Richard D. Wemer^*, Michael Detamore, Robert A. Weatherly

Abstract: The objective of this study was to immunohistochemically elucidate the major extracellular matrix constituents of rabbit tracheal cartilage. The impetus for this project is the need for crucial design and validation criteria for tissue engineering juxtaposed with the conspicuous lack of trachea extracellular matrix data in the literature. Tracheal tissue specimens were harvested from New Zealand White rabbits, and were immunostained for collagen I, collagen II, aggrecan and decorin; and a Verhoeff-Van Gieson stain was performed to visualize elastin. The most striking result was the highly organized relationship between distinct fibrous (containing collagen I, decorin and elastin) and hyaline-like (containing collagen II and aggrecan) regions of the tracheal wall. The tracheal cartilage stained strongly with collagen II throughout, with periodic bands of aggrecan in the tracheal arches, meaning that there were areas void of aggrecan immunostaining alternating with areas with strong aggrecan immunostaining. In contrast, the periphery of the cartilage and the perichondrium itself exhibited strong collagen I staining and no collagen II staining. Elastin fibers and decorin were also detected along the periphery of the cartilage in the perichondrium and corresponded highly with the distribution of collagen I staining. The body of the rabbit trachea is therefore composed of a hyaline-cartilage structure primarily made of collagen II and bands of aggrecan, surrounded by a fibrous region composed of elastin and collagen I, indicative of a flexible tissue with distinct regions of compressive integrity. This information will be a valuable reference to future tissue engineering efforts in the creation of a biosynthetic substitute for laryngotracheal reconstruction.

Keywords: Trachea; Collagen; Aggrecan; Decorin; Elastin.

Cite this paper: nullWemer, R. , Detamore, M. and Weatherly, R. (2010) Immunohistochemical characterization of the rabbit tracheal cartilages. Journal of Biomedical Science and Engineering, 3, 1007-1013. doi: 10.4236/jbise.2010.310131.

References

[1]   Tan, Q., Steiner, R., Hoerstrup, S.P and Weder, W. (2006) Tissue-engineered trachea: History, problems and the future. Eur J Cardiothorac Surg, 30, 782-786.

[2]   Kojima, K. and Vacanti, C.A. (2004) Generation of a tissue-engineered tracheal equivalent. Biotechnol Appl Biochem, 39, 257-262.

[3]   Bucheler, M. and Haisch, A. (2003) Tissue engineering in otorhinolaryngology. DNA Cell Biol, 22, 549-564.

[4]   Grillo, H.C. (2002) Tracheal replacement: a critical review. Ann Thorac Surg, 73, 1995-2004.

[5]   Grillo, H.C. (2003) Tracheal replacement. J Thorac Cardiovasc Surg, 125, 975.

[6]   Grillo, H.C. (2005) Bioengineered airway tissue. J Thorac Cardiovasc Surg, 129, 1208.

[7]   Birchall, M. and Macchiarini, P. (2008) Airway transplantation: a debate worth having? Transplantation, 85, 1075-1080.

[8]   Doss, A.E., Dunn, S.S., Kucera, K.A., Clemson, L.A. and Zwischenberger, J.B. (2007) Tracheal replacements: Part 2. ASAIO J, 53, 631-639.

[9]   Kucera, K.A., Doss, A.E., Dunn, S.S., Clemson L.A. and Zwischenberger J.B. (2007) Tracheal replacements: Part 1. ASAIO J, 53, 497-505.

[10]   Macchiarini, P., Jungebluth, P., Go, T., Asnaghi, M.A., Rees, L.E., Cogan, T.A., Dodson, A., Martorell, J., Bellini, S., Parnigotto, P.P., Dickinson, S.C., Hollander, A.P., Mantero, S., Conconi, M.T. and Birchall, M.A. (2008) Clinical transplantation of a tissue-engineered airway. Lancet.

[11]   Steger, V., Hampel, M., Trick, I., Muller, M. and Walles, T. (2008) Clinical tracheal replacement: transplantation, bioprostheses and artificial grafts. Expert Rev Med Devices, 5, 605-612.

[12]   Asnaghi, M.A., Jungebluth, P., Raimondi, M.T., Dickinson, S.C., Rees, L.E., Go, T., Cogan, T.A., Dodson, A., Parnigotto, P.P., Hollander, A.P., Birchall, M.A., Conconi, M.T., Macchiarini, P. and Mantero, S. (2009) A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: from concept to clinical trial. Biomaterials, 30, 5260-5269.

