**1. Introduction**

End‐to‐end anastomosis after an extensive tracheal resection is the method of choice for tracheal pathologies such as benign stenosis secondary to post‐intubation injury, trauma, congenital, iatrogenic, or neoplasm causes. It is successfully performed in defects affecting up to 50% of the trachea in adults and 33% in infants. More extensive lesions are not amenable to conventional surgical intervention. Tracheal reconstruction after extensive resection is unresolved and remains one of the most important challenges in tracheal surgery [1]. In the search for alternative methods of replacing long tracheal segments, segmental trachea substitution using tracheal segments biopreserved by cryopreservation and lyophilization has been attempted; however, the clinical application of the procedure has been limited due to the fact that contradictory results have been reported [1–3]. A series of interconnected cartilage rings maintain the tubular shape of the trachea, allowing the passage of air. Chondrocytes are the resident cells of the tracheal cartilage. They reside in cavities in the matrix called cartilage lacunae. Chondrocyte connections to each other are crucial for adequate matrix balance and function, determining the tracheal cartilage stiffness of the biopreserved tracheal allograft. The biopreservation of the cartilage must therefore be investigated in basic research models of chondrocyte injuries. Necrosis, or irreversible cell death, is characterized by nuclear swelling, pyknosis, karyorrhexis, karyolysis, and cytoplasmic eosinophilic staining. Dehydration, rehydration, freezing, and thawing result in increased cell death by both necrosis and apoptosis.

Caspase‐3 is the primary inducer of cell death by apoptosis. It is implicated in tissue damage due to mechanical ventilation [4], ischemia‐reperfusion [5, 6], orotracheal cannulation due to the tidal volume effect [7], and freezing and thawing processes inherent in the cryopreservation of different tissues and cells [1].

The aim of this study was to compare the effect of two preservation methods (lyophilization and cryopreservation) on the canine tracheal cartilage by microscopic evaluation of necrotic cell death.
