The data obtained here suggest that age has a significant effect on cellular repopulation of the ACL wound site treated with a collagen-platelet composite, even at only a few weeks after surgery. There was no significant effect of age on wound site vascular density in the first 4 weeks of ligament healing. Immature animals had higher cell densities within the wound sites compared to Adolescent and Adult animals, although the latter did catch up partially during the course of the experiment. In general, the observed processes were very similar to what has been reported previously as “ligamentization,” a term for the changes observed histologically when a tendinous ACL graft transforms into a ligamentous, ACL-like tissue.18–20
However, while the distinct phases of initial necrosis, proliferation, and remodeling/ligamentization can be identified in ACL grafts, we observed no necrosis in the healing ligaments studied here.
In all of the age groups, the central wound site was populated with large, ovoid fibroblasts at 1 week. These fibroblasts had characteristics of activated fibroblasts, suggesting they were actively participating in the invasion, population, and remodeling of the wound site. Over the 4 weeks of the experiment, the cell nuclei in all groups gradually became more fusiform; however, this occurred 2 weeks later in the Immature group than in the Adolescent or Adult groups. To the best of our knowledge, age-related differences in the processes of ligament healing are being reported for the first time. As all wound sites were repopulated by the 1-week time point, additional studies at even shorter time points may help to delineate if patient age significantly effects this initial invasion of the wound site.
We found cellular population to be very similar to the events previously described for incorporation of ACL grafts,21,22
healing of the medial collateral ligament,23
and the initial histological response of an ACL to injury.8
While there were generally large increases in cell numbers in all groups, the fibroblast density as a function of position remains the same regardless of the age of the animal, with the Immature group showing the highest numbers with significantly more cells than the Adult group. However, higher proliferative activity should not be confused with higher biosynthetic activity, i.e., a higher cell number is not necessarily a predictor of a better outcome. As a matter of fact, compared to the Adult animals, the faster proliferating Immature and Adolescent animals showed a less uniformly distributed matrix in our histological assessment. Similar patterns of rapidly proliferating cells, which eventually resulted in a poorly differentiated and mechanically inferior scar tissue, has been previously observed in an animal model of rotator cuff repair.24
However, at 4 weeks, we saw comparable levels of collagen organization in all groups.
In addition, it seems that the cellular repopulation originated from the center of the ACL stumps. Previous studies have shown that there is a large, mobile population of fibroblasts in the ACL stump and that these cells readily migrate into a collagenous biomaterial.25
It is possible that cells intrinsic to the ACL and located in the proximal and distal stump may participate in the populating of the provisional scaffold material, stimulated by the growth factors from the platelet concentrate. Pluripotential mesenchymal cells have been hypothesized to reside in the perivascular region of multiple tissues as pericytes.26,27
In this study, the hypertrophy of the cells in these perivascular areas, both between ruptured ligament fascicles (endoligament) and in the tissue covering the ligament (epiligament) may be one possible source of the cells invading the ACL wound site. High cell density within the wound site has been previously observed to occur before an increase in healing ligament strength,14
suggesting that the cellular invasion occurs, until some type of contact inhibition occurs at a high cell density. Once this occurs, there appears to be a shift toward collagen synthesis and improving regenerate tissue strength. This interpretation is supported by the observations in this article; namely, by the increasing cell numbers from either side, the presence of highly metabolically active fibroblasts, demonstrated by large nuclei, in the center of the implant, and the beginnings of an organized collagen structure several weeks later.
The increased cellular density in the younger animals is consistent with earlier in vitro studies of cellular proliferation and migration in our lab which show that the ACL cells from younger animals have greater proliferation and migration potential than those from Adolescent or Adult animals.17
Both the proliferation and migration of intrinsic ACL cells has been cited as a prerequisite for proper wound healing,28,29
and thus our findings might be a reason why Immature animals could be able to heal more quickly and efficiently than older age groups.
The second wound repair mechanism investigated in this study was revascularization of the ACL wound site. The small differences in vascular density noted at 1 week are more likely related to a higher vascular density in the intact Immature ACL given the location of the vessels in the proximal and distal ACL remnants. Other than this finding, there were no significant differences between the age groups in vascular density at any location or time point. This is likely due at least in part to the fact that the study was of relatively short duration. Recently, work in the Yorkshire pig model of the ACL healing, revascularization has been noted to occur between 6 and 9 weeks.14
During ligamentization of an ACL graft, revascularization occurs between 3 and 8 weeks in the rabbit model,30
and 6 and 20 weeks in the canine model,18
so the time points studied here may be too short to determine the full extent of vascular change that will be seen in this model. However, the observed increase in arterioles along the edges of the scar region in the epiligament is consistent with the findings during ligamentization20,31
and ACL remodeling.8
Additional studies at time points longer than 4 weeks are needed to determine if revascularization of the central wound site is significantly effected by patient age.
This study was a qualitative histologic analysis of healing ACL tissue. This type of study is limited in its ability to provide quantitative data, as can be done with biochemical assays on biopsies of the healing tissue or digests of the entire ligament. The numbers obtained in the histomorphometric analysis are also subject to the selection of the sites for analysis, a potential source of error which is only partly addressed by standardizing the locations of analysis and blinding the reviewers. However, there are also advantages of a histologic study when beginning to look at questions such as the effect of age on ligament healing. Entire sections of the ligament can be analyzed and regional variations determined. Features of the tissues, such as blood vessel location and overall organization and nonhomogeneity of the ligament can be appreciated in whole organ sections. Use of these studies as a first line analysis can guide future studies by providing a map for obtaining biopsies for biochemical or cell-based assays. For example, the regional variation seen in the cell density along the length of the ACL might suggest that the cranio-caudad location of a biopsy could influence the results and that future studies might wish to normalize the biopsy location for this reason.
The porcine model approximates the human knee in terms of biomechanics and wound healing attributes; however, the chief limitation of this model is the inability to restrain the animals postoperatively. The animals in this study were allowed to be weight-bearing as tolerated, and most animals were weight-bearing on the operative knees within a few hours of surgery. This may have placed excessive early stress on the repairs which would not be seen in a human knee when the patient was on crutches postoperatively. Future studies to evaluate the effect of rehabilitation on ACL healing are warranted in a model where rehabilitation may be better controlled such as the canine model.
In our gradual translation from the “bench” to the “bedside,” we have used various models, from 2D cell culture, to 3D hydrogels in vitro, to a central defect wound in vivo, and now to a complete transection. These models are all stepping stones with gradations of cost, complexity, and animal lives. As we optimize solutions in earlier models, we can then apply them to more complex models. In this article, we used ACL transection as a simplified model for ACL injury. In a true ACL injury, the ligament tissue likely has a broader zone of injury than that in the model used here. In addition, subchondral bone, articular cartilage, meniscus and capsular injury are also present. Future studies which add these degrees of complexity to the model will be warranted if we can demonstrate treatment efficacy in this simpler model.
In conclusion, our study showed that Immature animals repopulate a wound site more densely than Adolescent or Adult animals. A collagen-platelet composite is well incorporated and supports ACL cell migration, proliferation, and collagen production in the first few weeks of healing. Whether this increased cell response is beneficial for functional healing of the ligament requires further longer-term studies.