Background
The current standard of care to treat a ruptured anterior cruciate ligament (ACL) is ACL reconstruction (ACLR), which involves replacing the torn ligament with a tendon graft. The implanted tendon graft undergoes a graft-maturation process known as ligamentization. The influx of host fibroblasts into the necrotic area of the grafted tendon occurs early during the graft-healing phase, and remodeling of the graft follows. However, the synthesis of type III collagen, typically found in weaker scar tissue, continues at higher concentrations, and the heterogeneous composition of collagen fibers of varying diameters of native ACL is never restored.
Purpose
We performed in vitro and in vivo studies of the outcomes of ACLR with decellularized tendon grafts reseeded with ACL-derived cells combined with acellularized cruciate ligament matrix (ACLM) powder as a biological scaffold. We hypothesized that this tissue-engineered construct would enhance the graft maturation process and mechanical properties of the graft following ACLR.
Methods
ACL-derived cells were harvested and isolated from remnants of ruptured ACLs within 4 weeks of injury in patients who had undergone ACLR. Cells were cultured at passage 3. An ACLM was prepared from human ACL and posterior cruciate ligament tissues. Forty-five Sprague Dawley rats were randomly divided into 3 groups: a standard allograft group, an ACLM powder-only injection group, and an ACL-derived cells and ACLM powder co-injection group (e.g., experimental group). A total of 3.75 × 105 ACL-derived cells (passage 3) was mixed with ACLM powder at a concentration of 30 mg/mL into a 15 µL cell-scaffold suspension and then injected into the decellularized tendon graft before ACLR. The rats were euthanized postoperatively at 4 and 8 weeks for histologic analysis, biomechanical testing, and micro-computed tomography (M-CT) analysis. A 2-tailed paired t-test and 1-way and 2-way analysis of variance were used to compare the data from the experimental and control groups at each time point.
Results
In vitro experiments found that combining ACLM powder and ACL-derived cells improved survival and integration of reseeded cells in the tendon extracellular matrix, resulting in the successful transplantation of ACL-derived cells (p < 0.001). Animal ACLR experiments confirmed histologically improved structural maturation in cellularity, metaplasia, and restoration of collagen crimping in the graft reseeded with ACL-derived cells and ACLM powder. Enhanced gene expression of type I collagen (p < 0.005) resulted in superior mechanical properties of the tendon graft (p < 0.05) compared with the control groups. M-CT analysis revealed a significant increase in the average rate of formation of new bone volume at the femoral (95.3% increase, p = 0.021) and tibial (48.4% increase, p = 0.011) tunnels compared with a standard allograft group.
Conclusion
Implantation of a tendon graft reseeded with culture-expanded ACL-derived cells, and ACLM powder enhanced the tendon graft's maturation process and mechanical properties. We provide in vitro and in vivo proof-of-concept evidence supporting the efficacy of reseeding a tendon graft with ACL-derived cells combined with ACLM powder as a biological scaffold for ACL reconstruction.