Collagen deposition (collagen/tracheal tissue, mg) in the membranous portions of the resected tracheal rings (presurgery) and the healed anastomosis were biochemically evaluated according to the Woessner method [22 (link)]. This approach determines the concentration of hydroxyproline per gram of tracheal tissue. The samples were weighed and placed in an oven at 80°C until the dry weight was obtained and hydrolysed in 1.5 ml of 6 N hydrochloric acid for 36 h. Subsequently, the samples were filtered and placed in an evaporator until the excess acid was removed. In the obtained residue, the pH was adjusted to 7.0, and each sample was reconstituted with distilled water to 10 ml. From this last dilution, an aliquot of 100 μl was obtained to determine the concentration of hydroxyproline. For the evaluation of hydroxyproline, a standard curve was prepared with a solution containing 1 mg of hydroxyproline/1 ml of water and adjusted for the hydroxyproline standards at concentrations of 200, 400, 600, 800, and 1000 μl of hydroxyproline; a blank containing distilled water was included. Subsequently, the following were added in all tubes: 1 ml of 0.05 M chloramine T dissolved in methyl cellosolve, water, and citrate buffer (pH 6.0); 1 ml of 3.0 N perchloric acid; and 1 ml of a solution of 20% paradimethylaminobenzaldehyde in methyl cellosolve. Next, the samples were incubated for 20 min at 60°C, and the absorbance of each sample was detected using a spectrophotometer (DU 640; Beckman Coulter Inc., Brea, CA, USA) at a wavelength of 560 nm. All chemicals were purchased from Sigma-Aldrich (Hydroxyproline Assay Kit; Sigma-Aldrich, St. Louis, MO, USA).
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