Mechanically superior matrices promote osteointegration and regeneration of anterior cruciate ligament tissue in rabbits

The gold standard treatment for anterior cruciate ligament (ACL) reconstruction is the use of tendon autografts and allografts. Limiting factors for this treatment include donor site morbidity, potential disease transmission, and variable graft quality. To address these limitations, we previously developed an off-the-shelf alternative, a poly(l-lactic) acid (PLLA) bioengineered ACL matrix, and demonstrated its feasibility to regenerate ACL tissue. This study aims to 1) accelerate the rate of regeneration using the bioengineered ACL matrix by supplementation with bone marrow aspirate concentrate (BMAC) and growth factors (BMP-2, FGF-2, and FGF-8) and 2) increase matrix strength retention. Histological evaluation showed robust tissue regeneration in all groups. The presence of cuboidal cells reminiscent of ACL fibroblasts and chondrocytes surrounded by an extracellular matrix rich in anionic macromolecules was up-regulated in the BMAC group. This was not observed in previous studies and is indicative of enhanced regeneration. Additionally, intraarticular treatment with FGF-2 and FGF-8 was found to suppress joint inflammation. To increase matrix strength retention, we incorporated nondegradable fibers, polyethylene terephthalate (PET), into the PLLA bioengineered ACL matrix to fabricate a “tiger graft.” The tiger graft demonstrated the greatest peak loads among the experimental groups and the highest to date in a rabbit model. Moreover, the tiger graft showed superior osteointegration, making it an ideal bioengineered ACL matrix. The results of this study illustrate the beneficial effect bioactive factors and PET incorporation have on ACL regeneration and signal a promising step toward the clinical translation of a functional bioengineered ACL matrix.

The goal of developing a bioengineered anterior cruciate ligament (ACL) matrix is to provide an off-the-shelf product that is functionally superior to autografts and allografts currently used for ACL reconstruction surgeries. There is clear need for advancement in this area as 30% of young active patients reinjure their ACL after surgery (1). Furthermore, athletes in the National Basketball Association and National Football League have a return-to-sport time after ACL reconstruction of 11.6 and 10.8 mo, respectively (2, 3). This lengthy period of rehabilitation has spurred interest in bioengineered ACL matrices that can accelerate and enhance ACL regeneration, so that all patients can return to their preinjury performance level faster.Our group previously fabricated a poly(l-lactic) acid (PLLA) bioengineered ACL matrix and evaluated its performance in rabbit ACL reconstruction models (4–7). The bioengineered ACL matrix resulted in excellent tissue regeneration, while experiencing a 41 to 66% rupture rate in vivo (4, 5). The cause of these ruptures was likely due to the interplay between the rate of tissue regeneration and matrix fatigue. Thus, this study aims to 1) accelerate ACL regeneration by supplementing the bioengineered ACL matrix with bone marrow aspirate concentrate (BMAC) and growth factors (bone morphogenetic protein 2 [BMP-2], fibroblast growth factor 2 [FGF-2], and FGF-8); and 2) increase the strength retention of the bioengineered ACL matrix by incorporating nondegradable polyethylene terephthalate (PET) yarns.BMAC is a promising translational stem cell therapy as it can be harvested and applied during surgery and is not regulated by the US Food and Drug Administration (FDA) (8, 9). The ability of BMAC to enhance the repair of damaged rotator cuff (10) and meniscus (11) tissues has been demonstrated in rabbit models. Thus, we hypothesized that the application of BMAC would serve as a source of progenitor cells and bioactive factors that would accelerate ACL regeneration. This report evaluates the feasibility of obtaining BMAC in a rabbit ACL reconstruction model and its regenerative potential.Growth factors have been widely investigated to accelerate bone and ligament regeneration through the proliferation and differentiation of progenitor cells (12). FGFs have been shown to stimulate the proliferation of cells and enhance tissue healing. In particular, FGF-2 has been shown to accelerate ligament healing (13), and a member of the FGF-8 subfamily has been shown to stimulate cartilage healing in a clinical study (14). Furthermore, the synergistic application of FGF-2 and FGF-8 induced dedifferentiation of mature cells in axolotls (15). Given the evidence supporting the proregenerative qualities of FGF-2 and FGF-8, we chose to apply FGF-2 and FGF-8 simultaneously in the intraarticular space. We hypothesized that the combinatorial application of FGF-2 and FGF-8 would accelerate ligamentization of the bioengineered ACL matrix by promoting the proliferation of progenitor cells and dedifferentiation of mature cells in the synovial environment.To accelerate bone regeneration, we utilized bone morphogenetic protein 2 (BMP-2), which is approved by the US FDA for a range of lumbar spinal fusion procedures and has been shown in ACL reconstruction models to enhance osteointegration of tendon grafts (16–18). In our previous study, we demonstrated that BMP-2 saline injections could enhance osteoid seam width and reduce bone tunnel cross-sectional area, a sign of bone regeneration (5). However, the effect was limited, likely due to the lack of a drug carrier. In this study, we hypothesized that the addition of a drug delivery carrier, fibrin glue (19–22), would potentiate the effect of BMP-2 and promote bone formation (23).The first iteration of the bioengineered ACL matrix, termed the “L-C ligament,” was completely biodegradable and composed of only PLLA yarns (4). The high rupture rate found in our previous study motivated us to modify the material composition of the bioengineered ACL matrix to reduce its mechanical fatigue rate. A compelling polymer to reduce fatigue rate is PET, a biocompatible nondegradable polymer with high tensile strength that has previously been utilized for orthopedic applications (24). To date, no study has investigated the use of a composite PLLA and PET bioengineered ACL matrix for ACL reconstruction. Following the patented design by Laurencin and colleagues (25), a composite bioengineered ACL matrix, termed the “tiger graft,” composed of 20 PLLA yarns and 4 PET yarns, was fabricated. The PLLA facilitates a greater volume of tissue regeneration as it gradually degrades, while the PET bolsters the mechanical strength of the matrix during the early phases of healing. We hypothesized that the tiger graft would have increased mechanical strength retention over the implantation period.The overall goal of this study was to accelerate ACL regeneration of a bioengineered ACL matrix by supplementation with BMAC and growth factors (BMP-2, FGF-2, and FGF-8) and by modulating the material composition of the matrix (Fig. 1). We evaluated the ligamentization and osteointegration of the bioengineered ACL matrices histologically. Microcomputed tomography (µCT) was performed to evaluate bone tunnel regeneration. The inflammatory and remodeling state of the synovial fluid was evaluated using an enzyme-linked immunosorbent assay (ELISA). Finally, biomechanical testing was conducted to determine the strength retention of the bioengineered ACL matrices.Open in a separate windowFig. 1.Fabrication of the bioengineered ACL matrix and implantation in a rabbit ACL reconstruction model. (A) Depiction of the braiding machine used to fabricate the bioengineered ACL matrices and the resulting biphasic structure of the matrix. Each matrix was composed of 24 yarns. (B) For the L-C ligament, 24 yarns of PLLA were braided together. For the tiger graft, 20 yarns of PLLA and 4 yarns of PET were braided together. Experimental groups were evaluated at 12 wk, and the L-C ligament (control) was further evaluated at 24 wk. (C) View of implanted bioengineered ACL matrix at the time of surgery (Left) and the application of fibrin glue (Right). BMAC or growth factors were mixed with fibrin glue for the experimental groups. (D) Representative image demonstrating the implantation of a fibrin gel in the tibial bone tunnel (Left, blue arrow) and subsequent fixation of a titanium suture button (Right).