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Objectives To assess the potential of infrared fiber-optic spectroscopy to evaluate the compositional properties of human tracheal cartilage. Study Design Laboratory-based study. Methods Twenty human cadaveric distal tracheas were harvested (age range 20-78 years; 6 females, 14 males) for compositional analysis. Histologic staining, Fourier transform infrared imaging spectroscopy data on collagen and proteoglycan (PG) content, and near-infrared (NIR) fiber-optic probe spectroscopic data that reflect protein and water content were evaluated. NIR fiber-optic probe data were also obtained from the proximal trachea in 4 human cadavers (age range 51-65 years; 2 females, 2 males) in situ for comparison to distal trachea spectral data. Results In the distal trachea cohort, the spectroscopic-determined ratio of PG/amide I, indicative of the relative amount of PG, was significantly higher in the tissues from the younger group compared to the older group (0.37 ± 0.08 vs 0.32 ± 0.05, P = .05). A principal component analysis of the NIR spectral data enabled separation of spectra based on tracheal location, likely due to differences in both protein and water content. The NIR-determined water content based on the 5200-cm -1 peak was significantly higher in the distal trachea compared to the proximal trachea ( P < .001). Conclusions Establishment of normative compositional values and further elucidating differences between the segments of trachea will enable more directed research toward appropriate compositional end points in regenerative medicine for tracheal repair.

Tissue decellularization allows isolation of tissue extracellular matrix components, enabling bioengineering of native tissue microenvironments with minimal antigenic components. Here we describe a method to harvest decellularized cartilage tissue from donor trachea using a series of chemical and enzymatic washes and incorporating the extracellular matrix in gelatin methacrylate hydrogels. This decellularized cartilage tissue is easily incorporated into a variety of hydrogels to create a cartilage tissue scaffold.

Objectives/hypothesis: The trachea is essential to respiratory function and is a mechanically and biochemically complex composite tissue. Tissue-engineering approaches to treat tracheal diseases require detailed knowledge of the native mechanical and biochemical properties of the trachea. Although the porcine trachea represents an excellent preclinical model, relevant mechanical and biochemical composition are incompletely characterized., Study Design: Experimental. The mechanical and biochemical properties of 12 intact porcine tracheas were determined to characterize their compliance, as well as the aggregate modulus, bidirectional elastic modulus, hydraulic permeability, and biochemical characteristics of individual cartilage rings., Results: Data demonstrate the glycosaminoglycan content of tracheal rings was (mean ± standard deviation) 190 ± 49 μg/mg. Hydroxyproline content was 8.2 ± 3.2 μg/mg, and DNA content was 1.3 ± 0.27 μg/mg, a four-fold difference between circumferential elastic modulus (5.6 ± 2.0 megapascal [MPa]) and longitudinal composite elastic modulus (1.1 ± 0.7 MPa, P < 0.0001) was also observed. Aggregate modulus (stiffness) of porcine tracheal rings was 1.30 ± 0.28 MPa, and inflationary compliance was 0.00472 ± 0.00188 cmH2 O(-1) ., Conclusion: This study presents a comprehensive characterization of the relevant biochemical and mechanical properties of porcine tracheal cartilage, which is considered an excellent candidate for xenogenic tracheal graft and a source for tissue-engineered tracheal reconstruction. The range of parameters characterized in this study agrees with those reported for hyaline cartilage of the airway in other species. These characteristics can be used as quantitative benchmarks for tissue-engineering approaches to treat tracheal disease., Level of Evidence: NA. Laryngoscope, 126:E325-E331, 2016., (© 2015 The American Laryngological, Rhinological and Otological Society, Inc.)

Tracheal cartilage has been considered as permanent cartilage without endochondral ossification. We examined ossification of the tracheal cartilage in 25 adults (mean age 67.8 years; range 20-87 years; male:female = 17:8). Thirteen cases (52%) demonstrated ossification of the tracheal cartilage, accompanied by the formation of lamellar bones with fatty bone marrow. Ossification was observed at the lateral peripheral region of the tracheal cartilage, while vascular invasion into the cartilage was seen even where no ossification was present. Twenty-two cases (88%) showed marked hyalinization in the central region of the tracheal cartilage. Type II collagen was immunolocalized in the cartilage matrix, except for in the central hyalinized area, while type I collagen was immunolocalized around the perichondrium and ossified areas in the tracheal cartilage. Type X collagen immunolocalized on territorial matrices around the lacunae near the ossified regions. These results suggest that tracheal cartilage in aged humans frequently ossifies physiologically, and that aging promotes osteogenesis, even in permanent cartilage.

Intact keratan sulfate chains derived from bovine tracheal cartilage have been examined using both one-dimensional methods and the two-dimensional experiments COSY-45 and TOCSY for homonuclear shift correlations and a modified COLOC (correlated spectroscopy for long-range couplings) approach for 13C-1H shift correlations. Partial 1H and 13C NMR signal assignments for residues within the intact polymer chain are reported; data derived from the repeat region signals and from chain cap residues are assigned by comparison with published data derived from oligosaccharides obtained through cleavage of keratan sulfate polymer chains using keratanase and keratanase II and are discussed in detail. The one-dimensional spectra for both 1H and 13C nuclei contain highly crowded signal clusters for which data analysis is not directly possible. COSY-45 analysis allow the correlation and assignment of many proton resonances located within the 3.4-4.8 p.p.m. chemical shift region while from the C/H correlation spectrum data are assignable for some signals within the complex set of carbon resonances which fall in the region between 68 and 86 p.p.m., This work using material from tracheal cartilage has permitted the first detailed combined 1H and 13C NMR examination of the primary keratan sulfate polymer structure; this sequence forms the basis for the more complex members of the keratan sulfate family present in other tissues such as articular cartilage and cornea where further residues such as (alpha1-3)-linked fucose and (alpha2-6)-linked N-acetylneuraminic acid are also present. This nondestructive method of analysis complements the currently available degradative methods for structure determination which may then subsequently be utilized.

