The overall objective of the proposed work is to identify effective and cost efficient solutions for functional reconstruction of equine upper airway cartilage.  For that purpose we need to identify a laryngeal cartilage replacement (LCR) that has immediate strength and results in a return to functionality within 1-4 months. We propose to use an extracellular matrix based graft material derived through cartilage decellularization, a process rendering the cartilage tissue non immunogenic, and which will result in a graft which possesses immediate strength upon implantation. We hypothesize, based upon published data and preliminary pilot studies, that implantation of this extracellular matrix (ECM) will result in incorporation within 2-4 weeks after implantation and be an effective LCR in the long term. The proposed work aims to: (1) investigate three decellularization methods for the production of acellular, mechanically robust, and geometrically accurate scaffolds from the equine epiglottic/arytenoid cartilages while retaining the tissue specific ultrastructure and functional composition of the source tissues; (2) determine the ability of these acellular scaffold materials, either alone or seeded with bone marrow derived cells, to incorporate with adjacent cartilage and integrate and function as laryngeal implants for the reconstruction or reinforcement of the equine upper airway following partial resection of the epiglottis and arytenoid cartilage in vivo.

 To fulfill Aim 1 we will collect laryngeal cartilage at post mortem from horses euthanized for reasons other than deformity or disease of the laryngeal cartilages. These larynges will be decellularized using 3 methods previously demonstrated to decellularize cartilaginous tissue. The resulting laryngeal tissue (ECM) will then be examined to determine which method yielded the best degree of decellularization, the most accurate 3D anatomical geometry, and the most similar microscopic and ultrastructural appearance of the tissue. In addition, the biochemical content of the ECM as well as its biomechanical strength will be determined and used to identify the most optimal method of decellularization. To complete Aim 2, the best LCR resulting from Aim 1 will be used to reconstruct the epiglottis or arytenoid following excision of the rostral third of the epiglottic cartilage and a 1 cm³ section of each arytenoid cartilage. We will implant a cell-seeded and a non-seeded LCR in the rostral third of the epiglottic cartilage and into a 1 cm section of the arytenoid cartilage. Additionally, the implants will be covered by a second form of ECM (derived from porcine urinary bladder) shown to promote the rapid formation of an epithelial surface in order to prevent infectious deterioration of the LCR. The horses will be assessed endoscopically on a weekly basis for the first month and during exercise at either 2, 3, or 4 months after implantation. Patency of the upper airway will be assessed on a high speed treadmill. Shortly thereafter the horses will be euthanized and histological, biochemical and biomechanical properties will be assessed and compared with native laryngeal cartilage.