Platelet concentrates, as a source for growth factors, are very popular tools in regenerative medicine. Their power has successfully been employed in tissue-engineering enhanced primary ACL repair6; 13; 14
, in enhanced ACL reconstruction6; 15
, as well as in other fields of surgery15
. However, recent studies have discovered negative effects of PRP and attaining a more differentiated view of what PRP is and does seems prudent and necessary7; 8
. In this study we asked whether reducing the platelet concentration in PRP used for primary ACL repair would affect outcomes in two categories, biomechanics and histology. Not only would such a reduction in platelet concentration be expected to cause a substantial reduction in risk of negative effects, it would also decrease the amount of blood that has to be drawn from the patient..
At 13 weeks, we found no evidence that reducing the platelet concentration from 5X to 3X compromises mechanical outcome after tissue-engineered enhanced primary ACL repair. Stiffness, which might very well be the most important biomechanical parameter, showed virtually identical results. Displacement and load, maximum load and yield load, showed only small differences in absolute values. Interestingly, these means for yield and maximum load showed a slight trend in favor of the lower PRP concentration group, although these differences had no statistical significance. It is noteworthy that the absolute values of the mean differences were very small, suggesting widely identical clinical outcomes for both PRP concentrations.
In the histological analysis, we observed significant differences in cellular density at the central wound site and the femoral insertion, with significantly larger cell numbers per area in the 5X group. Findings based on the Ligament Maturity Index highlighted significant histological differences between the 5X and 3X groups. The cellularity subscore showed not only the increased cellularity but the difference in cell shape. 5X fibroblasts were more elongated whereas the 3X wound area was populated by a mix of both fusiform and spheroid cell shapes. The fibroblasts in the 5X specimens were also better organized within the larger collagen bundles compared to the cells in the 3X specimens ().
The relationship between tissue cellularity and biomechanical function has yet to be clarified. In a recent series of studies of the effects of skeletal maturity on ligament healing, the young animals had a greater cellularity of the wound site at 1 to 2 weeks after injury in comparison with adult animals16; 17
, and the yield load of the ligaments in that age group were significantly higher than the adults at the 15 week time point11
. This suggests that a higher cellularity early in the wound healing process may be beneficial in the porcine model. In addition, a recent study in equine tendon healing reported that use of PRP resulted in both an increase in tendon cellularity and tendon strength at 24 weeks after injection18
, and a second study of MCL healing in rabbits found that use of porcine SIS resulted in both increased cellularity and increased mechanical properties of the ligament after 12 weeks of healing. This suggests that a higher cellularity early in the wound healing process may be beneficial in some situations and animal models. Other in vivo studies of tendon and ligament healing have not found an association between cellularity and biomechanical properties19; 20
. Thus, the relationship between cellularity and mechanical properties is not as yet completely defined.
In this study, while we did find significant differences for secondary histological outcomes, we found no statistically significant difference between the groups in biomechanical outcomes. Having a larger sample size or smaller standard deviation might have led to statistically different result; but would have been less likely to show a clinically meaningful decrease in mechanical properties with a reduction in platelet concentration. This is due to the fact that for most outcomes, the differences in the means of the two groups were relatively small, and in all cases, the differences favored the group with a lower platelet concentration. While the differences were not statistically significant, this trend makes it even less likely that increased sample sizes would reveal a detrimental effect of reducing the platelet concentration.
ACL healing has been shown to go through a revascularization phase between six and nine weeks after suture repair, during which time, the biomechanical properties of the healing ligament hit a low point13
. For this study, we selected a time point that was beyond this nadir in tissue properties. Differences at earlier time points would not necessarily correlate to survival of the repairs through the revascularization process, and thus, results at those time points might be mechanistically interesting, but less translational in their applicability. We have used a similar time point in previously published studies for this reason9; 13; 21
. The study of longer time points would have been more justifiable, again, if the decrease in platelet concentration were seen to be significant at 13 week time point.
This study used a randomized complete block experimental design to minimize inter-animal variability, a problem which plagues in vivo studies. We have used this approach with success in other in vivo studies13; 22
. The strength of this matched design is that it allows us to limit the number of animal lives required to answer specific questions while maintaining strict control over confounding biologic variables (i.e. weight, sex, activity level). However with this design we are inherently limited to study only one research question per experiment (in this case, 3X vs. 5X concentration of platelets).
In conclusion, our data suggest that there is little functional difference in ligament healing with the use of 3X or 5X PRP. The use of a lower concentration of platelets allows for a proportionally smaller amount of blood required to make the PRP, thus making the procedure easier for the patient.