Spectroscopic Analysis of Human Tracheal Tissue during Decellularization

Abstract

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.

Development and Characterization of a Porcine Liver Scaffold

“…However, FTIR can assess the composition of the extracellular matrix [31] and can produce standardised repeatable results. To date, it has been used to assess the ECM finger print in fibrocartilage bio-scaffold for bone-tendon interface [32] and in the evaluation of de-cellularised human tracheal samples [33]. It has not to our knowledge been used to determine the presence of any residual detergent or enzymes remaining in biological tissue following de-cellularisation.…”

Tissue-Engineered Grafts from Human Decellularized Extracellular Matrices: A Systematic Review and Future Perspectives

“…In fact, as regards tracheal cartilage, Macchiarini et al [28], Gonfiotti et al [37], Baiguera et al [38], and Elliott et al [30] experienced a detergent enzymatic approach by means of 4% sodium deoxycholate, dH 2 O, and 2000 KU (Kunitz Units) DNase-I in 1 M NaCl followed by further washes in dH 2 O. A slight change to this protocol was recently reported; in fact Tint et al [39] worked with tracheas which were previously freezed in liquid nitrogen and rinsed in a solution of 2 mM CaCl 2 and 1.3 mM MgSO 4 after DNase treatment. However, in all the reported experiences, a complete decelluarization of human trachea required several cycles for a period of three to eight weeks.…”

Optimization of a decellularization protocol of porcine tracheas. Long-term effects of cryopreservation. A histological study

“…Among them, 4% sodium deoxycholate (SDC) in combination with DNase is the most commonly used. [4][5][6][7][8][9][10][11][12] Some authors have reported acceptable degrees of decellularization using other detergents such as sodium dodecyl sulfate (SDS), Triton X-100 or 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) alone or in combination. [13][14][15][16][17][18][19][20] However, in many cases the authors have directly extrapolated the decellularization protocols reported in the literature, without considering important aspects such as differences in the composition of the extracellular matrix that exist between species.…”