The Harry M. Zweig Memorial Fund for Equine Research

Tissue Engineered Cartilage in the Equine Airway

- Dr. Normand G. Ducharme

DucharmeDr. Normand G. Ducharme

Upper airway obstruction is a common cause of poor performance in horses and several responsible diseases are arytenoids chondritis, epiglottic entrapment, and dorsal displacement of the soft palate. At the opening of the larynx (voicebox), arytenoid cartilages (flappers) serve two functions. They hold the airway open during exercise and they close the airway during swallowing to protect it. Disease of the arytenoid cartilages, known as chondritis, is common and causes physical enlargement of the cartilage. The only available treatment is to remove the affected cartilage. Many important surgical structures, which hold the airway open, attach to the arytenoid and removing it can lead to these structures collapsing into the airway. In addition, the protective swallowing mechanism is lost and this can lead to coughing and aspiration. Another cause is inflammation, shortening, and deformation of the epiglottic cartilage which causes permanent displacement of the soft palate due to the loss of restraining stiffness by the epiglottis. In addition, dorsal displacement of the soft palate occurs due to loss of support within the palate itself.

The aim of this project is to use tissue engineering to rebuild these structures and reduce the obstructions in the airway. In our current investigation of a laryngeal biomechanics in horses, we are developing a structural model of the equine larynx to determine the stiffness of the cartilage and measure the effects the physiological airway wall pressures have on the laryngeal cartilages. We have used medical reconstruction software on 3D (CT and MRI) imaging to generate a geometrically accurate biomechanical model of the equine upper airway, and we are obtaining the tissue mechanical properties of the laryngeal cartilage to build the structural aspects of the model. Thus the biomechanical model provides the essential building blocks of cartilage shape and structural composition. We will take size, shape and stiffness information from the structural model and use it to direct the tissue engineering steps. The epiglottis and palate can also be derived from the medical imaging data, so we will further characterize the biochemical and biomechanical properties of these tissues.

These blocks form a foundation to apply new technologies developed within tissue engineering. This field of biomedical engineering is focused on combining engineering principles of design and function with increasing control of cellular processes. A basic description of tissue engineering is “the concept that the repair and regeneration of biological tissues can be guided through application and control of cells, materials, and proteins.” (Bonassar 1998). The design process in tissue engineering is to characterize the native tissue to be replaced, determine a carrier material that meets that function in the short-term, but then allows for the growth of the cells of that tissue at the site of repair. Thus the carrier material must provide a structural scaffold (e.g. for structural tissues like cartilage) while it is seeded with cells and proteins that encourage growth and integration at the repair site. This field has had a large impact in the repair of joint cartilage for diseases like arthritis, and we are seeking to apply these principles to cartilage in the equine airway.

Our approach will follow established protocols for creating these tissue-engineered constructs for tracheal, articular, and meniscal cartilage. The structural scaffold of choice will be comprised of alginate gel due to its formability for injection molding. Injection molding is fabrication process whereby the structural material is initially in a highly viscous state to be injected into a mold of the desired shape. With application of hardening agents (an idea similar to epoxy), the material retains the mold shape after extraction. Alginate gels retain suitable shape after crosslinking for cartilage-like geometries and stiffness, plus the gels can be directly mixed with cultured cartilage cells to provide a combined product for injection into the mold. Thus, the finished tissue-engineered construct meets some initial requirements of shape, cell density, protein levels, and stiffness. The goal with incubation, and subsequent implantation, is that the cellular components within the construct eventually ‘repair’ the implant to near-native form and function. To our knowledge, this has not been pursued at the opening to the larynx, although tissue engineering practices are being applied for tracheal reconstructions.

The native material properties and 3D model mentioned above are the starting points for this approach. Given these targets, a major challenge in this proposal is establishing the tissue engineering protocols specific to laryngeal cartilages. Yet, given the success of tissue engineering approaches for other cartilage reconstructions, these challenges can be met, or at least, better characterized in our study. The primary aims follow from our starting points: develop the targets for our TECs; develop and create molds and cultures, using harvested cartilage cellular material and an alginate gel; and evaluate their ability to restore structural function.