a rabbit model, Spindler et al reported that the addition of TGF-b2 to the wound site resulted in an increase in scar size, but not scar strength39.
Healing of intra-articular defects using substitute provisional scaffolds has been a recent area of interest. Defects in the ACL have been treated with synthetic scaffolds loaded with growth factors including TGF-b41 and also with hyaluronic acid42. These techniques have had limited success, but unfortunately continue to have problems with implant-host integration, cell survival after transplantation, and degradation with time. Better results have been obtained with the use of collagen scaffolds loaded with platelets.
In a central defect rabbit model, Wiig et al reported improved covering of a central defect in the ACL with intra-articular injection of hyaluronic acid in a rabbit model42. In that study, a greater angiogenic response was seen in the group of HA treated ligaments. More Type III collagen was produced in the ligaments treated with HA than in the group of ligaments treated with saline. No biomechanical testing was reported for that study.
Recent studies have detailed the outcomes of treating a complete ACL transection with a suture repair augmented with a substitute scaffold. The complete transection model in the pig results in instability of the knee joint itself and provides a harsher healing environment that closely mimics the condition of the human knee after ACL rupture. In this study, bilateral ACL transections were performed in seven 30 kg Yorkshire pigs and repaired with a four stranded, absorbable suture repair 43(FIGURE 1). In five animals, one of the repairs was augmented with placement of a collagen-platelet composite at the ACL transection site, while the contralateral knee had suture repair alone. No postoperative immobilization was used. After a four week healing period the animals underwent in vivo magnetic resonance imaging followed by euthanasia and immediate biomechanical testing. Six control knees with intact ACLs from three additional animals were used as an intact ACL control group. Supplementation of suture repair with a collagen-platelet composite resulted in formation of a large scar mass in the region of the ACL. Load at yield, maximum load and ACL tangent modulus were all significantly higher in the suture repairs augmented with collagen-platelet composite than in repairs performed with suture alone. This paper concluded that biomechanical healing of the porcine ACL after complete transection and immediate suture repair can be enhanced at an early time point with use of a collagen-platelet composite placed in the wound site at the time of primary repair43.
The results of this study demonstrated that primary repair using the collagen-platelet composite resulted in healing ligament strength which was over 50% of the intact ligament strength at 4 weeks after repair. This strength compares is favorably with the current gold standard of ACL reconstruction, where strengths at 3 to 4 weeks are under 25% of the intact ligament strength (Figure 8, Hunt et al, 200544).
This first demonstration of histologic and biomechanical healing of the ACL will likely lead to entirely new fields of inquiry surrounding treatment of this important structure. Hypotheses regarding the influence of patient age or gender on repair outcome become interesting and important, as do questions regarding the ability to maintain “mechanical homeostasis” by stimulating sufficient anabolic cellular responses to generate wound strength, while at the same time, encouraging gradual catabolism of the provisional scaffold. It is also likely that the concentration of cellular components of the platelet-rich plasma (specifically platelets and leukocytes) significantly effect the cellular proliferation and migration into the wound site, as might the concentration of the extracellular matrix molecules within the substitute scaffold. One might hypothesize that changes in these fundamental cellular behaviors may result in significant changes in the mechanical, and functional, properties of this ligament. These are just a few of the questions that will likely be investigated over the next few years.
The last decade of research into the potential of primary ACL repair has resulted in discovery of a novel mechanism to explain the failure of the ACL to heal and the validation of several large animal models to test new techniques of repair in vivo. Further optimization of these techniques into a successful surgical procedure has the potential to alter the clinical treatment of ACL injuries.
Funding was received from NIH grants AR054099, AR052772 and AR049346.
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