Preservation of micro-architecture and angiogenic potential in a pulmonary acellular matrix obtained using intermittent intra-tracheal flow of detergent enzymatic treatment

Abstract

Tissue engineering of autologous lung tissue aims to become a therapeutic alternative to transplantation. Efforts published so far in creating scaffolds have used harsh decellularization techniques that damage the extracellular matrix (ECM), deplete its components and take up to 5 weeks to perform. The aim of this study was to create a lung natural acellular scaffold using a method that will reduce the time of production and better preserve scaffold architecture and ECM components. Decellularization of rat lungs via the intratracheal route removed most of the nuclear material when compared to the other entry points. An intermittent inflation approach that mimics lung respiration yielded an acellular scaffold in a shorter time with an improved preservation of pulmonary micro-architecture. Electron microscopy demonstrated the maintenance of an intact alveolar network, with no evidence of collapse or tearing. Pulsatile dye injection via the vasculature indicated an intact capillary network in the scaffold. Morphometry analysis demonstrated a significant increase in alveolar fractional volume, with alveolar size analysis confirming that alveolar dimensions were maintained. Biomechanical testing of the scaffolds indicated an increase in resistance and elastance when compared to fresh lungs. Staining and quantification for ECM components showed a presence of collagen, elastin, GAG and laminin. The intratracheal intermittent decellularization methodology could be translated to sheep lungs, demonstrating a preservation of ECM components, alveolar and vascular architecture. Decellularization treatment and methodology preserves lung architecture and ECM whilst reducing the production time to 3 h. Cell seeding and in vivo experiments are necessary to proceed towards clinical translation.

Enhanced Re-Endothelialization of Decellularized Rat Lungs

“…In line with previous reports, 5,24,25 our work demonstrates that following gentle decellularization by detergent perfusion 26 the vascular compartment of the DLS remains largely intact, and capable of supporting perfusion of the entire organ, although some leakage of perfused proteins through FIG. 8.…”

Revascularization of decellularized lung scaffolds: principles and progress

“…However, the same combination of TX and SDC consistently depletes ENs and GAGs (58). Comparing the studies listed Table 1 we conclude that nondenaturing, nonionic detergents (i.e., TX/ SDC) have the least damaging effect on collagens, ENs, and LNs, whereas denaturing and zwitterionic detergents (i.e., SDS and CHAPS) deplete these critical structural ECM/BM proteins in both the vascular and airway compartments.…”

“…1) Morphology shows significant alterations in either the alveolar size or parenchymal volume surrounding the vasculature, but the decellularized organ macroscopically resembles the normal lung tissue (9). 2) Histological analysis shows insignificant loss of collagens but 30 -60% loss of elastin and/or GAGs (58,69).…”

“…Although proteomic analysis of the whole DLS has shown a significant loss of EN (79) following decellularization, histological analysis focused on the vascular compartment suggests that this loss occurs mainly from the airways, pleura, and parenchyma, but not from the vascular compartment: in rodent (21,74,83,93), nonhuman primate (10), and human (119) DLS, the internal elastic lamina of large diameter arteries is maintained. Morphological analysis of the DLS by scanning electron microscopy has shown persistence of hollow tubular structures resembling macrovasculature and, to a lesser extent, also the microvasculature down to the capillary level, thus leaving conduits for blood perfusion largely intact morphologically, yet unable to prevent leakage/hemorrhage (58). Transmission electron microscopic studies following lung decellularization revealed thin, dense ECM structures resembling the respiratory basement membrane as well as cross sections of collagen fibrils supporting the alveolus, indicating fairly faithful preservation of ECM architecture (113).…”

“…In addition, maintaining physiological perfusion pressure will be critical for preserving collagens and EN within the pulmonary vascular tree of bioengineered DLS. Future protocols may also consider significantly shortening the time of detergent exposure, e.g., using intermittent flow regimen (58) in combination with the use of protease inhibitors (128) and/or autologous serum (38), to decrease the loss of critical matrix components within the pulmonary vasculature.…”