This report is the first to show progressive functional healing of the ACL with improved yield load, displacement at yield, and linear stiffness properties over time. The changes in structural properties were accompanied by significant changes in the ligament tissue biology. In addition, the placement of a CPC in the wound site resulted in repairs with improved structural properties, greater cellularity, and a more fusiform cell shape. The use of a CPC has been found to stimulate ACL graft healing after ACL reconstruction.10
The present study is different in that CPC is stimulating healing of the ACL rather than replacing it.
This study is the first to show functional healing with primary ACL repair at 3 months in any animal model. Studies examining primary repair methods in humans have found this treatment gives results no better than those of nonoperative treatment, with most patients experiencing pain and instability.31,40
Regarding other ligaments, previous studies have documented the healing of the medial collateral ligament (MCL) as occurring in 3 phases: hemostasis and inflammation, cellular proliferation and matrix deposition, and matrix deposition and long-term remodeling.12
On the basis of the results reported here, it appears that after suture repair, the phases of healing in the ACL follow a similar course, with the phases being (1) inflammation, (2) cellular proliferation, (3) vascular proliferation, (4) vascular pruning, and (5) collagen remodeling. Although these phases were present in both groups (suture and CPC), they resulted in a more functional result (improved yield load and stiffness of the repairs) when a bioactive scaffold supplemented the sutures.
The first phase noted here was a cellular proliferation phase, which occurred between 4 and 6 weeks after injury. This phase was characterized by repair tissue with a cellular density more than 3 times that of the intact ACL, and it activated ovoid fibroblasts throughout the wound site. Some new vessels were noted during this phase, but no significant organization of collagen into bundles could be identified. Woo et al,49
studying changes in the MCL with a combined ACL and MCL injury, found that MCL cellularity is at its maximum at 6 weeks after transection (although the 4-week time point was not examined). Frank et al,12
studying dissection of the rabbit MCL, observed a decrease in cell number from 3 to 6 weeks, similar to what this study found. Thus, the cellular proliferation phase noted in the healing ACL is consistent with that observed in other models of soft tissue repair.
The vascular proliferation phase of ACL repair was noted at the 6-week time point, and it was characterized by increasing vascularity of the wound site (more than 20-fold greater than that in the intact ligament) and by decreasing cellular density. During this phase, the increased vessel number in the wound site was predominantly composed of small blood vessels (ie, capillaries), whereas the regions of ligament near the bone had fewer larger vessels (arterioles and venules). As a comparison, the MCL healing study by Frank et al12
found an increase in vascularization up until 3 weeks. Arnoczky et al4
examined the revascularization of a patellar tendon graft used to replace the canine ACL and so observed an increase in vascularization up until 20 weeks. Rougaff and Shelbourne37
conducted biopsies of human patellar tendon autografts used for ACL repair. They found that capillary invasion into the patellar tendon started at 3 weeks and increased in prevalence up to 8 weeks after surgery. Thus, the increase in vascularity at 6 weeks, as observed in the healing ACL, is consistent with the healing of other ligaments and so may be faster than the revascularization of ACL grafts in animal models. Note that platelet-rich plasma has been associated with increases in myofibroblast formation and vascularization, although this study did not note a significant difference produced by CPCs.5,6,22
Vascular pruning was noted between 6 weeks and 3 months as the large number of disorganized capillaries in the wound site changed to smaller numbers of arterioles, with an increasing degree of organization of the vessels into paths that were parallel to the newly deposited collagen bundles. In a study looking at the vascular invasion of a canine patellar tendon graft, vascular response subsided by 26 weeks.4
In human ACL rupture, the vascularity decreased from the 16- to 20-week time point to the 52- to 104-week time point.27
Thus, the observation of a decrease in vascularity as healing proceeds is consistent with prior reports, although this phase occurred earlier here than what was reported in ACL grafts and nonhealing ACL tissue.
At 3 months, collagen deposition and remodeling were noted in the healing ACLs. During this phase, many collagen bundles were aligned in the ACL, but they were not as yet uniformly aligned with the longitudinal axis as were those in the intact ligament. Other studies have shown that collagen fiber orientation is the best predictor of strength.8,11,26,33
Therefore, with time, additional alignment of the collagen could result in increased yield loads and stiffness of the healing ligament tissue. This remodeling phase was also characterized by a decrease in hypercellularity and hypervascularity, as seen in the earlier time points; however, cell and vessel density remained higher than that in the intact ligaments, thereby suggesting that healing is not yet complete for these parameters. Frank et al12
found that collagen remodeling took place from 6 to 14 weeks but that little changed between 14 and 40 weeks. Woo et al,50
in a rabbit MCL study, stated that collagen remodeling begins several weeks after injury and may continue for months or even years. In this model of healing, collagen remodeling takes place between 6 weeks and 3 months, and it likely continues beyond the final time point noted in this study.
It is important to note the decrease in structural properties at the 6-week time point, when revascularization is occurring. In a study on the patellar tendon of the rabbit, Tohyama and Yasuda43
noted a 42% decrease in mechanical strength at the time of maximal revascularization, or 6 weeks. In sheep ACL grafts, the highest vascularity was seen between 6 and 12 weeks, which was noted as the nadir in strength for the grafts.47
Thus, the revascularization period in several soft tissues apparently occurs between 6 and 12 weeks, and it is a phase accompanied by a decrease in strength (). This factor is an important one to take into account when designing postoperative rehabilitation protocols so that undue stress is not placed on the healing tissue during this phase.