[13]   Jungebluth, P., Go, T., Asnaghi, A., Bellini, S., Martorell, J., Calore, C., Urbani, L., Ostertag, H., Mantero, S., Conconi, M.T. and Macchiarini, P. (2009) Structural and morphologic evaluation of a novel detergent-enzymatic tissue-engineered tracheal tubular matrix. J Thorac Cardiovasc Surg, 138, 586-593.

[14]   Grimmer, J.F., Gunnlaugsson, C.B., Alsberg, E., Murphy, H.S., Kong, H.J., Mooney, D. J. and Weatherly, R.A. (2004) Tracheal reconstruction using tissue-engineered cartilage. Arch Otolaryngol Head Neck Surg, 130, 1191- 1196.

[15]   Robey, T.C., Eiselt, P.M., Murphy, H.S., Mooney, D.J. and Weatherly, R.A. (2000) Biodegradable external tracheal stents and their use in a rabbit tracheal reconstruction model. Laryngoscope, 110, 1936-1942.

[16]   Robey, T.C., Valimaa, T., Murphy, H.S., Tormala, P., Mooney, D.J. and Weatherly, R.A. (2000) Use of internal bioabsorbable PLGA “finger-type” stents in a rabbit tracheal reconstruction model. Arch Otolaryngol Head Neck Surg, 126, 985-991.

[17]   Hallers, E.J., Rakhorst, G., Marres, H.A., Jansen, J.A., Kooten, T.G., Schutte, H.K., Loon, J.P., Houwen, E.B. and Verkerke, G.J. (2004) Animal models for tracheal research. Biomaterials, 25, 1533-1543.

[18]   Buhler, R.B., Sennes, L.U., Mauad, T., Melo, E.C., Silva, L.F. and Saldiva, P.H. (2008) Collagen fiber and versican distribution within the lamina propria of fetal vocal folds. Laryngoscope, 118, 371-374.

[19]   Butler, J.E., Hammond, T.H. and Gray, S.D. (2001) Gender-related differences of hyaluronic acid distribution in the human vocal fold. Laryngoscope, 111, 907-911.

[20]   Hammond, T.H., Gray, S.D. and Butler, J.E. (2000) Age- and gender-related collagen distribution in human vocal folds. Ann Otol Rhinol Laryngol, 109, 913-920.

[21]   Gray, S.D., Titze, I.R., Alipour, F. and Hammond, T.H. (2000) Biomechanical and histologic observations of vocal fold fibrous proteins. Ann Otol Rhinol Laryngol 109, 77-85.

[22]   Gray, S.D., Titze, I.R., Chan, R. and Hammond, T.H, (1999) Vocal fold proteoglycans and their influence on biomechanics. Laryngoscope, 109, 845-854.

[23]   Hammond, T.H., Zhou, R., Hammond, E.H., Pawlak, A. and Gray, S.D. (1997) The intermediate layer: a morphologic study of the elastin and hyaluronic acid constituents of normal human vocal folds. Journal of Voice, 11, 59-66.

[24]   Pawlak, A.S., Hammond, T., Hammond, E. and Gray, S.D. (1996) Immunocytochemical study of proteoglycans in vocal folds. Ann Otol Rhinol Laryngol, 105, 6-11.

[25]   Chan, R.W., Fu, M., Young, L. and Tirunagari, N. (2007) Relative contributions of collagen and elastin to elasticity of the vocal fold under tension. Ann Biomed Eng, 35, 1471-1483.

[26]   Melo, E.C., Lemos, M., Aragao, X.F.J., Sennes, L.U., Nascimento, S.P.H. and Tsuji, D.H. (2003) Distribution of collagen in the lamina propria of the human vocal fold. Laryngoscope, 113, 2187-2191.

[27]   Sivasankar, M. and Ivanisevic, A. (2007) Atomic force microscopy investigation of vocal fold collagen. Laryngoscope, 117, 1876-1881.

[28]   Tateya, T., Tateya, I. and Bless, D.M.(2006) Collagen subtypes in human vocal folds. Ann Otol Rhinol Laryngol, 115, 469-476.

[29]   Tateya, T., Tateya, I. and Bless, D.M. (2007) Immuno-scanning electron microscopy of collagen types I and III in human vocal fold lamina propria. Ann Otol Rhinol Laryngol, 116, 156-159.

[30]   Sakae, F.A,, Imamura, R., Sennes, L.U., Mauad, T., Saldiva, P.H. and Tsuji, D.H. (2008) Disarrangement of collagen fibers in Reinke’s edema. Laryngoscope, 118, 1500-1503.

