Ashley N. Mastrangelo

Collagen-Platelet Composite Enhances Biomechanical and Histologic Healing of the Porcine Anterior Cruciate Ligament

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.

Biomechanical outcomes after bioenhanced anterior cruciate ligament repair and anterior cruciate ligament reconstruction are equal in a porcine model.

PURPOSE The objective of this study was to compare the biomechanical outcomes of a new method of anterior cruciate ligament (ACL) treatment, bioenhanced ACL repair, with ACL reconstruction in a large animal model. METHODS Twenty-four skeletally immature pigs underwent unilateral ACL transection and were randomly allocated to receive bioenhanced ACL repair with a collagen-platelet composite, allograft (bone-patellar tendon-bone) reconstruction, or no further treatment (n = 8 for each group). The structural properties and anteroposterior laxity of the experimental and contralateral ACL-intact knees were measured 15 weeks postoperatively. All dependent variables were normalized to those of the contralateral knee and compared by use of generalized linear mixed models. RESULTS After 15 weeks, bioenhanced ACL repair and ACL reconstruction produced superior biomechanical outcomes to ACL transection. However, there were no significant differences between bioenhanced ACL repair and ACL reconstruction for maximum load (P = .4745), maximum displacement (P = .4217), or linear stiffness (P = .6327). There were no significant differences between the 2 surgical techniques in anteroposterior laxity at 30° (P = .7947), 60° (P = .6270), or 90° (P = .9008). CONCLUSIONS Bioenhanced ACL repair produced biomechanical results that were not different from ACL reconstruction in a skeletally immature, large animal model, although the variability associated with both procedures was large. Both procedures produced significantly improved results over ACL transection, showing that both were effective treatments in this model. CLINICAL RELEVANCE Bioenhanced ACL repair may one day provide an alternative treatment option for ACL injury.

Reduced platelet concentration does not harm PRP effectiveness for ACL repair in a porcine in vivo model.

Enhanced primary repair of the ACL using a collagen scaffold loaded with platelets has been shown to improve the functional healing of suture repair in animal models. In this study, our objectives were to determine if lowering the platelet concentration would reduce the structural properties of the repaired ACL and increase postoperative knee laxity. Eight Yucatan mini‐pigs underwent bilateral suture repair. In one knee, the repair was augmented with a collagen scaffold saturated with platelet‐rich plasma (PRP) containing five times the systemic baseline of platelets (5×) while the contralateral knee had a collagen scaffold saturated with PRP containing three times the systemic baseline of platelets (3×). After 13 weeks of healing, knee joint laxity and the structural properties of the ACL were measured. The 3× platelet concentration resulted in a 24.1% decrease in cellular density of the repair tissue (p < 0.05), but did not significantly decrease the structural properties [3× vs. 5×: 362 N vs. 291 N (p = 0.242) and 70 N/mm vs. 53 N/mm (p = 0.189) for the yield load and linear stiffness, respectively]. The 3× platelet concentration also did not significantly change the mean anteroposterior knee laxity at 30° and 90° of flexion [5× vs. 3×: 3.5 mm vs. 5.1 mm (p = 0.140), and 6.1 mm vs. 6.3 mm (p = 0.764)] but did result in a lower AP laxity at 60° [5× vs. 3×: 8.6 mm vs. 7.3 mm (p = 0.012)]. The decrease in platelet concentration from 5× to 3× to enhance suture repair of the ACL did not significantly harm the mechanical outcomes in this animal model.

The Effect of Skeletal Maturity on Functional Healing of the Anterior Cruciate Ligament

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.

The Effect of Skeletal Maturity on the Regenerative Function of Intrinsic ACL Cells

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.

