The results of this study show that the use of an atelocollagen scaffold alone to enhance primary suture repair of the ACL was not effective. There were no significant improvements in any of the evaluated biomechanical parameters (AP laxity or structural properties). It should be noted that the AP laxity of the intact adolescent Yucatan minipig knee is approximately 2 mm using the testing methods reported here (unpublished data). In the present study, the average values for the SUTURE and SCAFFOLD repairs were 350% of that value. The average value of AP laxity at 30° was 34% higher in the SCAFFOLD group than in the SUTURE group, a difference that did not reach statistical significance, likely due in part to the small sample size of our study. The AP laxity at 30° is likely dominated by the posterior capsular constraints and the effect of scaffold placement on those constraints deserves further study. However, the measured difference in means of AP laxity at 60° and 90° of flexion (where the ACL repair tissue would be expected to play a larger role) were far smaller at these more physiologic testing conditions. In addition, the maximum tensile load of an intact ACL of an adolescent Yucatan minipig is approximately 1600N (unpublished data). Thus, the maximum failure loads of the SUTURE and SCAFFOLD knees on average only reached 25% of the intact ACL load. As material properties were not evaluated in this study, it is possible that the tissue quality was different between the two groups; however, as the structural properties dictate joint function and laxity, these were chosen as our primary outcome measures. Future studies including material property measurements would be useful, particularly for evaluating repair techniques that result in significant improvements in AP laxity or structural properties.
In this study, there were no significant differences in either the mechanical or histologic outcomes when a collagen scaffold was added to the suture repair. In prior studies of the healing ACL at similar time points9
when a functional improvement in mechanical properties is seen with treatment with a collagen-platelet composite, this has been associated with an increase in the cellular density of the repair tissue. Similarly, when improvements in ACL graft strength are noted with the addition of a collagen-platelet adjunct, histologic differences are noted between groups, with elimination of the avascular areas of the graft10
. However, the specific cellular mechanisms which are responsible for the improvements in mechanical properties with biologic enhancement of the healing ACL remain unknown. Future studies to investigate these mechanisms are warranted for treatments which result in improved functional outcomes.
Atelocollagen is more resistant to synovial fluid degradation than a fibrin clot; however, there were no signs of residual scaffold at 13 weeks. Therefore, the lack of improvement in the functional properties could be due to the premature loss of the scaffold. This could also be the explanation for the lack of improvement seen with use of an atelocollagen hydrogel in our earlier study1
. However, other prior studies using acellular implants have also shown failure to improve failure loads in tendon repair23–30
, where other studies utilizing collagen scaffolds combined with mesenchymal stem cells (MSCs) as a biologic stimulus have been effective in stimulating tendon repair24–30
. For example a collagen matrix seeded with MSCs reached 75% of the maximum force of the intact tendon, whereas the control group of the collagen matrix without the MSCs only reached 40% strength of the maximum force.26
Based on this collection of results, one might hypothesize that platelets or MSCs add a biologic stimulus, while the collagen scaffold provides the structural support, and both are required to stimulate ligament or tendon healing.
Prior studies of the tensile properties of ligaments have typically applied a small pre-load to the bone-ligament-bone construct to ensure consistency in testing.31
However, in prior studies of repaired ACLs,10
we noted differences in the amount of displacement of the joint surfaces with the application of the small pre-load between specimens. As one of the principal functions of ligaments is to maintain a specific bone-to-bone distance relationship, we felt that this displacement difference from tibiofemoral contact (−5 N compression) to a low tensile “pre-load” (+5 N tension) was worth recording as an additional outcome measure. A repaired ligament with a long slack region that allows the joint to shift by 10 mm before restraining joint motion is likely to be non-functional, even if it can eventually support high yield and failure loads.
Complete ACL transection and repair in the larger porcine model offers multiple advantages. The large animal model allows for easy identification of the structures of interest, both at the time of ligament transection and retrieval, and relative ease of structural repair for surgeons. The biomechanical assays are well established for knees of this size. The porcine model was selected since it is anatomically similar to the human knee,32
is dependent on its ACL for joint stability,33
and it has hematologic characteristics that are similar to the human particularly important for wound healing studies involving platelets.34
Adolescent animals were selected for study because they represent the human population at greatest risk for ACL injury. The adolescent minipigs had ascertained 95% of their skeletal growth and were sexually mature. Over the period of the study, the animals increased in weight by an average of only 1.5 Kg.
The porcine model has a few limitations common to large animal studies in quadrupeds. Their gait pattern is different from humans due to the use of four limbs for weight bearing rather than two. While the pig model was selected due to its anatomical and biomechanical similarities to the human knee, there may be differences in gait and rehabilitation which cannot be reliably controlled. Furthermore, there may be subtle differences in the wound healing cascade that are not yet appreciated.
There are also limitations to the histomorphometric approach used in this study. While large differences in cellularity or vascularity can be appreciated using this method, there may be smaller differences between groups that would not be apparent with this qualitative method.
This study was designed to evaluate short-term healing in the porcine model. In a recent evaluation of the porcine model, we determined that the nadir in ligament healing strength occurs between 6 and 9 weeks after surgery.35
For the present study, we selected a time point after this window (i.e. 13 weeks) where an improvement in the structural properties could be detected. Whether differences would occur with additional healing time remains unknown.
In our previous large animal studies, a relatively large variability between animals was observed.10,12,22,35
In order to increase the power of the study, we utilized paired comparison between the two treatments within each animal. This paired design enabled us to control the variability inherent to animal, knee, and ligament size, and growth rate. However, the effects of bilateral surgery (i.e. immediate weight bearing on both repaired joints and the potential for a higher spike in inflammatory response due to bilateral trauma) must be acknowledged.
Due to the expenses of large animal studies, the number of knees in each study group was relatively low (n=8). This can raise concerns about low power in a study in which no significant difference is detected between groups. However, the differences between the SCAFFOLD and SUTURE groups were not only statistically insignificant, they were likely to be clinically insignificant given the small changes between means of the two groups. For example, the 5% improvement in linear stiffness or maximum failure load with use of the collagen scaffold would unlikely justify the expense and complexity of an augmented repair procedure. Nonetheless, the study was more than 80% powered to detect if the enhanced ACL repair would have a return of mechanical strength similar to the successfully healing MCL at a similar time point (60% of normal).36
To determine if the small change noted in maximum load (~5%) of the present study were statistically significant, the study would require over 800 animals to complete.
The goal of this study was to determine if the collagen component of a collagen-platelet composite (CPC) was responsible for enhancing ACL wound healing. The CPC combination has been previously shown to improve wound healing.9–11
In the present study, we determined that the collagen scaffold alone was not sufficient for stimulating the healing process. When taken together, these results suggest that there are at least two core components required to enhance healing of an ACL tear; a collagen scaffold and a stable source of platelets. It is likely that fibroblasts need a scaffold that remains situated between the torn ligament ends and growth factor stimulation by platelets are needed to initiate and drive wound healing. These two factors may work synergistically to simulate the wound healing process that occurs naturally in extra-articular joint healing. Future work will be aimed at methods to stimulate biological activity in collagen scaffolds to enhance intra-synovial soft tissue repairs.