[31]   Sato, K., Hirano, M. and Nakashima, T. (2003) 3D structure of the macula flava in the human vocal fold. Acta Otolaryngol, 123, 269-273.

[32]   Sato, K., Hirano, M. and Nakashima, T. (2002) Age- related changes of collagenous fibers in the human vocal fold mucosa. Ann Otol Rhinol Laryngol, 111, 15- 20.

[33]   Sato, K. and Hirano, M. (1995) Histologic investigation of the macula flava of the human vocal fold. Ann Otol Rhinol Laryngol, 104, 138-143.

[34]   Hirano, S., Minamiguchi, S., Yamashita, M., Ohno, T., Kanemaru, S.I. and Kitamura, M. (2008) Histologic characterization of human scarred vocal folds. Journal of Voice, Epub.

[35]   Ximenes, F.J.A, Tsuji, D.H., Nascimento, P.H. and Sennes, L.U. (2003) Histologic changes in human vocal folds correlated with aging: A histomorphometric study. Ann Otol Rhinol Laryngol, 112, 894-898.

[36]   Kamel, K.S., Beckert, L.E. and Stringer, M.D. (2009) Novel insights into the elastic and muscular components of the human trachea. Clin Anat, 22, 689-697.

[37]   Al-Zghoul, M.F., Ismail, Z.B., Al-Rukibat, R.K. and Al- Majali, A.M. (2006) A quantitative study on the trachea of young Arabian camels (Camelus dromedarius). J Camel Pract Res, 13, 129-133.

[38]   Cozzi, B., Bagnoli, P., Acocella, F. and Costantino, M.L. (2005) Structure and biomechanical properties of the trachea of the striped dolphin Stenella coeruleoalba: evidence for evolutionary adaptations to diving. Anat Rec A Discov Mol Cell Evol Biol, 284, 500-510.

[39]   Pierson, D.J. (2009) The physiology of dinosaurs: circulatory and respiratory function in the largest animals ever to walk the earth. Respir Care, 54, 887-911.

[40]   Evans, M.J., Fanucchi, M.V., Baker, G.L., Winkle, L.S., Pantle, L.M., Nishio, S.J., Schelegle, E.S., Gershwin, L.J., Miller, L.A., Hyde, D.M. and Plopper, C.G. (2004) The remodelled tracheal basement membrane zone of infant rhesus monkeys after 6 months of recovery. Clin Exp Allergy, 34, 1131-1136.

[41]   Evans, M.J., Winkle, L.S., Fanucchi, M.V., Toskala, E., Luck, E.C., Sannes, P.L. and Plopper, C.G. (2000) Three-dimensional organization of the lamina reticularis in the rat tracheal basement membrane zone. Am J Respir Cell Mol Biol, 22, 393-397.

[42]   Evans, M.J., Fanucchi, M.V., Baker, G.L., Winkle, L.S., Pantle, L.M., Nishio, S.J., Schelegle, E.S., Gershwin, L.J., Miller, L.A., Hyde, D.M., Sannes, P.L. and Plopper, C.G. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. Am J Physiol Lung Cell Mol Physiol , 285, L931-939.

[43]   Amiri, M.H. and Gabella, G. (1988) Structure of the guinea-pig trachea at rest and in contraction. Anat Embryol (Berl), 178, 389-397.

[44]   Maki, J.M., Sormunen, R., Lippo, S., Kaarteenaho, W.R, Soininen, R. and Myllyharju, J. (2005) Lysyl oxidase is essential for normal development and function of the respiratory system and for the integrity of elastic and collagen fibers in various tissues. Am J Pathol, 167, 927-936.

[45]   Sasano, Y., Takahashi, I., Zhu, J.X., Ohtani, H., Mizoguchi, I. and Kagayama, M. (2001) Gene and protein expressions of type I collagen are regulated tissue-specifically in rat hyaline cartilages in vivo. Eur J Morphol, 39, 149-154.

[46]   Sasano, Y., Takahashi, I., Mizoguchi, I., Kagayama, M., Takita, H. and Kuboki, Y. (1998) Type X collagen is not localized in hypertrophic or calcified cartilage in the developing rat trachea. Anat Embryol (Berl), 197, 399-403.

[47]   Sasano, Y., Mizoguchi, I., Furusawa, M., Aiba, N., Ohtani, E., Iwamatsu, Y. and Kagayama, M. (1993) The process of calcification during development of the rat tracheal cartilage characterized by distribution of alkaline phosphatase activity and immunolocalization of types I and II collagens and glycosaminoglycans of proteoglycans. Anat Embryol (Berl), 188, 31-39.

