What was needed after the in vitro proof of principle was confirmation in an in vivo model that closely mimicked that human knee joint. Among the available large animal models, canine models have a long track record for ligament repair models, but procedures that are routinely successful in humans fail in canines, making it a less clinically relevant model. Pigs and sheep are closest in anatomy and biomechanics and are validated and economical models for knee surgery and ligament repair and were therefore chosen for this purpose. The development of the model itself, however, was a two-stage procedure. First, the therapeutic potential of enhanced healing had to be tested in a stable, central, defect before a second variable – mechanical stress – could be introduced in the second step, the complete rupture model.
The central defect model, designed to test the biologic healing capacity of enhance repair, was a canine model. In a randomized trial, a standardized. 3.5mm central defect was created in the central portion of the ACL and either left untreated as a control, or treated with a collagen-platelet-composite (CPC)24; 35
. Comparing this central defect model to the biological gold standards of ligament and tendon healing, the medial collateral ligament (MCL) and patellar tendon, the model confirmed the poor healing capacity of the untreated central defect of the ACL and excellent healing of the untreated defect in the MCL and patellar tendon24
. In addition, a procedure where the defect was filled in with a collagen-platelet composite (CPC) resulted in healing of the ACL defect that was histologically similar to the MCL and patellar tendon. Further histology and immunohistochemistry of the defect site showed a significant increase in tissue filling, and growth factor expression at 3 and 6 weeks post-op with use of CPC24
. Finally, MRI analysis and biomechanical testing was done at 6 weeks post-op to complement the description of the repair tissue's histologic profile35
. These analyses showed a significant increase in ACL strength after treatment with CPC, and a significant association of this increase in strength with the amount of wound filling on MRI35
The stable defect model showed that treatment with CPC resulted in more defect fill and a stronger repair tissue, similar to the tissue seen in the healing MCL. However, a more clinically relevant model would be a complete ACL rupture. In the second stage of in vivo testing, a complete ACL transection model was validated in pigs. A new problem in this model was how to deliver and maintain the CPC in the defect site. In the most recent modification of the technique, a specially processed collagen scaffold soaked with PRP is used as a delivery system.
Briefly, for descriptive purposes, the procedure can be divided into two aspects. The central mechanism is suture bridge which is stabilized proximally with an Endobutton (Smith and Nephew, Andover, MA) on the proximal lateral femoral cortex and then sutures from the Endobutton are brought through the knee and tied over a extracortical button on the anteromedial aspect of the tibia to stabilize the knee during the early post-op period A second set of sutures coming from the Endobutton are ties to the tibial stump of the ACL in an attempt to recreate the initial trajectory of the torn ACL. To accomplish this, six suture limbs, run from the Endobutton through a 4.5 mm tunnel into the knee joint. Four of the suture limbs are threaded through the collagen scaffold and continue to leave the knee through a 2.4 mm tunnel from the tibial ACL insertion onto the medial tibial cortex where they are tied, under tension control, over an extracortical button. These sutures serve to pull the scaffold, soaked with PRP hence malleable, into the knee and to afford initial antero-posterior stability of the joint. Since these sutures are absorbable and lose roughly 25% of their initial strength per week, there is an increasing amount of mechanical stress, or stimulus, on the repair tissue over time until the suture is completely absorbed after approximately 63 days on average (Ethicon, Somerville, NJ). This is the first aspect of the procedure, creating a temporarily stable scaffold for repair in perfect alignment with the original ACL. With two of the three sutures used for scaffold fixation and antero-posterior stability, the remaining two suture limbs running from the femoral Endobutton are tied to a whipstitch running up and down the anterior and posterior side of the tibial ACL stump, pulling the ACL tibial stump into the scaffold. This second aspect of the repair procedure with aligns and stabilizes the distal ACL stump to allow for cell migration into the collagen sponge. At this point, the repair construct is left untouched for 10 minutes before wound closure to allow for complete clotting of the PRP.
The use of three sutures begs the question whether stability in biomechanical testing is confounded by this material, rather than afforded by the repair tissue. However, when compared with suture repair alone, suture repair augmented with a collagen-platelet composite resulted in significant improvement in repair strength at 4 weeks (the time when suture strength approaches 0), and 3 months36; 37
. At the same time, there was a significant increase in cellularity with the use of CPC, i.e. a strong, ongoing regenerative response, suggesting even better biomechanical outcomes once these cells produced an organized extracellular matrix36
A potential shortcoming of these studies is that the porcine model builds on an ACL transection rather than a complete, traumatic rupture. However, it should be remembered that even in a clinically complete rupture some fiber bundles as well as the synovial sheath are oftentimes preserved. Also, given the anatomy of the porcine ACL, which is a broad ligament that flatly lies on the tibial plateau rather than obliquely crossing the notch, the “transection” is more of a staged process than a single cut, usually resulting in a combination of numerous cuts at various levels of height as well as quite a few ruptured fibers. Finally, it seems worthwhile to consider that tunnels are drilled through both physes, femoral and tibial, to pass sutures. Such tunnels potentially might jeopardize the growth plate and affect growth. However, the tunnels used for this technique are rather small both in absolute diameter and in relation to physeal area, centered, and bear only little tension, i.e. as much tension as four 0.35mm thick sutures are able to withstand. All these parameters have been shown to reduce the risk of growth disturbances38-43