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Collagen is an important biomaterial in intra-articular tissue engineering, but there are unanswered questions about its safety. We hypothesize that the addition of type-I-collagen for primary repair of the Anterior Cruciate Ligament (ACL) might result in a local and systemic reaction in a porcine model after 15 weeks as demonstrated by joint effusion, synovial thickening, elevated intraarticular and systemic leukocyte counts. Further, this reaction might be aggravated by the addition of a platelet concentrate. Eighteen porcine ACLs were transected and repaired with either sutures (n=6), a collagen sponge (n=6), or a collagen-platelet-composite (CPC; n=6). Twelve intact contralateral knees served as controls (n=12). No significant synovial thickening or joint effusion was seen in the collagen-treated knees. Synovial fluid leukocyte counts showed no significant differences between surgically treated and intact knees, and no differences were seen in leukocyte counts of the peripheral blood. The addition of a platelet concentrate to the knee joint resulted in lower serum levels of IL-1ß, but serum levels of TNF-α were not significantly different between groups. In conclusion, the presence of collagen, with or without added platelets, did not increase the local or systemic inflammatory reactions following surgery, suggesting that Type I collagen is safe to use in the knee joint.

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Magnetic resonance imaging (MRI) could potentially be used to non-invasively predict the strength of an ACL graft after ACL reconstruction. We hypothesized that the volume and T2 relaxation parameters of the ACL graft measured with MRI will predict the graft structural properties and anteroposterior (AP) laxity of the reconstructed knee. Nine goats underwent ACL reconstruction using a patellar tendon autograft augmented with a collagen or collagen-platelet composite. After 6 weeks of healing, the animals were euthanized, and the reconstructed knees were retrieved and imaged on a 3T scanner. AP laxity was measured prior to dissecting out the femur-graft-tibia constructs which were then tested to tensile failure to determine the structural properties. Regression analysis indicated a statistically significant relationship between the graft volume and the failure load (r(2)=0.502; p=0.049). When graft volume was normalized to the T2 relaxation time, the relationship was even greater (r(2)=0.687; p=0.011). There was a significant correlation between the graft volume and the linear stiffness (r(2)=0.847; p<0.001), which remained significant with T2 normalization (r(2)=0.764; p=0.002). For AP laxity at 30° flexion, there was not a significant correlation with graft volume, but there was a significant correlation with volume normalized by the T2 relaxation time (r(2)=0.512; p=0.046). These results suggest that MRI volumetric measures combined with graft T2 properties may be useful in predicting the structural properties of ACL grafts.

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A number of recently published studies have established a substantial age dependence of the response of ACL fibroblasts to stimulation by platelet-rich plasma (PRP). Further in-depth research of this age dependence revealed negative effects on both histological and biomechanical results in a large animal model. However, while it has been postulated that this association could affect potential human applications negatively too it remains to be proven that the same effects occur in human cells. Thus it was the objective of this study to search for age dependence in human fibroblasts before further human experiments are done. Human fibroblasts were obtained from 10 immature and adolescent patients, based on a-priori power calculations, and cultured in a collagen-PRP composite. Three parameters that are pivotal for defect remodeling and wound healing-cell migration, cell proliferation, and scaffold contraction-were chosen as endpoints. Both migration and proliferation were significantly higher in immature cells, but no differences were seen in wound contraction. The former findings suggest that immature patients respond more favorably to treatment with PRP, which consequently might translate into better results in ACL tissue engineering.

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BACKGROUND: The effects of skeletal maturity on functional ligament healing are unknown. Prior studies have suggested that ligament injuries in skeletally mature animals heal with improved mechanical properties. In this study, we hypothesized that skeletally immature animals have improved functional healing compared with skeletally mature animals. METHODS: Twenty-one Yucatan minipigs (eight juvenile, eight adolescent, and five adult animals) underwent bilateral anterior cruciate ligament transection. On one side, the ligament injury was left untreated to determine the intrinsic healing response as a function of age. On the contralateral side, an enhanced suture repair incorporating a collagen-platelet composite was performed. Biomechanical properties of the repairs were measured after fifteen weeks of healing, and histologic analysis was performed. RESULTS: Anterior cruciate ligaments from skeletally immature animals had significantly improved structural properties over those of adult animals at three months after transection in both the untreated and repair groups. Use of the enhanced suture technique provided the most improvement in the adolescent group, in which an increase of 85% in maximum load was noted with repair. The repair tissue in the adult tissue had the highest degree of hypercellularity at the fifteen-week time point. CONCLUSIONS: Functional ligament healing depends on the level of skeletal maturity of the animal, with immature animals having a more productive healing response than mature animals.

