Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery · 2018
Spectroscopic Analysis of Human Tracheal Tissue during Decellularization.
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Tint Derrick, Stabler Collin T, Hanifi Arash, Yousefi Farzad, Linkov Gary, Hy Kenneth, Soliman Ahmed M S, Pleshko Nancy
Аннотация
OBJECTIVE: To use mid-infrared (IR) spectroscopy to assess changes in the cartilaginous framework of human trachea during decellularization.
STUDY DESIGN: Laboratory-based study.
SETTING: Research laboratory.
METHODS: Six cadaveric human tracheas were decellularized using a detergent enzymatic method (DEM). Tissue samples were obtained from each specimen after 0, 1, 10, and 25 DEM cycles for histologic and spectroscopic analysis. Decellularization was confirmed using hematoxylin and eosin (H&E) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) staining. Changes in cartilaginous framework were examined using Fourier transform infrared imaging spectroscopy (FT-IRIS) and an attenuated total reflectance (ATR) probe in the mid-IR frequencies. Results were statistically analyzed using 1-way analysis of variance (ANOVA) and principal component analysis (PCA).
RESULTS: Six decellularized tracheal scaffolds were successfully created using a DEM protocol. Histologic examination showed near-complete nuclear loss following 25 DEM cycles. As observed with FT-IRIS analysis, the collagen absorbance signal (1336 cm-1) was predominantly in the perichondria and remained stable after 25 DEM cycles ( P = .132), while the absorbance from sugar rings in proteoglycans and nucleic acids in hyaline cartilage (1080 cm-1) showed a significant decrease after 1 DEM cycle ( P = .0007). Examination of the luminal surface of the trachea with an ATR probe showed raw mid-IR spectra consistent with cartilage. PCA showed significant separation of spectra corresponding to treatment cycle along the principal components 1 and 2.
CONCLUSION: Mid-IR spectroscopy is a viable method of monitoring changes in extracellular matrix components during the decellularization of human trachea.
STUDY DESIGN: Laboratory-based study.
SETTING: Research laboratory.
METHODS: Six cadaveric human tracheas were decellularized using a detergent enzymatic method (DEM). Tissue samples were obtained from each specimen after 0, 1, 10, and 25 DEM cycles for histologic and spectroscopic analysis. Decellularization was confirmed using hematoxylin and eosin (H&E) and 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) staining. Changes in cartilaginous framework were examined using Fourier transform infrared imaging spectroscopy (FT-IRIS) and an attenuated total reflectance (ATR) probe in the mid-IR frequencies. Results were statistically analyzed using 1-way analysis of variance (ANOVA) and principal component analysis (PCA).
RESULTS: Six decellularized tracheal scaffolds were successfully created using a DEM protocol. Histologic examination showed near-complete nuclear loss following 25 DEM cycles. As observed with FT-IRIS analysis, the collagen absorbance signal (1336 cm-1) was predominantly in the perichondria and remained stable after 25 DEM cycles ( P = .132), while the absorbance from sugar rings in proteoglycans and nucleic acids in hyaline cartilage (1080 cm-1) showed a significant decrease after 1 DEM cycle ( P = .0007). Examination of the luminal surface of the trachea with an ATR probe showed raw mid-IR spectra consistent with cartilage. PCA showed significant separation of spectra corresponding to treatment cycle along the principal components 1 and 2.
CONCLUSION: Mid-IR spectroscopy is a viable method of monitoring changes in extracellular matrix components during the decellularization of human trachea.