The addition of a CPC to the repair site of the ACL induced histologic changes and improved the functional performance of the healing tissue. At the 3-month time point, the addition of CPC resulted in an overall increase in ligament cellularity, and more of the cells had a fusiform cell shape. The increase in cellularity is likely to have resulted in the presence of a greater number of cells that might be capable of producing and organizing collagen. In addition, the fusiform shape of the fibroblast has been associated with increased collagen secretion and crimp formation.1,7,46
One or both of these findings could in turn explain the increase in yield load seen in the CPC ligaments at the 3-month time point.
One of the 6-week animals showed very little scar formation in both the suture knee and the CPC-treated knee. This paucity of ACL tissue may have been due to a failure in the neutralization or setting of the hydrogel or to an early forceful kicking motion in the animal postoperatively; however, that it occurred in both knees suggests that this animal may have had some defect in its intrinsic wound-healing capability.
This study agrees with the findings in previous research on CPC-enhanced primary ACL repair in that the structural properties of CPC-treated ligaments increase.29
The current study further advances the use of CPC to treat ACL injuries in a number of ways. This study examined the healing knees up to 3 months postoperatively; it examined the histologic appearance of the ligament; and it demonstrated an increase in vascularity matched by a loss of strength that had not been previously noted.
These studies compared the use of suture repair alone with suture repair supplemented with CPC. The observation that CPC improved the biomechanical function of the repairs at 15 weeks is interesting. Prior studies have demonstrated that the use of platelet-rich plasma alone28
or the use of a collagen scaffold alone (B.C.F. et al, unpublished data) is ineffective at improving the biomechanical properties of an ACL repair. This finding suggests that the combination of a provisional scaffolding (collagen in this experiment) and a source of stimulatory cytokines (as found in platelets) may be necessary to achieve enhanced functional healing and that the use of one component or the other alone may not produce the same result.
The principal weakness of this study is the limited time points studied. Selecting 4 weeks as the minimum time point means that the acute inflammatory phase of healing was not evaluated in this work. Similarly, by not having a time point greater than 3 months, remodeling was not yet complete. Whereas studying ligaments 6 months after surgery would provide further knowledge on augmented healing, the current funding for this area of research unfortunately limits the length of study. Although additional studies at earlier and later time points are needed to completely define ACL healing, this study provides data about the cellular proliferation, vascular proliferation, vascular pruning, and early remodeling phases in the functionally healing ACL. In addition, this study was performed in an immature large-animal model. Working with immature animals allows us to focus on the population that stands to suffer the longest period of disability owing to premature osteoarthritis after an ACL tear. The applicability of these results to adults is not known, because the effects of skeletal maturity on ligament healing have yet to be defined. In addition, whereas large-animal models have clear advantages over smaller models, particularly when vascularization is of interest, these models are clearly different from the human condition; the animals are quadruped and there is no ability to control the postoperative rehabilitation. Also, although the anatomy and morphology of the pig knee are similar to those of the human,51
there may be subtle differences that are not yet appreciated. However, these models provide a place to study ACL healing with traditional methods that require destruction of the healing tissue (biomechanical testing and histology). Animal models also required that histologic analysis be performed on tissues that were biomechanically tested. Biomechanical testing would not alter the main features of cellularity and vascularity, and although crimp could be affected, there is no a reason to believe that it would have disproportionately affected one group. This experimentation clearly cannot be performed in a clinical trial; thus, it provides key insights into the ACL healing process that may contribute to our understanding of how to encourage and enhance ACL healing in the future. In addition, with the relatively large interanimal variability often encountered in these large-animal models, the use of a bilateral model greatly amplifies the power of the study to determine significant differences between treatment groups. In the groups where each animal had one experimental knee and an associated internal control, the power of the study is significantly increased to detect treatment effects. However, this approach limits the number of treatments to one experimental side and one control side. The choice of what might be the optimal contralateral control is often a difficult one. We had recently completed a study demonstrating the use of a collagen scaffold alone (ie, without concentrated platelets), which did not result in a significant improvement in the biomechanical properties of the simpler suture repair (B.C.F. et al, unpublished data); as such, we elected in the current study to continue with the consistent use of a suture-only control. Finally, at least some of the kinematic abnormalities that occur in the ACL-deficient and ACL-reconstructed knee are inherently rotational.3,42,45
However, testing methods to measure these types of abnormalities in the porcine knee are not yet standardized and were thus not included in this study.
In summary, this study for the first time documents healing of the ACL over a 3-month time course, with significant improvements in ACL function noted with the addition of a CPC. The histologic changes that occurred with the use of CPC—namely, greater cell density in the wound site and a more fusiform cell shape—help to characterize enhanced ACL repair. Although additional studies at earlier and later time points are clearly needed to completely define the entire process of ACL healing, this model of enhanced suture repair provides important insights that support the possibility of treating ACL injuries with enhanced primary repair methods.