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Platelet-rich plasma (PRP) has shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, in animal models, the healing potential of the ACL is dependent on animal age. In this study, we hypothesized that there are age-dependent differences in ACL cell metabolism, collagen gene expression, and the ability of the cells to respond to growth factors in PRP. To test this hypothesis, ACL cells were obtained from skeletally immature, adolescent and adult pigs, and cultured in a collagen type I hydrogel with or without PRP for 14 days. When cultured in collagen-only hydrogel, ACL cells from adult pigs had a 19% lower apoptotic rate as compared to immature pigs (p = 0.001) and a 25% higher cellular metabolic activity as compared to adolescent pigs (p = 0.006). The addition of PRP to the collagen hydrogel resulted in a significantly increased cellular metabolic activity, reduced apoptotic rate, and stimulation of collagen production in the cells from the immature and adolescent animals (p < 0.05 for all comparisons) but had less effect on adult cells. These findings suggest that skeletal maturity may influence ACL cells' metabolic activity, apoptosis, collagen production, and response to PRP. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:79–85, 2012
The anterior cruciate ligament (ACL) is one of the four major ligaments of the knee and serves as the primary stabilizer of anteroposterior knee translation. The ACL is susceptible to injuries that can cause pain and discomfort, joint instability, and eventually degenerative joint disease. The prevalence of ACL injury is high, especially in active, adolescent athletes.1 However, unlike extra-articular ligaments, the injured ACL does not heal spontaneously, possibly due to the poor vascularization of the ligament and the unfavorable nature of the intra-articular environment.1, 2 While there are several methods of ACL reconstruction under investigation (including the use of anatomic landmarks for graft placement3 and double bundle surgery4–6), no current surgical procedure has been proven to reliably and completely restore long-term knee function after an ACL tear.7–9 In addition, the rate of progression to premature osteoarthritis is relatively high after an ACL injury, even with the current gold standard of treatment.10 An ultimate goal of ACL research should be help to diminish this osteoarthritis progression.
Recently, enhancing healing of ligaments using bioactive substances has received increasing interest. Growth factors have been found to influence chemotaxis, differentiation, proliferation, and synthetic activity of ACL cells and may potentiate the healing of ligaments.11–14 Platelet-rich plasma (PRP) is a fraction of whole blood which contains an increased concentration of platelets over baseline.15 The growth factors released from platelets, a few of which include platelet-derived growth factor (PDGF-AA, AB, and BB), transforming growth factor (TGF)-β1 and β2, platelet-derived angiogenesis factor (PDAF), insulin growth factor-1 (IGF-1), and platelet factor-4 (PF-4) are known to play a pivotal role in wound healing.16 Additionally, PRP contains a multitude of other growth factors, as well as multiple plasma proteins, a few of which are fibrin, fibronectin, vitronectin, and thrombospondin, which are known to act as cell adhesion molecules important for osteoblasts, fibroblasts, and epithelial cells.17–19 Therefore, the bioactive substances included in PRP may activate cells involved in ACL healing.
Previous work has shown that PRP can enhance cell viability and promote collagen types I and III expression by ACL cells in three-dimensional (3D) in vitro culture.14 In vivo studies have also shown that PRP can stimulate ACL healing at early time points in large animal models.12–14 These findings imply that positive clinical results may be expected in patients treated with PRP-enhanced ACL repair. However, characteristics of patients who may be good candidates for stimulating healing of the ligament have not yet been defined. Conventional orthopedic wisdom states that “children heal faster than adults” for fractures, and basic science studies in animals appear to support this clinical wisdom.15, 36 As patients with open physes are vulnerable to ACL injury, and stand to have the longest period of disability if premature osteoarthritis occurs, it is clinically important to begin to examine the effect of skeletal maturity on the ability of PRP to stimulate functional healing in the ACL.
To address the question of age-dependence on ACL cell response to PRP, an in vitro study was designed to evaluate the influences of age and skeletal maturity on ACL cell activity and response to PRP. Our first hypothesis was that ACL cell viability and collagen gene expression in a 3D scaffold would be dependent on animal age. Prior studies have investigated the effect of age on both human and animal ACL cell metabolism and proliferation.20 In those studies, cell proliferation and migration were both found to be higher in skeletally immature animals,20 two factors which may have contributed to the earlier wound population found in the younger animals,21 and the improved biomechanical response to healing in skeletally immature animals.22 These changes may be due to a decrease in growth factor receptor number on the ACL cells with age.23 We now wished to expand on this finding to determine whether these same effects would be seen in a 3D culture, and what the effect of PRP would be on these findings.
Our second hypothesis was that the ability of PRP to preserve cell viability and enhance collagen production for ACL fibroblasts would also be age-dependent. To test these hypotheses, ACL cells were cultured in a 3D scaffold with or without PRP, for 14 days. A collagen type I hydrogel was selected as scaffold because collagen is a known platelet activator16 and also because collagen type I is a major component of ligament tissue.24, 25 DNA content, cellular metabolic activity, and apoptosis in the scaffold were measured after 14 days in culture. Collagen expression was also assessed using quantitative RT-PCR.
