The addition of blood platelets to the collagen scaffold (CPC group) resulted in significant reduction in AP knee laxity at 6 weeks after autograft patellar tendon ACL reconstruction but did not appear to have an effect on the structural properties of the graft. The large reduction (~50%) in early postoperative AP laxity warrants continued study. In addition, the systemic platelet count of the animals correlated directly with the maximum load for both the experimental group (CPC) and the control group (COLL) and inversely correlated with AP laxity. These findings were consistent across groups, which could be caused by intraarticular bleeding. Intraartciular bleeding could potentially have introduced platelets into the control hydrogel before it set. Nonetheless, these findings highlight the important role blood platelets may play in ACL reconstruction graft healing.
The improvement in AP laxity was most evident when the knees were tested in 30° of flexion (approximately full extension in the caprine knee). At this position, ACL reconstructed knees treated with CPC were on average approximately half the AP laxity at 30° versus the COLL (8.3 vs. 15.0 mm; ). At 60° of knee flexion the CPC had less AP laxity; however, the difference between groups was smaller and not significant (12.0 vs. 17.4 mm for the CPC and COLL groups, respectively). The large increases in AP laxity seen in the COLL group are consistent with those of previous studies of ACL reconstruction with autogenous patellar tendon grafts using the goat model.16–19
After 6 weeks of healing, Papageorgiou et al. reported laxity values of 13.2 mm and 16.2 mm with the ACL reconstructed knee at 30° and 60° of flexion, respectively, compared to the contralateral control (3.9 mm).18
The improvement seen by the addition of CPC is also a marked improvement from results published previously where the AP laxity of the knee increased from 2.0 ± 0.7 mm in the intact ovine knee to 8.3 ± 2.3 mm in the ACL reconstructed ovine knee after 6 weeks.4
The mechanism causing the decrease in AP laxity was not determined in our experiment. Some plausible explanations include recruitment of myofibroblasts into the healing ACL graft, “tightening” of the knee capsule, scarring within the joint, and flexion contractures. However, no adhesions to the capsule other than the scar mass surrounding the graft were apparent upon dissection, and there were no perceived differences in the range of flexion–extension motion in either group.
When platelets come into contact with collagen in a wound site in vivo, they are known to degranulate and release growth factors in the local wound environment. This same effect has been noted previously using a CPC in both in vitro and in vivo studies. In vitro, PDGF-AB has been found to be released in relevant quantities from a similar CPC, suggesting platelet activation and cytokine release.20
In vivo, high levels of FGF-2, PDGF-AB, and TGF-b are found to be localized in the area of CPC implantation for up to 3 weeks after gel implantation, suggesting sustained presence of these platelet-related growth factors in the wound site after platelet activation.5
To our knowledge, this is the first report of a correlation between systemic platelet count and ACL graft strength in an animal model. This finding is supported by other studies detailing the role of specific growth factors released by platelets, such as IGF-1, TGF-β1 VEGF, PDGF, and FGF-2, in the healing of tendon and ligament.21
ACL cell migration in vitro has been stimulated by TGF-β1, while PDGF-AB and FGF-2 can stimulate ACL cell proliferation in a 3D collagen scaffold.22
Platelet-rich plasma has also been reported to stimulate healing of bone both in animal models23
Platelet gels have been used to improve the strength of the abdominal wall fascia after hernia repair in rats.25
However, the use of platelets to stimulate tendon and ligament healing directly has been less widely reported,26
and is a relatively new field of inquiry.
Previous attempts to accelerate the revascularization phase with VEGF in a sheep model found increased vessels but decreased linear stiffness and increased laxity.27
By applying platelets in a stabilized collagen hydrogel we have simulated a more “natural” environment with the natural occurrence of platelets deposited in a fibrin clot as in extraarticular healing. Further, platelets contain a multitude of growth factors in addition to TGF-β1 and PDGF seemingly through natural selection in the appropriate concentration for extraarticular healing. Highly relevant to clinical application is that the cost to apply autologous platelets from peripheral blood is much less than a recombinant TGF, PDGF, or EGF.