[48]   Tamechika, I., Itakura, M., Saruta, Y., Furukawa, M., Kato, A., Tachibana, S. and Hirose, S. (1996) Accelerated evolution in inhibitor domains of porcine elafin family members. J Biol Chem, 271, 7012-7018.

[49]   Suzuki, Y., Furukawa, M., Abe, J., Kashiwagi, M. and Hirose, S. (2000) Localization of porcine trappin-2 (SKALP/elafin) in trachea and large intestine by in situ hybridization and immunohistochemistry. Histochem Cell Biol, 114, 15-20.

[50]   Kusafuka, K., Yamaguchi, A., Kayano, T. and Takemura, T. (2001) Ossification of tracheal cartilage in aged humans: A histological and immunohistochemical analysis. J Bone Miner Metab, 19, 168-174.

[51]   Roberts, C.R., Rains, J.K., Pare, P.D., Walker, D.C., Wiggs, B. and Bert, J.L. (1998) Ultrastructure and tensile properties of human tracheal cartilage. J Biomech, 31, 81-86.

[52]   Rao, R.A., Mehta, M.R. and Toussaint, K.C. (2009) Fourier transform-second-harmonic generation imaging of biological tissues. Opt Express, 17, 14534-14542.

[53]   Maki, K., Hayashi, S., Nishioka, T., Kimura, M. and Noguch, T.: A new type of matrix vesicles is found in fetal bovine tracheal cartilage. Connect Tissue Res, 41, 109-115.

[54]   Detamore, M.S. and Athanasiou, K.A. (2003) Structure and function of the temporomandibular joint disc: implications for tissue engineering. J Oral Maxillofac Surg, 61, 494-506.

[55]   Poole, A.R., Webber, C., Pidoux, I., Choi, H. and Rosenberg, L.C. (1986) Localization of a dermatan sulfate proteoglycan (DS-PGII) in cartilage and the presence of an immunologically related species in other tissues. J Histochem Cytochem, 34, 619-625.

[56]   Danielson, K.G., Baribault, H., Holmes, D.F., Graham, H., Kadler, K.E. and Iozzo, R.V. (1997) Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. Journal of Cell Biology, 136, 729-743.

[57]   Teng, Z., Ochoa, I., Li, Z., Lin, Y., Rodriguez, J.F., Bea, J.A. and Doblare, M. (2008) Nonlinear mechanical property of tracheal cartilage: A theoretical and experimental study. J Biomech, 41, 1995-2002.

[58]   Miller, T.L., Altman, A.R., Tsuda, T. and Shaffer, T.H. (2007) An ultrasound imaging method for in vivo tracheal bulk and Young’s moduli of elasticity. J Biomech, 40, 1615-1621.

[59]   Rains, J.K., Bert, J.L., Roberts, C.R. and Pare, P.D. (1992) Mechanical properties of human tracheal cartilage. J Appl Physiol, 72, 219-225.

[60]   Jayaraju, S.T., Paiva, M., Brouns, M., Lacor, C. and Verbanck, S. (2008) Contribution of upper airway geometry to convective mixing. J Appl Physiol, 105, 1733-1740.

[61]   Russo, J., Robinson, R. and Oldham, M.J. (2008) Effects of cartilage rings on airflow and particle deposition in the trachea and main bronchi. Med Eng Phys, 30, 581-589.

[62]   Rakesh, V., Rakesh, N.G,. Datta, A.K., Cheetham, J. and Pease, A.P. (2008) Development of equine upper airway fluid mechanics model for Thoroughbred racehorses. Equine Vet J, 40, 272-279.

[63]   Nazridoust, K. and Asgharian, B. (2008) Unsteady-state airflow and particle deposition in a three-generation human lung geometry. Inhal Toxicol, 20, 595-610.

[64]   Isaacs, K.K., Schlesinger, R.B. and Martonen, T.B. (2006) Three-dimensional computational fluid dynamics simulations of particle deposition in the tracheobronchial tree. J Aerosol Med, 19, 344-352.

[65]   Martonen, T.B., Zhang, Z., Yu, G. and Musante, C.J. (2001) Three-dimensional computer modeling of the human upper respiratory tract. Cell Biochem Biophys, 35, 255-261.

[66]   Delaere, P., Vranckx, J., Verleden, G., Leyn, P. and Raemdonck, D. (2010) Tracheal allotransplantation after withdrawal of immunosuppressive therapy. N Engl J Med, 362, 138-145.