Currently, there is no adequate treatment available for the replacement of tracheal segments larger than 6 cm. The goal of these studies is to develop and fabricate a functional trachea replacement in a rabbit model using tissue engineering principles, and to also fabricate a human-sized neotrachea using human cells in an athymic rat model. This laboratory has developed the methodology to produce large scaffold-free cartilage sheets, which when implanted in vivo and surrounded by fascia or muscle, produces a vascularized neotracheal construct. Rabbit ear and shoulder chondrocytes will be tested and optimized for cartilage sheet production and will be tested for their long-term stability and function in a trachea segmental defect model. The sheets are first fabricated in a custom double diffusion bioreactor and then implanted proximal to the future segmental repair site in the trachea where it is allowed to mature for approximately 12 weeks prior to segmental tracheal reconstruction. The neotrachea is transplanted along its vasculature into segmental tracheal defect and will be harvested at 4, 8 and 12 weeks postimplantation. Three distinct surgical formats will be tested including the direct transplant alone, transplant with a T-tube (to assist breathing and promote repair), and transplant with T-tube along with cheek mucosal free transplant. The harvested neotracheas are assessed by histologic and immunohistochemical methods including staining for glycosaminoglycans, and immunohistochemistry for collagens type I, II, and X, and elastin, examined, histologically, for structure, tissue integration, epithelialization, and are evaluated for biomechanical strength. Particular attention will be paid to the fibrosis, the formation of a mucous membrane, and also whether bone forms within the neotrachea. Alternative approaches to apply epithelial cells to the engineered tissue may be employed. The long-term goals are to develop the methodology to produce a functional trachea to repair segmental defects in rabbits, and to translate this technology to human derived cells. To this end, in parallel with the optimization study of rabbit chondrocytes, human chondrocytes will be obtained from the trachea, ear, nose and from articular surfaces and will be optimized for cartilage sheet formation by first testing a bank of growth factors and cytokines using a chondrogenesis aggregate culture assay system. Human chondrocyte-derived sheets will then be tested in an athymic rat model for their ability to form a human-sized neotrachea in vivo, and will be assessed by the histologic, biochemical and mechanical assays.

Public Health Relevance

Currently, there is no adequate treatment for large (6 cm or greater) trachea defects. The focus of this project is to use tissue engineering principles to fabricate a neo-trachea that can repair segmental defects in rabbits and to then apply these principles to fabricate a neo-trachea using human chondrocytes. The long-term goal this project seeks to develop a tissue engineering method to repair damaged tracheas in people.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE015322-09
Application #
8385489
Study Section
Musculoskeletal Tissue Engineering Study Section (MTE)
Program Officer
Lumelsky, Nadya L
Project Start
2003-07-01
Project End
2014-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
9
Fiscal Year
2013
Total Cost
$427,442
Indirect Cost
$189,842
Name
Benaroya Research Institute at Virginia Mason
Department
Type
DUNS #
076647908
City
Seattle
State
WA
Country
United States
Zip Code
98101
Kean, Thomas J; Mera, Hisashi; Whitney, G Adam et al. (2016) Disparate response of articular- and auricular-derived chondrocytes to oxygen tension. Connect Tissue Res 57:319-33
Whitney, G A; Mansour, J M; Dennis, J E (2015) Coefficient of Friction Patterns Can Identify Damage in Native and Engineered Cartilage Subjected to Frictional-Shear Stress. Ann Biomed Eng 43:2056-68
Kean, Thomas J; Dennis, James E (2015) Synoviocyte Derived-Extracellular Matrix Enhances Human Articular Chondrocyte Proliferation and Maintains Re-Differentiation Capacity at Both Low and Atmospheric Oxygen Tensions. PLoS One 10:e0129961
Whitney, G Adam; Jayaraman, Karthik; Dennis, James E et al. (2014) Scaffold-free cartilage subjected to frictional shear stress demonstrates damage by cracking and surface peeling. J Tissue Eng Regen Med :
Whitney, G Adam; Mera, Hisashi; Weidenbecher, Mark et al. (2012) Methods for producing scaffold-free engineered cartilage sheets from auricular and articular chondrocyte cell sources and attachment to porous tantalum. Biores Open Access 1:157-65
Henderson, James H; Ginley, Nell M; Caplan, Arnold I et al. (2010) Low oxygen tension during incubation periods of chondrocyte expansion is sufficient to enhance postexpansion chondrogenesis. Tissue Eng Part A 16:1585-93
Gilpin, David A; Weidenbecher, Mark S; Dennis, James E (2010) Scaffold-free tissue-engineered cartilage implants for laryngotracheal reconstruction. Laryngoscope 120:612-7
van Osch, Gerjo J V M; Brittberg, Mats; Dennis, James E et al. (2009) Cartilage repair: past and future--lessons for regenerative medicine. J Cell Mol Med 13:792-810
Weidenbecher, Mark; Tucker, Harvey M; Awadallah, Amad et al. (2008) Fabrication of a neotrachea using engineered cartilage. Laryngoscope 118:593-8
Weidenbecher, Mark; Henderson, James H; Tucker, Harvey M et al. (2007) Hyaluronan-based scaffolds to tissue-engineer cartilage implants for laryngotracheal reconstruction. Laryngoscope 117:1745-9

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