Matrilin 1, or cartilage matrix protein, is a member of a novel family of extracellular matrix proteins. To date, four members of the family have been identified, but their biological role is unknown. Matrilin 1 and matrilin 3 are expressed in cartilage, while matrilin 2 and matrilin 4 are present in many tissues. Here we describe the generation and analysis of mice carrying a null mutation in the Crtm gene encoding matrilin 1. Anatomical and histological studies demonstrated normal development of homozygous mutant mice. Northern blot and biochemical analyses show no compensatory up-regulation of matrilin 2 or 3 in the cartilage of knockout mice. Although matrilin 1 interacts with the collagen II and aggrecan networks of cartilage, suggesting that it may play a role in cartilage tissue organization, studies of collagen extractability indicated that collagen fibril maturation and covalent cross-linking were unaffected by the absence of matrilin 1. Ultrastructural analysis did not reveal any abnormalities of matrix organization. These data suggest that matrilin 1 is not critically required for cartilage structure and function and that matrilin 1 and matrilin 3 may have functionally redundant roles.

The action of sodium hypochlorite on N-acetylglucosamine, N-acetylgalactosamine, chondroitinsulfate and hyaluronic acid was studied by 1H-nuclear magnetic resonance (1H-NMR) in order to model some aspects of degradation processes caused by neutrophils on carbohydrate polymers of cartilage in rheumatoid arthritis. N-Acetyl side groups of carbohydrate monomers and chondroitinsulfate yield a resonance at 2.01-2.04 ppm in proton NMR-spectra. This resonance is observed in hyaluronic acid solutions only after a prolonged incubation to yield shorter polymeric chains. Sodium hypochlorite causes a continuous decrease of the line for N-acetyl groups. Two new resonances appear in the 1H-NMR spectra. An intermediate product, assumed as a chlorinated product of N-acetyl side chains, shows a chemical shift of about 2.35 ppm. This intermediate is hydrolyzed to a carbohydrate ring and acetate (1.90 ppm). Sodium hypochlorite acts in all systems investigated mainly on N-acetyl groups. Only small effects on the carbohydrate ring were found under our experimental conditions.

The rat tracheal cartilage was shown to calcify during development. The process of calcification was characterized in terms of distribution of alkaline phosphatase (ALP) activity and alterations to immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans during the development of the tracheal cartilage, in comparison with calcification of the epiphyseal growth plate cartilage. ALP activity was not identified in the tracheal cartilage in the course of calcification, which therefore differed from that in the growth plate. The tracheal cartilage matrix was not resorbed or invaded by type I collagen during calcification. This suggests that no osteogenesis is involved in calcification of the cartilage. Immunoreactivity for type II collagen became weaker in the central region of the tracheal cartilage during development. No net loss of proteoglycans was identified with Alcian blue staining after calcification of the tracheal cartilage. Immunoreactivity for chondroitin 4-sulphate increased in the calcified tracheal cartilage, while reactivity for chondroitin 6-sulphate was weaker in the calcified area than in the surrounding uncalcified region of the tracheal cartilage. The alteration of the extracellular matrices during development may be involved in the calcification of the rat tracheal cartilage.

Keratan sulphate chains were isolated from bovine tracheal ring cartilage (15-18-month-old animals) after papain digestion of the tissue followed by ethanol fractionation, chondroitinase ABC digestion and alkaline borohydride reduction. The keratan sulphate chains were further purified by anion-exchange chromatography on a Pharmacia Mono-Q column in order to remove any contaminating chondroitin sulphate and O-linked oligosaccharides. The chains were then treated with keratanase and the digest was subjected to alkaline borohydride reduction, producing oligosaccharides with galactitol at their reducing ends. The reduced digest was chromatographed on a Nucleosil 5 SB anion-exchange column and individual oligosaccharides were isolated. One of these, oligosaccharide (I), was shown by 500 MHz 1H-n.m.r. spectroscopy to have the following structure: NeuAc alpha 2-3Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (I) The structure of this oligosaccharide shows that keratan sulphate chains from bovine tracheal ring cartilage may be terminated with N-acetylneuraminic acid linked alpha (2-3) to an unsulphated galactose. Keratan sulphate chains were also isolated from bovine femoral head cartilage (15-18-month-old animals) using an identical protocol, but with keratanase which was subsequently shown to have sialidase activity. This yielded oligosaccharide (II), the unsialyated version of (I): Gal beta 1-4GlcNAc(6SO4) beta 1-3Gal-ol (II).

High-field 1H-n.m.r.-spectroscopic studies supported by chemical carbohydrate analyses show that skeletal keratan sulphates (KS-II) of bovine origin may be sub-classified into two groups. Keratan sulphate chains from articular and intervertebral-disc cartilage (KS-II-A) contain two structural features, namely alpha(1----3)-fucose and alpha(2----6)-linked N-acetyl-neuraminic acid residues, that are absent from keratan sulphates from tracheal or nasal-septum cartilage (KS-II-B).