The addition of PRP alone to a suture repair of the ACL was not sufficient to enhance any of the outcome measures evaluated here, including AP laxity of the knee and the tensile properties of the repair. It should be noted that the experimental design was designed with 80% power to detect a 30% improvement in biomechanical results. Therefore, it is unlikely that a clinically meaningful result was missed.
In this study, the porcine model was chosen because of its size, its dependence on the ACL for function,23
its similarity with human gait biomechanics,24
and the similarity of the baseline coagulation values and platelet sedimentation characteristics to human blood.25
Platelet counts in the pig averaged 482 ± 95 k/mm3
, a value just above the normal range reported for humans (150 to 450 k/mm3
However, the Yorkshire pig grows at an extremely fast rate, and in this study the animals almost doubled in size from 30 to 60 kg over the three months of the study and during this same period, the maximum load of the intact ACL almost triples (data not shown). Therefore, the chief limitation of this model is the fact that it may be problematic to use for a study designed to go longer than 3 months. The use of minipigs obviates this rapid growth issue, but in our experience, there are two problems that prevent the minipig from being a good animal for large-scale testing. First, the purchase price of these animals is typically 10 times greater than that of the Yorkshire pig, and second, age-, weight-, and gender-matched groups of animals are more difficult to find due to their limited availability compared to the young Yorkshire animals. As noted in the results, the animals used in this study had the advantages of very little interanimal variation in range of motion, joint size or laxity, and weight at the beginning of the study. Thus, for initial studies designed to go 3 months or less, the skeletally immature Yorkshire pig continues to be a good model for relatively large-scale studies, whereas the minipig may be more conducive to, longer term studies with fewer experimental groups.
In addition, the use of a skeletally immature large animal model likely provides the harshest testing environment for ligament healing studies. Prior work examining partial ACL transection and patellar tendon healing suggest that the scar formed in skeletally immature animals is functionally inferior to that that formed in skeletally mature animals.4,9
Thus, if we can eventually be successful in stimulating healing in this more difficult population, the results may be more effectively translated to the mature knee; whereas development of techniques in the mature knee may not hold up in the skeletally immature knee.
An additional limitation of this model is that is that bilateral surgeries were performed on each animal. Thus, there may be effects on activity limitations imposed by this model may be more severe than that seen in a unilateral model. To mitigate this concern, animals were monitored closely for any signs of persistent ambulatory defects during the first 2 weeks after surgery. No evidence of limp, disinterest in food or water or lack of activity were noted to persist longer than 1 week in any of the animals.
The final limitation of this model is that although it is less expensive than the minipig models, large animal studies still remain inherently more costly than studies with smaller animals. Therefore, the number of animals that can be included in each group is far less than might be accomplished with a mouse or rabbit model with the same budget. As in this study, this can lead to a relatively low power to detect small differences between groups. However, the number of animals for this study was calculated based on a power analysis for the percent difference between groups that we felt would be clinically significant (30% improvement) and the previously determined standard deviation for primary suture repair in the pig model (20%). Using these values, our standardized effect size was 1.5. Our experimental sample size of five animals provided 80% power to detect this size of a difference for each of the failure and laxity variables based on a paired t-test and our prior standard deviations in this model. Our actual standard deviations in the model were higher than 20%, and thus our power lower for the outcome measures that estimated in the sample size calculations—for example, the power to detect a 30% difference in AP laxity at 30° was only 71%, whereas at 60° it remained over 80%. Post hoc analysis demonstrated that particularly for failure load, the power was relatively low (26%) to determine if the observed 23 N higher mean obtained in the PRP group was significant. Based on the observed variability, and a subsequent effect size of 0.5 (using the lowest observed SD and the mean difference between groups), we would have needed approximately 28–32 animals to achieve 80% power for detecting whether this relatively small difference was statistically significant.
However, this model has actually fairly low variability within groups compared to the knee laxity differences that would need to be achieved to make primary repair clinically relevant. For example, the standard deviation in AP laxity was 2.2 mm at 30° and 3.3 mm at 60° for the suture repair group, whereas the difference in AP laxity between the suture repair group and the AP laxity at 60° of flexion in a historical group of intact pig ACLs ligaments was 8.9 mm.19
To achieve a clinically significant improvement of 6 mm of laxity (thus bringing the repaired knee to within 3 mm of the intact knee laxity), the effect size would be approximately 6 mm/3 mm or 2.0, and based on repeated-measures ANOVA with knee as the repeated-measures fixed factor, a sample size of five pigs provided 80% probability to detect this magnitude of a difference. Thus, we are confident in concluding that effects this large or larger as a result of adding PRP are unlikely. With the data obtained in this study, we can also now predict that with similar standard deviations in future studies, this model will provide 90% power to detect a difference of 6 mm in AP laxity if six animals are in each group, and 95% power if n
=7. Thus, this may be a useful model in future studies given the relatively low standard deviations, likely made possible by the availability of age-, gender- and weight-matched animals.
This is the second study using the porcine complete ACL transection model. In the first study, collagen–PRP hydrogels were used to stimulate repair of the ACL and the results evaluated at 4 weeks after surgery. In that study, significant increases in maximum load and linear stiffness with the addition of a collagen–PRP hydrogel were reported. In this study, using PRP alone without the collagen scaffold, no significant effects using PRP were noted on the same mechanical properties. The maximum load of the suture-only repairs in this study, at 3 months, was almost twice as high as that previously reported at 4 weeks, suggesting some additional gain in strength may occur even in the ACLs repaired with sutures and no collagen or PRP. However, the mean maximum load at 14 weeks for ACLs treated with suture and PRP did not achieve the same value as that previously reported for collagen–PRP-enhanced repairs at 4 weeks (103 ± 27 N).
The immature pig knee has several clinically important similarities with the human condition with regard to ACL injury and healing. There was no significant spontaneous improvement in knee laxity with suture repair over the 3 months, a finding similar to that in humans after ACL repair. In addition, the addition of PRP alone did not significantly improve healing of the ligament. In comparing these results with historic controls, the AP laxity of the knee at 14 weeks after suture repair of the ACL with or without PRP was almost three times higher than that previously reported for intact knees19
(4.9 ± 0.9 mm, mean ± SD; p
<0.0001; ), but remained well below that previously reported in the ACL deficient porcine knee (32 mm; n
Even with the addition of PRP, there was little evidence in this group of the two ends of the ACL reconnected with a sufficient scar mass. This is consistent with prior reports suggesting that even after acute ACL injury in human patients, the bleeding from the ligament and surrounding tissues is not enough to encapsulate the ends of the ligament in a fibrin clot.10
In this study, it would appear the same is true, and that the fibrin and platelets of the PRP need to be immobilized by a scaffold material within the ACL wound site to be effective as was seen in the prior short-term study in this model using a collagen scaffold to “capture” the platelets in the wound site.7
In conclusion, the skeletally immature Yorkshire pig represents an animal model that is conducive to studies of stimulating ACL repair. The knee is large enough for standard surgical equipment and techniques to be used, these animals are readily available in age-, weight-, and gender-matched groups to minimize heterogeneity. In addition, the biomechanics of the knee and response to suture repair are similar to that of the human knee. Thus, for short-term (<3 month) studies of ACL repair, the skeletally immature pig model is recommended. However, the use of PRP alone as an adjunct to suture repair did not improve ACL healing in this model, and currently there is no translational animal model evidence to support its use. We hypothesize that PRP would need to be combined with a scaffold to stabilize it within the wound site in order for it to be effective.