*4.1. Tibial Compression Overload*

*C57Bl*/*6J* mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; Stock No: 000664) at four weeks of age and randomized into experimental groups (*n* >= 4). The antibiotic (AB) -treated group received treatment (ampicillin [78,79] (1.0g/L; Sigma; A9518-25G; St. Louis, MO, USA); neomycin [80–82] (0.5 g/L; Sigma; N1876-25G; St. Louis, MO, USA) in drinking water starting at weaning (four weeks of age) for six weeks; the untreated group (VEH) was provided with regular drinking water. Five days prior to injury at 10 weeks of age, cohorts of mice were separated into three groups (VEH, AB, and lipopolysaccharide (LPS)). The LPS group received an intraperitoneal (IP) injection of LPS (1 mg/kg; Sigma; L6529-1MG; St. Louis, MO, USA), while the VEH and AB groups received an IP injection of saline of an equivalent volume. On the day of injury, all groups were subjected to non-invasive ACL rupture using a single dynamic tibial compressive overload using an electromagnetic material testing system (ElectroForce 3200, TA Instruments, New Castle, DE, USA) as previously described [35,74,83]. Cohorts were placed under anesthesia using isoflourane prior to injury [84]. ACL injury was performed by placing the mouse in the system and applying a compressive load at 1 mm/s until ACL rupture (typically 12N-14N); the uninjured group was placed in the system and received a non-injury compressive force (2N-3N). After injury, mice cohorts were given saline (0.05 mL) and buprenorphine (0.05 mg/kg) and returned to normal cage activity as previously described [34–36,74]. All animal experiments were approved by the Lawrence Livermore National Laboratory and University of California, Davis Institutional Animal Care and Use Committee (approved on 14/07/2016), and conformed to the Guide for the care and use of laboratory animals under protocol 250.
