Randall selitto paw pressure test apparatus

The gastrocnemius muscle of adult mice was injured by applying 3500 to 3700 grams of force with a Randall Selitto Paw Pressure Test Apparatus (IITC Life Science). Care was taken to avoid incidental contact with the tibia and fibula, which was verified by μCT. Injuries were performed while mice were under isoflurane anesthesia. Treated mice received a single subcutaneous dose of ActA-mAb (10 mg/kg) and/or a single i.p. dose of the anti-ACVR1 monoclonal antibody JAB0505 (10 mg/kg) on the day of injury. Histological analyses were performed on days 6 and 14 after injury, flow cytometry analyses were performed on days 5 and 10 after injury, and μCT imaging was conducted on days 14, 21, 28, and 35 after injury.
To produce a subclinical injury that does not cause HO in this model, the tibialis anterior muscle was injected with 50 μL of 2.5% methylcellulose (Sigma-Aldrich) in sterile PBS. JAB0505 administration was as above. methylcellulose-injected mice were imaged by μCT at 15 and 22 days after injury.

FACS-isolated and expanded FAPs were resuspended in 50 µL of ice-cold 1X Dulbecco's Phosphate-Buffered Saline (DPBS; Gibco, Billings, MT) and injected into the gastrocnemius muscle of SCID mice as previously described (Lees-Shepard et al., 2018 (link)). In most cases, both gastrocnemius muscles of an individual mouse were injected. For a given treatment group, variation in HO volume within and between mice was not statistically different, and each injection was treated as an independent event for statistical analysis. The gastrocnemius muscle was pinch-injured the day of transplantation using 3500 – 3700 grams of force applied with a Randall Selitto Paw Pressure Test Apparatus (IITC Life Science, Woodland Hills, CA). Treated SCID host mice received a single subcutaneous dose of ActA-mAb (10 mg/kg) on the day of injury, or daily IP injections of 1.47 mg/kg palovarotene or vehicle, beginning 3 days prior to injury and transplantation.

The gastrocnemius muscle of adult mice was injured either by forceps pinch or by cardiotoxin injection. For pinch injury, the gastrocnemius and covering skin was gripped at the approximate midbelly of the muscle with 2-mm wide tissue forceps and pressure was applied for 5 s. Care was taken to avoid breaking the skin and incidental contact with the tibia and fibula; the latter was verified by µCT. In subsequent testing with a Randall Selitto Paw Pressure Test Apparatus (IITC Life Science), similarly sized lesions were observed following application of 2200–2700 g of force to the gastrocnemius muscle. For cardiotoxin injury, 100 µL of 10 μM cardiotoxin (Sigma) in PBS was injected into the midbelly of the muscle. Injuries were performed while mice were under isoflurane anesthesia.