Histoacryl

All animal and surgical procedures were performed in accordance with the Institutional Animal Care and Use Committee at Johns Hopkins University School of Medicine. For all animal experiments female NOD-scid IL2Rgnull (NSG) immunodeficient mice (Jackson Lab, Bar Harbor, ME, USA) aged 2–4 months (n = 9) were used to allow for implantation of human cells. Sub-critical “pocket” defects (~5–13% of TA muscle) were performed, as previously described [3 (link)]. Briefly, following isoflurane anesthetization, the TA was exposed, and part of the muscle was removed. Engineered muscle grafts were then placed in the defect site (2 skeletal muscle grafts per defect) and sutured to the remaining muscle using nonabsorbable sutures (6–0 Nylon, Express Medical Supplies). Implanted scaffolds were grown for 9–12 days prior to implantation. Sutures, as well as surgical glue (Histoacryl, B Braun Medical, Bethlehem, PA, USA), were also used to close the skin and Rimadyl (Patterson Veterinary, Greeley, CO, USA) (5 mg/kg) was injected subcutaneously after the surgery for pain management. The mice were sacrificed by isoflurane overdose and cervical dislocation 1 to 2 weeks after the surgery. Immediately following this, the TA muscle, including the implanted scaffolds, was removed and cryopreserved for sectioning.

Animals were anesthetized using fentanyl, medetomidine, and midazolam (50 µg/kg, 5 mg/kg, and 0.5 mg/kg, delivered i.p., respectively), and analgesia was provided with carprofen (7 mg/kg delivered s.c.). Body temperature was maintained using a thermostatically regulated heating pad. Respiration rate and depth of anesthesia was monitored throughout the procedure. Following opening of the skin and removal of connective tissue overlying the sagittal suture and parietal bones, the skull was cleaned with H₂O₂ (3%). A custom-made implant, consisting of a flat circular attachment surface for attachment to the skull, and implant body with three anti-rotation pins and a magnet (Figure 7A–B), was fixed to the dried skull using a UV-curing dental adhesive (Optibond FL, Kerr Corporation, Orange, California, USA) and a UV-curing dental composite (Charisma, Kulzer GmbH, Hanau, Germany). The implant attachment surface and body were made from light-weight, bio-compatible dental resin (Dental SG, Formlabs, Germany). Skin margins were closed with 5/0 Vicryl sutures (Ethicon Inc, Somerville, NJ, USA) and a cyanoacrylate adhesive (Histoacryl, B.Braun, Melsungen, Germany). The injectable anesthetic combination was antagonized with naloxone, atipamezole and flumazenil (respectively 1.2 mg/kg, 0.5 mg/kg and 0.75 mg/kg, delivered i.p.), and the animal was allowed to recover.

The eye cameras for oculo-videography were mounted on mounting arms which were attached to a baseplate with complementary holes to the anti-rotation pins on the implant and fitted with a magnet of complementary polarity. During positioning of the head-camera, mice were anesthetized with isoflurane (induction: 3–5% isoflurane, maintenance: 2.0% isoflurane in air). Anesthetic depth and body temperature were monitored as above. The cameras were positioned to have a sharp image of the entire eye, with the mounting arms adjusted such that the cameras and mounting system caused minimal disruption to the mouse’s lateral and frontal field of view. Mounting arms were secured with cyanoacrylate adhesive glue (Histoacryl, B.Braun, Melsungen, Germany). The eye-camera system was then removed and the animal allowed to recover.