The results of this in vitro study suggest that in two large animal models, porcine and ovine, cells taken from skeletally IMMATURE animals have greater proliferation and migration potential than ADOLESCENT and ADULT cells. These results support previous reports that the migratory and proliferative ability of fibroblasts is age dependent.18–23
Both the proliferation and migration of intrinsic ACL cells has been cited as a prerequisite for proper wound healing20,24
and thus our findings might be a reason why IMMATURE animals could be able to heal more quickly and efficiently than older age groups.
Invasion of the wound site by fibroblasts is critical to the formation of fibrovascular scar.25
As an early response to injury, fibroblasts will migrate toward the wound site.26
Previous studies have shown that defective wound healing occurs when migration potential is less than optimal.27,28
The decrease in migration rates seen in adolescence and adulthood could thus potentially also represent a mechanism for decreased healing potential of the ACL after minor partial injuries that may occur as a normal part of daily “wear and tear.” If such a deficiency in repair were present, it could lead to an imbalance in the normal tissue remodeling that occurs in all tissues, an imbalance toward continual breakdown with insufficient repair and gradual weakening of the tissue until failure.
Platelets were used in this study in an attempt to try to simulate the clinical situation where a fibrin platelet clot forms in a wound site as the first step in wound healing. Prior in vitro experiments have demonstrated that the CPC releases growth factors important in wound healing over a 10-day period.29,30
Thus, it is possible that the platelets used in this in vitro experiment also released these growth factors and subsequently produced a growth factor environment more similar to that seen in the in vivo healing wound than constructs without platelets or cultured with static concentrations of growth factors in PBS. Certainly, the in vitro nature of our study limits is direct application to clinical use, and this is the primary limitation of our study. Another limitation is that we did not study older adult animals. The ages of animals used are more likely comparable to younger age groups of humans—the widely open physes of the IMMATURE animals would correspond to a human patient less than 10 years of age, the closing physes of the ADOLESCENT animals would be similar to that seen in humans between 12 and 16 years of age, and the relatively recently closed physes in the ADULT group would correspond to young adults in their third and fourth decade of life.
In addition to migration, proliferation and contraction of cells at the wound site characterizes a major component of wound healing.24,26
Increased cellular proliferation is required early in the repair process of the ACL for appropriate stages of wound healing to take place. Fibroblast proliferation is often accompanied by matrix synthesis, which together mark the onset of the proliferation phase. Histological analysis has shown that the fibroblast becomes the most dominant cell type 3 weeks postinjury, allowing for the wound to heal, and is then followed by a rapid decline at 6 weeks, marking the remodeling phase.31–33
The values of the 2-day MTT assay were similar in all three age groups for both species. This suggests that the initial seeding density was similar for each age group in the proliferation experiments. The higher values seen in the immature animals at later time points thus suggests a higher rate of cell proliferation by cells from the younger animals. These results are consistent with prior studies of fibroblast proliferation where fibroblasts from older donors show a decrease in replication rates and lower cell yields at cellular confluency in vitro.34
Taken in total, these results suggest that an increase in age results in lower rates of cell migration, proliferation, and contraction, and potentially negatively impact the capacity of a wound to heal after injury.
Interestingly, the same effect of age was seen in both species, where skeletally immature animals had the highest rates of migration and proliferation, and adult animals had the lowest. The similarity in their skeletal maturity status and the similarity in the findings in both species do make it more likely that these findings are applicable across species rather than species-specific. If these findings are applicable across species, and perhaps even applicable in the human situation, it might suggest that one reason for the relatively accelerated healing noted in children after injury when compared with adults is the enhanced ability of cells from immature tissues to migrate and proliferate to and within the wound site. Clearly, further studies are needed to validate this hypothesis. In addition, the age-related differences in cell proliferation and scaffold contraction were significant at day 14. Whether these differences persist at longer time points is as yet unknown. Hydrogel studies of this type are often limited in the length of time that can be studied, as reorganization and contraction of the hydrogels can lead to premature failure of the scaffolds and invalidate the study. Thus, for studying these effects at longer time points, future studies using an in vivo model of ACL repair may be needed.
Although the differences between age groups for the two species were consistent, there were observed differences in the magnitude of the measured cellular proliferation between porcine and ovine assays. These differences could be due to at least two different factors: first, that sheep cells migrate much faster than the porcine cells or second, that each sheep cell that migrates takes up a greater amount of dye than each porcine cell. We do not know if it is one of these factors, or another unidentified factor which is responsible for this observation. However, as the principal goal of our study was to determine not the difference between species, but the differences as a function of skeletal age, it was gratifying to note that the trends in cellular proliferation and migration observed as a function of age were similar in both species, suggesting that the age dependence may not be true only in one animal model, but across species.
It is interesting to note that although patients who are skeletally immature and prepubescent are certainly active, the incidence of ACL tears in these athletes is far lower than that in adolescents or adults.35
Perhaps some of these differences in catastrophic injury rates can be attributed to intrinsic factors, such as faster migration of cells to wound sites, which may allow these equally active patients to more effectively repair microstrain or individual fascicle disruption which may occur semi-regularly, thus avoiding accumulation of these small defects and eventual coalescence into a complete disruption.
One limitation to this study is the use of animal models. As it is very difficult to obtain healthy ACL cells from a patient with no knee injury, we worked to mitigate this limitation by selecting two animal models that are thought to most closely approach the human condition. The age groups chosen for the porcine and ovine models used in this study are anatomically similar in their epiphyseal (growth) plate growth. The time points chosen correlate for both species so that the immature animals have an open growth plate, adolescent is partially open and adults have a closed growth plate. The porcine model has the most similar healing characteristics to human in skin. It is commonly used for wound healing studies36
and also has similar anatomy and biomechanics to the human knee,37
whereas sheep are one of the most common models for ACL reconstruction, 38,39
and the placement of its anteromedial and posterolateral bundles are similar to that of a human model.40
The fibroblasts cultured from these two animals were passaged twice prior to the experiment. We chose a low passage because it has been shown that late passaged fibroblasts display an increased nuclear size, which correlates with slow and nondividing cells and thus, higher passage cells may be a less accurate representation of the in vivo condition.34,41
We used two large animal models, as we felt that if similar results were seen in both models that the results were less likely to be species-dependent. If different results were noted, we might have tested the results in a third species or looked for other physiologic reasons the responses were different (different weight gain curves, different timing of sexual maturity in relationship to skeletal maturity, etc). The similarity in their skeletal maturity status and the similarity in the findings in both species do make it more likely that these findings are applicable across species rather than species-specific, but further studies would be required to validate that hypothesis.
The chief limitation of this study is its in vitro nature. The environment of the culture dish is very different from that of the in vivo environment, and although the in vitro model is useful to begin to understand intrinsic ACL cell migration behaviors, there are likely many other cell types involved in governing the wound response, including inflammatory cells found in the blood. These cells are excluded from this in vitro assay, and therefore future studies should include in vivo studies where the effects of the intrinsic ACL cells are more difficult to isolate and ascertain, but the cumulative invasion characteristics of all the cells in the in vivo wound environment can be determined as a function of animal age. Although additional studies are needed, these experiments suggest the role of skeletal maturity may influence the repair capacity of intrinsic ACL cells.