Computed tomography-guided tissue engineering of upper airway cartilage

Abstract

Normal laryngeal function has a large impact on quality of life, and dysfunction can be life threatening. In general, airway obstructions arise from a reduction in neuromuscular function or a decrease in mechanical stiffness of the structures of the upper airway. These reductions decrease the ability of the airway to resist inspiratory or expiratory pressures, causing laryngeal collapse. We propose to restore airway patency through methods that replace damaged tissue and improve the stiffness of airway structures. A number of recent studies have utilized image-guided approaches to create cell-seeded constructs that reproduce the shape and size of the tissue of interest with high geometric fidelity. The objective of the present study was to establish a tissue engineering approach to the creation of viable constructs that approximate the shape and size of equine airway structures, in particular the epiglottis. Computed tomography images were used to create three-dimensional computer models of the cartilaginous structures of the larynx. Anatomically shaped injection molds were created from the three-dimensional models and were seeded with bovine auricular chondrocytes that were suspended within alginate before static culture. Constructs were then cultured for approximately 4 weeks post-seeding and evaluated for biochemical content, biomechanical properties, and histologic architecture. Results showed that the three-dimensional molded constructs had the approximate size and shape of the equine epiglottis and that it is possible to seed such constructs while maintaining 75%+ cell viability. Extracellular matrix content was observed to increase with time in culture and was accompanied by an increase in the mechanical stiffness of the construct. If successful, such an approach may represent a significant improvement on the currently available treatments for damaged airway cartilage and may provide clinical options for replacement of damaged tissue during treatment of obstructive airway disease.

FIG. 1.

Mold design and casting. Computed tomography reconstruction of equine airway tissues (A) was performed and then used to create a 3D Solidworks model of equine epiglottis (B). Injection molds were created from the Solidworks models through rapid prototyping (C) and used to create molded alginate constructs (preculture sample shown in D). Color images available online at www.liebertpub.com/tec

FIG. 2.

Gross morphologic appearance. Native equine epiglottis (A), molded construct before culture (B), and construct after 2 weeks in static culture (C) are shown demonstrating that constructs had correct anatomic shape and size both pre and postculture. Color images available online at www.liebertpub.com/tec

FIG. 3.

Live/dead staining of cultured constructs at 1 days (A, B), 3 days (C, D), 1 week (E, F), 2 weeks (G, H), 3 weeks (I, J), and 4 weeks (K, L). Results demonstrate high cellular viability of constructs at all time points. Percent viability (M) and DNA per construct dry weight (N) are shown. Scale bar=100 μm. Color images available online at www.liebertpub.com/tec

FIG. 4.

Histologic architecture. Safranin O staining of constructs cultured for 1–4 weeks (A–D). Picrosirius red staining of constructs cultured for 1–4 (F–I), with arrows indicating areas of collagen staining. Verhoeff's elastic staining of constructs cultured for 1–4 weeks (K–N), with arrows indicating areas of elastin staining. Accumulation of glycosaminoglycan (GAG), collagen, and elastin was observed with the greatest gains in GAG content over the culture period. Native epiglottis is shown for reference (E, J, O) Scale bar=100 μm. Color images available online at www.liebertpub.com/tec

FIG. 5.

Percent water content (A) as well as GAG (B), hydroxyproline (C), and elastin (D) content of cultured constructs in μg/mg construct dry weight are shown. GAG and collagen content were observed to increase over the 4-week period. Native tissue is shown for reference. Insets are used to demonstrate differences in week 1–4 values when not clearly distinguishable alongside native. All values are presented as mean±standard deviation. Groups not connected by the same letter are statistically significant, p<0.05.

FIG. 6.

Aggregate modulus of constructs after 1, 2, 3, and 4 weeks of static culture. Aggregate modulus was observed to increase over the 4-week culture period. Native tissue is shown for reference. All values are presented as mean±standard deviation. Groups not connected by the same letter are statistically significant, p<0.05.