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BACKGROUND: Enhanced primary anterior cruciate ligament repair, in which suture repair is performed in conjunction with a collagen-platelet composite to stimulate healing, is a potential new treatment option for anterior cruciate ligament injuries. Previous studies have evaluated this approach at the time of anterior cruciate ligament disruption. HYPOTHESIS: Delaying surgery by 2 or 6 weeks would have a significant effect on the functional outcome of the repair. STUDY DESIGN: Controlled laboratory study. METHODS: Sixteen female Yorkshire pigs underwent staged, bilateral surgical anterior cruciate ligament transections. Anterior cruciate ligament transection was initially performed on 1 knee and the knee closed. Two or 6 weeks later, enhanced primary repair was performed in that knee while the contralateral knee had an anterior cruciate ligament transection and immediate repair. Biomechanical parameters were measured after 15 weeks in vivo to determine the effect of delay time relative to immediate repair on the healing response. RESULTS: Yield load of the repairs at 15 weeks was decreased by 40% and 60% in the groups where repair was delayed for 2 and 6 weeks, respectively (P = .01). Maximum load showed similar results (55% and 60% decrease in the 2- and 6-week delay groups, respectively; P = .011). Linear stiffness also was adversely affected by delay (50% decrease compared with immediate repair after either a 2- or 6-week delay, P = .011). Anterior-posterior laxity after 15 weeks of healing was 40% higher in knees repaired after a 2-week delay and 10% higher in those repaired after a 6-week delay (P = .012) when tested at 30° of flexion, but was not significantly affected by delay when tested at 60° or 90° (P = .21). CONCLUSION: A delay between anterior cruciate ligament injury and enhanced primary repair has a significant negative effect on the functional performance of the repair. CLINICAL RELEVANCE: As future investigations assess new techniques of anterior cruciate ligament repair, the timing of the repair should be considered in the design and the interpretation of experimental studies.

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Primary suture anterior cruciate ligament (ACL) repair was abandoned in favor of reconstruction due to a high rate of clinical failures. However, the insertion of a collagen scaffold loaded with platelets into the wound at the time of suture repair ("enhanced primary repair") has been shown to improve functional healing in animal models. Our objectives were to determine if using a collagen scaffold alone (without platelets) would be sufficient to increase the structural properties of the repaired ACL and decrease postoperative knee laxity compared to suture repair without the scaffold. Eight Yucatan minipigs underwent bilateral ACL transection and suture repair. In one knee, the repair was augmented with a collagen scaffold (SCAFFOLD group) while the other had suture alone (SUTURE group). After 13 weeks of healing, knee joint laxity and the structural properties of the ACL were measured. The addition of the collagen scaffold to suture repair of a transected ACL did not significantly improve the mean anteroposterior knee laxity [SCAFFOLD vs. SUTURE: 6.1 +/- 1.4 vs. 4.4 +/- 2.0 mm (p = 0.07), 8.1 +/- 2.0 vs. 7.6 +/- 2.0 mm (p = 0.66), and 6.2 +/- 1.2 vs. 6.1 +/- 1.8 mm (p = 0.85) at 30 degrees, 60 degrees, and 90 degrees flexion, respectively]. Likewise, there were no significant differences in the structural properties [SCAFFOLD vs. SUTURE: 367 +/- 185.9 vs. 322 +/- 122.0N (p = 0.66) and 90.7 +/- 29.5 vs. 85.0 +/- 30.3N/mm (p = 0.74) for the yield load and linear stiffness, respectively]. The use of a collagen scaffold alone to enhance suture repair of the ACL was ineffective in this animal model. Future work will be directed at stimulating biological activity in the scaffold.

