Abstract
PurposeUpon anterior cruciate ligament (ACL) rupture, reconstruction is often required, with the hamstring tendon autograft as most widely used treatment. Post-operative autograft remodeling enhances graft rupture risk, which occurs in up to 10% of the patient population, increasing up to 30% of patients aged under 20 years. Therefore, this research aimed to identify potential biological predictors for graft rupture, derived from patient-specific tissue remodeling-related cell properties in an in vitro micro-tissue platform.MethodsHamstring tendon-derived cells were obtained from remnant autograft tissue after ACL reconstructions (36 patients, aged 12–55 years), and seeded in collagen I gels on a micro-tissue platform. Micro-tissue compaction over time – induced by altering the boundary constraints – was monitored. Pro-collagen I expression was assessed using ELISA, and protein expression of tenomodulin and α-smooth muscle actin were measured using Western blot. Expression and activity of matrix metalloproteinase 2 were determined using gelatin zymography.ResultsOnly micro-tissues corresponding to younger patients occasionally released themselves from the constraining posts. Pro-collagen I expression was significantly higher in younger patients. Differences in α-smooth muscle actin and tenomodulin expression between patients were found, but these were age-independent. Active matrix metalloproteinase 2 expression was slightly more abundant in younger patients.ConclusionsThe presented micro-tissue platform exposed patient-specific remodeling-related differences between tendon-derived cells, with the micro-tissues that released from constraining posts and pro-collagen I expression best reflecting the clinical age-dependency of graft rupture. These properties can be the starting point in the quest for potential predictors for identifying individual patients at risk for graft rupture.
Highlights
The anterior cruciate ligament (ACL) connects the femur to the tibia and is one of the most important ligaments for knee joint stability
Vascular endothelial growth factor (VEGF)-induced graft vascularization supplies cell nutrients, and combined with the cytokines secreted in the early graft healing phase, this stimulates myofibroblasts – characterized by α-smooth muscle actin expression – from the synovial fluid to invade the graft
The pellet containing cells and small tissue fragments was resuspended in medium consisting of high-glucose Dulbecco’s Modified Eagle Medium (HG-DMEM; Gibco, 42,430–025) and Nutrient Mixture F-12 Ham (F12; Gibco, 21,765–029) in a 1:1 (v/v) ratio, supplemented with 20% fetal bovine serum (FBS; Sigma-Aldrich, F7524) and 5% penicillinstreptomycin, and cultured for 7 days at 37 °C and 5% CO2 with 2–3 medium changes
Summary
The anterior cruciate ligament (ACL) connects the femur to the tibia and is one of the most important ligaments for knee joint stability It consists of a predominately anisotropic collagen I extracellular matrix populated by fibroblast-like cells [24]. After ACL reconstruction with an autograft, a remodeling process is initiated that transforms the tissue to match its new (mechanical) environment [16] This remodeling can roughly be divided in an early graft healing phase, a proliferation phase and a ligamentization phase [18, 38, 41]. The necrotic fibroblasts secrete cytokines (e.g., tumor necrosis factor-α, interleukin 6, vascular endothelial growth factor (VEGF)) and matrix metalloproteases (MMPs) [55]. It can take up to 2 years after surgery for patients to return to their pre-injury activity level [33], which is achieved by approximately 65% of recreational athletes [3]
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