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The results of this in vitro study suggest that skeletal maturity has significant effects on ACL cell activity and responsiveness to PRP. When cultured in a COL hydrogel without PRP, ACL cells from adult pigs had a lower apoptotic rate than those from immature pigs and a higher cellular metabolic activity than those from adolescent pigs, whereas the cells from adolescent pigs had a higher expression level of collagen type III mRNA than those from immature and adult pigs. The addition of PRP to the COL resulted in much greater stimulation of metabolic activity, including stimulation of collagen types I and III mRNA expression in the immature and adolescent ACL cells than in the adult cells. In addition, the addition of PRP led to a reduced apoptotic rate only in the cells from immature and adolescent pigs.
Without exposure to PRP, there were differences in the “baseline” behaviors of the cells from the different age groups in the 3D cultures. Cells from the adolescent animals had an overall lower metabolism, but a higher gene expression for types I and III collagen. In contrast, the immature cells had a significantly higher apoptotic rate than the other two cell types. The mechanisms behind these changes are not yet elucidated. Several reports have documented the presence of mesenchymal stem cells within the ACL26, 27 and it is well known that the number of mesenchymal stem cells decreased with age in other tissues.28 Therefore, although we used the same number of cells in each age group, it is possible that the differences seen between age groups may have been due to a preferentially MSC-enriched population of cells in the younger age groups. Further work to study the composition and cellular makeup of the ACL would be useful to clarify the mechanism behind the observations made here.
PRP seemed to have a more productive effect on the immature cells than on the adult cells. The immature and adolescent cells had the largest increase in metabolism and collagen gene expression with the addition of PRP and the immature cells were best protected by PRP against apoptosis. Cellular metabolism was increased in all groups with the addition of PRP, demonstrating that ACL cells from animals of all ages were capable of mounting some metabolic response to the cytokines and extracellular matrix proteins in the PRP. The immature and adolescent cells had an increase in MTT of over 50% with the addition of PRP, while the adult cells had a more modest increase of 35%. One possible reason the immature cells may have a more robust response to PRP is that immature cells have a higher concentration of growth factor receptors, which may make them more able to respond to high concentrations of the anabolic cytokines found in PRP.
The increase in collagen gene expression in the younger cells with the addition of PRP may be due to the fact that growth factors known to be released from PRP, such as PDGF and TGF-β, have the ability to promote the synthesis of collagen types I and III by fibroblasts.29, 30 However, the addition of PRP and the associated cytokines did not significantly promote the expression of collagen mRNA in adult pig fibroblast. The addition of PRP also appeared to slow the apoptotic rate of immature ACL cells to a greater degree than the adult cells. This may be due to the presence of platelet-released growth factors, such as PDGF and IGF, which have been shown to enhance cell viability and suppress cell apoptosis.31, 32 It may also be due to the presence of plasma proteins in the PRP, such as fibronectin, which have also been reported to have a protective effect against apoptosis.33 In the current study, we were careful to make sure the composition of the PRP was identical for the three age groups by using the same preparation of PRP for all experiments. Thus, it would appear that the differences in age observed in this experiment may be due to inherent differences in the response of the ACL cells themselves. The fact that both of these anabolic functions were stimulated more in the immature animals by identical PRP preparations may be related to the fact that mRNA expression of the receptors of some growth factors, such as PDGF and TGF-β, are known to be age-dependent and the expression level decreased in pig fibroblast with increasing age.33
Scaffold contraction was greater in the group of constructs cultured with ACL cells from immature animals than when cultured with ACL cells from older animals. This may be related to the fact that ACL cells from immature animals have a more vigorous proliferative and migration response in vitro than adult cells,20, 21, 34 possibly due to the increased number of growth factor receptors.23 In addition, all age groups had an increased contraction response when the cells were cultured in the presence of PRP. Previous work on collagen-platelet constructs has demonstrated that culture of the collagen-PRP scaffolds without cells results in little scaffold contraction, suggesting this observed phenomenon is fibroblast mediated rather than platelet mediated,35 which makes it more likely that the platelet action is on the fibroblasts themselves, rather than a platelet-mediated direct scaffold contraction. Scaffold contraction may be undesirable if it results in premature degradation and retraction of the provisional scaffold, two phenomena which may inhibit wound filling and healing. However, controlled shrinkage and degradation of these types of regenerative templates may be desirable, if the rates are slow enough to allow simultaneous tissue ingrowth.
The injured ACL does not heal spontaneously, due in part to poor vascularization and an intra-articular environment that is unfavorable for ligament regeneration. In the present study, to better mimic the in vivo environment, ACL fibroblasts were cultured in a 3D scaffold. The supply of oxygen to the cells in these in vitro statically cultured constructs is dependent on diffusion, and often demands of oxygen and nutrients far exceed the supply in the central construct. The finding that cellular metabolic activity of the Adult-COL group was higher than that of the Adolescent-COL group, as shown in Figure 2, indicates that fibroblasts from adult pigs might have a higher resistance to hypoxia than those from adolescent pigs. In addition, previous studies have shown that hypoxia is a sufficient trigger for cell apoptosis in vitro and in vivo.36, 37 In the current study, approximately 40% of the young-COL cells were apoptotic cells in the young-COL group and only 20% of the cells in the adolescent-COL and adult-COL groups, were also suggesting an improved resistance to the hypoxic conditions in the 3D scaffold and in the knee for adult cells.
In conclusion, the present in vitro study demonstrated that skeletal maturity has significant effects on ACL cell activity and the response of the ACL cell to PRP. This suggests that future studies should take animal age into account when interpreting results of ACL in vivo studies.