Vicryl plus sutures

Mouse or human PDGFRα cell subsets were assessed for ectopic bone formation. Mouse PDGFRα reporter⁻ or reporter⁺ cells (3.0 × 10⁶) were mixed with 45 mg of hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) mixture (w/w = 4:6, Zimmer Dental Inc., Carlsbad, CA). After anesthesia and analgesia, subcutaneous implants were placed on the dorsal surface of 8‐week‐old male NOD SCID mice (Stock # 001303, The Jackson Laboratory; 2 implants per mouse). The samples were analyzed after 8 weeks. An outline of the animals in each experimental group is provided in Supplementary Table S6.
Human PDGFRα⁻ or PDGFRα⁺ cells (3.0 × 10⁶) derived from the same human periosteum sample were resuspended in 40 μL of PBS and mechanically mixed with 50 mg of demineralized bone matrix (DBX) putty (morselized human cortical bone in sodium hyaluronate with a 31% bone content by weight, courtesy of Musculoskeletal Transplant Foundation, Edison, NJ). After anesthesia and analgesia, pockets were cut into the biceps femoris muscles, and cells mixed with DBX were intramuscularly implanted into the thigh muscle pouch of 8-week-old male NOD SCID mice. The muscle and skin were closed with 4–0 Vicryl*Plus sutures (Ethicon Endo-Surgery, Blue Ash, OH). An outline of the animals in each experimental group is presented in Supplementary Table S7.

Animals were housed and experiments were performed in accordance with institutional guidelines at Johns Hopkins University under ACUC approval. A DBX putty (courtesy of Musculoskeletal Transplant Foundation, Edison, NJ) was used for ectopic bone formation in mice. Briefly, CD107a^low or CD107a^high cells derived from the same human WAT sample at passage five were prepared at a density of 3 million total cells in 40 μl PBS and mechanically mixed with 45 mg DBX putty. DBX alone was used as an acellular control. The cell preparation was then implanted intramuscularly into the thigh muscle pouch of 8-week-old male NOD-SCID mice (RRID:IMSR_JAX:001303, The Jackson Laboratory, Bar Harbor, ME) as previously described with some modifications (James et al., 2012c (link)). Mice were anesthetized by isoflurane inhalation and premedicated with buprenorphine. Incisions in the hindlimbs were made, and pockets were cut in the biceps femoris muscles by blunt dissection, parallel to the muscle fiber long axis. Dissection methods and the surgical manipulation of tissues were kept as constant as possible across animals. The muscle and skin were each closed with 4–0 Vicryl*Plus sutures (Ethicon Endo-Surgery, Blue Ash, OH). See Supplementary file 5 for an outline of animals per experimental group. Surgical implantations and subsequent analyses were performed blinded to treatment group.