Immature animals have higher cellular density in the healing anterior cruciate ligament than adolescent or adult animals

There has been recent interest in the biologic stimulation of anterior cruciate ligament (ACL) healing. However, the effect of age on the ability of ligaments to heal has not yet been defined. In this study, we hypothesized that skeletal maturity would significantly affect the cellular and vascular repopulation rate of an ACL wound site. Skeletally Immature (open physes), Adolescent (closing physes), and Adult (closed physes) Yucatan minipigs underwent bilateral ACL transection and suture repair using a collagen‐platelet composite. The response to repair was evaluated histologically at 1, 2, and 4 weeks. All three groups of animals had completely populated the ACL wound site with fibroblasts at 1 week. The Immature animals had a higher cellular density in the wound site than the Adult animals at weeks 2 and 4. Cells in the Immature ligament wounds were larger and more ovoid than in the Adult wounds. There were no significant differences in the vascular density in the wound site. Animal age had a significant effect on the density of cells populating the ACL wound site. Whether this observed cellular difference has an effect on the later biomechanical function of the repaired ACL requires further study.

Delay of 2 or 6 Weeks Adversely Affects the Functional Outcome of Augmented Primary Repair of the Porcine Anterior Cruciate Ligament

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.

Safety of intra-articular use of atelocollagen for enhanced tissue repair.

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-1β, 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.

Injection temperature significantly affects in vitro and in vivo performance of collagen‐platelet scaffolds

Collagen‐platelet composites have recently been successfully used as scaffolds to stimulate anterior cruciate ligament (ACL) wound healing in large animal models. These materials are typically kept on ice until use to prevent premature gelation; however, with surgical use, placement of a cold solution then requires up to an hour while the solution comes to body temperature (at which point gelation occurs). Bringing the solution to a higher temperature before injection would likely decrease this intra‐operative wait; however, the effects of this on composite performance are not known. The hypothesis tested here was that increasing the temperature of the gel at the time of injection would significantly decrease the time to gelation, but would not significantly alter the mechanical properties of the composite or its ability to support functional tissue repair. Primary outcome measures included the maximum elastic modulus (stiffness) of the composite in vitro and the in vivo yield load of an ACL transection treated with an injected collagen‐platelet composite. In vitro findings were that injection temperatures over 30°C resulted in a faster visco‐elastic transition; however, the warmed composites had a 50% decrease in their maximum elastic modulus. In vivo studies found that warming the gels prior to injection also resulted in a decrease in the yield load of the healing ACL at 14 weeks. These studies suggest that increasing injection temperature of collagen‐platelet composites results in a decrease in performance of the composite in vitro and in the strength of the healing ligament in vivo and this technique should be used only with great caution.

Effect of anterior cruciate healing on the uninjured ligament insertion site

The effect of anterior cruciate healing on the uninjured ligament insertion site after enhanced suture repair with collagen‐platelet composites (CPC) has not yet been defined. In this study, we hypothesized that fibroblasts and osteoclasts would participate in generating histologic changes in insertion site morphology after transection and bioenhanced repair of the ACL, and that these changes would be age‐dependent. Skeletally immature, adolescent, and adult Yucatan mini‐pigs underwent ACL transection and bioenhanced suture repair. The histologic response to repair of the insertion site was evaluated at 1, 2, 4, and 15 weeks. In young and adolescent animals treated with bioenhanced suture repair with CPC, changes in the insertion site included: (1) fibroblastic proliferation with loss and return of collagen alignment in the fibrous zone; (2) osteoclastic resorption within fibrocartilage zones at 2–4 weeks; and (3) partial reappearance of fibrocartilage zones at 15 weeks. In adult animals; however, degenerative changes were noted by 15 weeks: (1) loss of parallel arrangement of collagen fibers in the fibrous zone; and (2) increasing disorganization and loss of columnation of chondrocytes in the fibrocartilage zone. These results suggest that fibroblasts and osteoclasts mediate histologic changes at the insertion site during bioenhanced suture repair of the ACL which may prevent insertion site degeneration, and that the magnitude of these changes may be a function of skeletal maturity.