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Anterior cruciate ligament (ACL) injuries are an important clinical problem, particularly for adolescent patients. The effect of skeletal maturity on the potential for ACL healing is as yet unknown. In this study, we hypothesized that fibroblastic cells from the ACLs of skeletally immature animals would proliferate and migrate more quickly than cells from adolescent and adult animals. ACL tissue from skeletally immature, adolescent, and adult pigs and sheep were obtained and cells obtained using explant culture. Cell proliferation within a collagen-platelet scaffold was measured at days 2, 7, and 14 of culture using AM MTT assay. Cellular migration was measured at 4 and 24 h using a modified Boyden chamber assay, and cell outgrowth from the explants also measured at 1 week. ACL cells from skeletally immature animals had higher proliferation between 7 and 14 days (p<0.01 for all comparisons) and higher migration potential at all time points in both species (p<0.01 for all comparisons). ACL cells from skeletally immature animals have greater cellular proliferation and migration potential than cells from adolescent or adult animals. These experiments suggest that skeletal maturity may influence the biologic repair capacity of intrinsic ACL cells.

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BACKGROUND: The anterior cruciate ligament (ACL) fails to heal after traumatic rupture. Furthermore, large-animal models have recently shown that 1-month functional ACL healing is augmented after suture repair when a bioactive scaffold is placed in the tear site. HYPOTHESIS: At the time of suture repair, placement of a bioactive scaffold in the ACL wound site would improve the structural properties of the tissue. STUDY DESIGN: Controlled laboratory study. METHODS: Twenty-seven knees in immature pigs underwent ACL transection and suture repair. A collagen-platelet composite (CPC) was used to supplement the repair in 14 knees. Knees were harvested at 4 weeks, 6 weeks, and 3 months. Mechanical testing and histologic analysis were performed. RESULTS: The addition of a CPC to a suture repair resulted in improvements in yield load and linear stiffness of the repair tissue at 3 months, as well as a significant increase in cell density. A reduction in yield load and stiffness occurred at the 6-week time point in both groups, a phase when revascularization was noted. CONCLUSION: The addition of a CPC to a suture repair enhanced the structural properties of the ACL, and the improvement was associated with increased cellularity within the healing ligament. CLINICAL RELEVANCE: The addition of a bioactive scaffold to the wound site improved the functional healing of the ACL after suture repair. The decreased repair strength during revascularization may indicate a need to protect the repair site through this period.

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BACKGROUND: The outcome of anterior cruciate ligament (ACL) reconstruction is variable, and many patients have increased joint laxity postoperatively. HYPOTHESIS: Placement of a collagen-platelet composite (CPC) around the graft at the time of ACL reconstruction decreases postoperative knee laxity and improves the structural properties of the graft compared with standard ACL reconstruction. STUDY DESIGN: Controlled laboratory study. METHODS: Thirteen immature pigs underwent unilateral ACL reconstruction with a bone-patellar tendon-bone allograft. In 6 pigs, a standard allograft was used to reconstruct the ACL. In 7 pigs, a CPC was placed around the allograft. After 15 weeks of healing, the animals were euthanized, and the anterior-posterior (AP) knee laxity and structural properties of the graft were measured. Qualitative histology of the grafts was also performed. RESULTS: The AP laxity values of the reconstructed knees, normalized to the contralateral control, were significantly reduced by 28% and 57% at 60 degrees and 90 degrees of knee flexion, respectively, with the addition of CPC (P < .001). Significant improvements in the graft structural properties were also found; the normalized yield (P = .044) and maximum failure loads (P = .025) of the CPC group were 60% higher than the standard ACL-reconstructed group. Although cellular and vessel infiltration were observed in the grafts of both groups, regions of necrosis were present only in the standard ACL-reconstructed group. CONCLUSION: These data demonstrate that the application of CPC at the time of ACL reconstruction improves the structural properties of the graft and reduces early AP knee laxity in the porcine model after 15 weeks of healing. CLINICAL RELEVANCE: Application of a CPC to an ACL graft at the time of surgery decreased knee laxity and increased the structural properties of the graft after 15 weeks of healing.

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