Gelfoam

Fertilized chicken eggs (Henry Stewart and Co., UK) were incubated at 37 °C. After 3 days of incubation, an approximately 3 cm diameter window was cut into the shell using dissecting scissors, revealing the embryo and CAM vasculature. Windows were sealed with tape and eggs were returned to the incubator for 5 days. At day 8 of incubation, decellularized human tracheal scaffolds (approximately 2–3 mm³) and pieces of absorbable gelatin sponge (Gelfoam; Pfizer; a negative control) were placed on the CAM between blood vessels. Scaffolds were checked daily for 6 days, when scaffolds were photographed in ovo with a stereomicroscope (Leica). Vessel ingrowth was quantified blindly as previously described [33] (link).

Placental histocultures and infection were carried out as described [25 (link),37 (link)] on first trimester placentas (4 placentas; mean = 13.11 ± 0.49 (SEM) weeks of amenorrhea, i.e., 11.11 ± 0.49 weeks of pregnancy; age of the women: mean = 23 ± 1.5 (SEM) year-old). Briefly, trophoblastic villi were dissected in small explants and infected or not by hCMV overnight before extensive washing and deposition on gelatin sponges (Gelfoam, Pfizer, New York, NY, USA) in Exofree medium. Conditioned medium was collected and renewed every 3 to 4 days. At 14 days of culture, collected medium was pooled for each condition and used to perform sEV preparation. Placental explants were weighed for normalization of resuspension volume.

In all cases, an endaural procedure with or without intercartilaginous incision was performed. Rosen’s incision was used to raise the endomeatal flap and after identification of the annulus fibrosis it was lifted. The ossicles, chorda tympani nerve and the facial nerve were identified. The surgeon inspected if the stapes was sclerotic and fixed. If so, the incudostapedial joint was cleaved, the stapedius muscle and the posterior crus of the stapes were transected and the anterior crus of the stapes was breached to remove the stapes superstructure. The stapes footplate was fenestrated with a KTP laser (Lumenis, Inc., Salt Lake City, Utah, USA), a Skeeter microdrill (Medtronic Xomed Inc, Jacksonville, Florida, USA), microinstruments or a combination of these. A Causse loop Teflon piston, a Kurz titanium piston or a Teflon wire piston was placed between the incus and the fenestration in the stapes footplate. Two groups of cases were identified: a group of cases that received a 0.4-mm-diameter piston and a group of cases that received a 0.6-mm-diameter piston. Depending on possible anatomical difficulties, the exact piston diameter (0.4 or 0.6 mm) and length were determined. Blood clots and/or allogeneic tissue (Gelfoam, Pfizer, New York, New York, USA) were used to seal the oval window.

Mechanical injury was inflicted by inserting an interdental brush (10 mm in width) through the right nostril [6 (link)]; an absorbable gelatin sponge (Gelfoam®; Pfizer Inc., New York, NY, USA) was then put in place (Figure 1). Group 1 (n = 8) was the sham-control group (no intervention). Groups 2 (n = 8) and 3 (n = 8) were the low- and high-curcumin groups, respectively. Rats in these groups received three drops daily of 5 mg/mL and 10 mg/mL solutions of curcumin (Sigma-Aldrich®, St. Louis, MO, USA; catalog number: C1386) in dimethyl sulfoxide (DMS) (Sigma-Aldrich; catalog number D8418), for 7 days. Group 4 (n = 8) was the DMS-only group. All rats were decapitated on day 15.

Each animal was placed in a prone position and, after sterilization, its skin was incised medially at T10. A laminectomy was carried out at T10 with scissors. The spinal cord was sharply cut with Iris scissors, and a piece of Gelfoam® (Pfizer Inc., New York, NY, USA) was intercalated between the both cut ends.

Female, 6-week-old Crl-scidBR mice (Charles River, Germany) were kept under pathogen-free conditions with water and food ad libitum. Animal experiments were performed in accordance with the German Animal Welfare Act and approved by the local government authorities, RP Oberfranken, Germany, 621-2531.1-13/03. Animals underwent surgery with implantation of 1.5 × 10⁵ SF together with healthy areas of human OA cartilage in a carrier matrix (Gelfoam, Pfizer, USA) with up to four cartilage implants per animal [20 (link)]. SCID mice were sacrificed after 60 days, and implants removed, snap-frozen, stained (H/E), and used for scoring [20 (link), 22 (link), 23 (link)].