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Objectives: After autograft hamstring harvesting for anterior cruciate ligament reconstruction (ACLR) there are several studies of semitendinosus (ST) and gracilis (G) tendon showing variation in muscle/tendon regeneration, muscle atrophy and myotendinous muscle retraction. There remains a gap in our knowledge on the extent of muscle function more than a decade after harvest. There is a paucity of long-term studies of hamstring muscle morphology following ACLR. There are no studies evaluating fatty infiltration, a known indicator of decreased muscle function. Likewise, the cross-sectional area of the hamstrings is a functional indicator of muscle strength. Finally, there are no comparative studies on hamstring and bone-patellar tendon-bone (BTB) autografts. In this study, we aim to determine the long-term function of the hamstring muscles as measured by quantitative magnetic resonance imaging (MRI) ten years after autograft hamstring ACLR by comparing to healthy controls and patients who received BTB autografts. Methods: The subjects were from the Multicenter Orthopaedic Outcome Network (MOON) nested cohort with at least ten-year follow-up and include 90 ACLR patients (50 hamstring autograft, 40 BTB autograft) and 21 healthy control participants. Inclusion criteria of the nested cohort were age 14-33 years old at index ACLR with unilateral ACL tear during a sport. Exclusion criteria were prior knee injury at enrollment or graft rupture during follow-up. Axial MR images of both legs from mid-thigh to knee were obtained for muscle cross-sectional area (CSA) and myotendinous junction position (MTJ) measurement. Six-point Dixon images for muscle %fat fraction measurement (%FF) were acquired at the same mid-thigh level as CSA measurements. The muscle CSA (cm2) of the semimembranosus (SM), semitendinosus (ST), gracilis (G), biceps femoris short head (Bshort), and biceps femoris long head (Blong) were measured on mid-thigh axial images by manual image segmentation. %FF maps were generated from the 6-point Dixon images and %FF of each hamstring muscle was calculated by overlying the muscle segmentation masks used for CSA measurement. ST and G MTJ distance (cm) to the joint line was measured manually using the axial and localizer images. For each graft type, a generalized linear model was first fit where the dependent variable was the biomarker measurement (separate models were fit for FF, CSA, and MTJ) and the independent variables were knee type (operated vs non-op), muscle, and their interaction. A transformation was used when the dependent variable was not normally distributed, as needed. Generalized Estimating Equations were used to account for the clustered nature of the data. A significance level of 0.05 was used to test the interaction term. If the interaction term was significant, then comparisons between operated and non-operated knees were performed for each muscle/tendon and graft type combination, using Holm's method to control the type I error rate. Results: Patient demographics: age 33.5 ± 5.3 years; 45 female; body mass index [BMI] 25.8 ± 5.1 kg/m²; Controls demographics: age 30.3 ± 7.8 years; 14 female; BMI 24 ± 5.1 kg/m². Mid-thigh muscle %FF (fat fraction (%)) values for hamstring autograft patients were significantly higher for the operated vs. non-operated limbs ST (15.2%±11.1% vs. 7.6%±5.2%) and G (13.1%±7.6% vs. 9.1%±4.4%) while the SM and biceps femoris showed no differences (Figure 1). In BTB autograft patients, there were no significant differences in muscle %FF between operated and non-operated limbs ST (7.3%±2.4% vs. 7.4%±2.5%) and G (10.1%±3.8% vs. 9.8%±3.9%) (Figure 1). For healthy controls overall, the left limbs had higher muscle %FF than the right limbs. Mid-thigh CSA (cross-sectional area (cm2)) measurements for hamstring autograft patients were significantly lower for the operated vs. non-operated limbs ST (6.7±3 vs 9.6±3.4) and G (3.7±1.8 vs. 4.5±1.6), while the SM and biceps femoris showed no differences (Figure 2). In BTB autograft patients, there were no significant differences in mid-thigh CSA between operated and non-operated limbs ST (10.2±3.7 vs. 10.4±4) and G (5.2±1.7 vs. 5.0±1.6) (Figure 2). For healthy controls, there were no significant differences in CSA between left vs. right limbs ST (7.9±2.6 vs. 7.9±2.4) and G (4.3±1.5 vs. 4.2±1.4). MTJ (myotendinous junction distance to the joint line (cm)) in hamstring autograft patients showed greater muscle retraction in the operated vs. non-operated limbs for ST (14.2±4.6 vs. 7.5±2.3) and G (9.9±4.4 vs. 6.4±1.6) (Figure 3). In BTB autograft patients, there were no significant differences in MTJ between operated vs. non-operated limbs ST (7.4±2.3 vs. 7.4±2) and G (6.2±1.5 vs. 6.2±1.4) (Figure 3). For controls there were also no significant differences in left vs. right limbs ST (5.7±2.5 vs. 5.6±2) and G (6.1±1.9 vs. 6.7±1.7). Conclusions: In long-term follow-up of over a decade following ACLR with autograft hamstring, the harvested hamstring muscles undergo significantly more fatty infiltration (higher muscle %FF), muscle atrophy (lower CSA) and myotendinous junction retraction (higher MTJ distance) than non-operated limbs in the same patients. Whereas the hamstring muscles remain symmetric as expected after ACLR with BTB autografts. No compensatory biceps femoris hypertrophy was found in hamstring autograft patients within this cohort. As a result of these three functional imaging measures (%FF, CSA, MTJ retraction) the harvested hamstrings do not resume normal indices. The biomechanical and functional consequences would need to be evaluated on an individual patient basis. One measure of harvest morbidity is the long-term persistence of significant changes in hamstring muscle functional imaging parameters after hamstring tendon harvesting for ACLR. [ABSTRACT FROM AUTHOR]