The 6-week time point is well recognized as a nadir of strength in ACL reconstruction for animal models. Failure load values in both our experimental and control ACL graft groups were approximately 10% of the intact ACL, which is slightly higher than previous reports of 3% in the sheep model,4
and typical for the goat model after 6 weeks of healing.16,18
Although this time point was useful in determining differences in the revascularization phase of healing of the ACL graft, longer term studies are required to evaluate the effects of platelets on graft strength and AP knee laxity later in the healing phase. As previous translational models of ACL graft healing with TGF-β/EGF6
demonstrated increased strength at 12 weeks, additional investigations examining higher platelet yields and the 12-week postoperative time point are planned.
One limitation of this study was that we were unable to perform platelet enrichment of the caprine blood. Centrifuge protocols that systematically changed the centrifugal force from 50 to 250 × g
, with various cycle times and combinations proved ineffective, and limited our ability to evaluate the role of a two- or fourfold platelet concentration on ACL graft healing, levels that have been shown to enhance primary ACL repair in canine and porcine models.10,11
This was likely due to the similarities in the sizes of the caprine erythrocytes and platelets.13
Although prior studies have reported the use of caprine PRP, three do not report platelet counts in both the whole blood and “PRP;”26,28,29
thus, platelet enrichment levels actually achieved in those studies cannot be evaluated. A fourth study reported TGF-β release from goat “PRP” at levels far below that for human PRP, making it unclear if they had actually produced functional goat PRP.13
An additional potential problem with the caprine model is that with the similar size of the erythrocyte and platelet, overlap between the platelet and erythrocyte cell populations when cell size is used to determine cell numbers is more likely to occur. Therefore, the platelet count measure that correlated with mechanical properties in this study is likely a combination of platelet and the smallest erythrocyte fraction. It is likely that the platelets in this fraction were the more active cellular component; however, further studies to evaluate the relative contributions of platelets and small erythrocytes would be needed to verify this.This problem could be avoided by using the canine or porcine models, which have a greater distinction in cell size and thus have cells that are more easily sorted. With the difficulties in reliably producing caprine PRP, we elected to use the more consistent whole blood for the CPC preparations. Unfortunately, we did not anticipate the great degree of difficulty in obtaining complete hemostasis at the operative site for the goats (which had been easy to do in our prior experience with ACL repair in the pig model), and thus, we observed some blood mixing with the injected gel in the COLL group, which likely added platelets to the hydrogel in the control animals. This makes it difficult to know if the observed effects were due to the systemic platelets, the collagen sponge alone, or the collagen sponge-infused platelet composite. Thus, to fully answer the questions regarding the effect of platelet concentration on ACL graft properties and knee laxity, a different animal model (either porcine or canine) where platelet and erythrocyte size are more distinct and thus the platelets can more easily be concentrated. Additional experiments using collagen scaffold only and controlling for systemic platelets will be necessary.10,11
Nonetheless, the findings reported here suggest that platelets may have an important role in intraarticular graft healing.
It is possible that the sponge, which was wedged between the graft and the notch, migrated after implantation. The sponge was relatively large so we would expect that it would remain in the proper position. Also, once the sponge was implanted and saturated with plasma, it was allowed to congeal for a minimum of 1 h before knee motion was allowed. Certainly, future studies that utilize a sponge that is wrapped around the graft might be worth pursuing given the results in this paper.
A final limitation of the study was the inability to control rehabilitation in the goat model (present in all large animal models of ACL reconstruction). Ruminants need to be standing within the first few hours of surgery, and therefore it is difficult to protect the graft during the initial healing stage. Bandaging and immobilization are effective with this animal model. Although slings could be used, this would result in complete nonweight bearing, which is not relevant to modeling the human postoperative rehabilitation scenario where patients are typically partially weight bearing or weight bearing as tolerated after surgery.
In summary, use of a CPC to enhance healing of an allograft ACL reconstruction correlated with early sagittal plane laxity and the systemic platelet count was highly predictive of ACL reconstruction graft strength and stiffness after 6 weeks of healing in the caprine model. We were not able to separate the relative importance of platelets in the sponge on the graft, platelet concentration in the sponge, and systemic platelet levels in affecting graft and joint properties. Therefore, further work investigating the possible role of a CPC on improving ACL reconstruction outcomes is warranted.