Hypertensive Rat Lungs Retain Hallmarks of Vascular Disease upon Decellularization but Support the Growth of Mesenchymal Stem Cells

Abstract

There are an insufficient number of donor organs available to meet the demand for lung transplantation. This issue could be addressed by regenerating functional tissue from diseased or damaged lungs that would otherwise be deemed unsuitable for transplant. Detergent-mediated whole-lung decellularization produces a threedimensional natural scaffold that can be repopulated with various cell types. In this study, we investigated the decellularization and initial recellularization of diseased lungs using a rat model of monocrotaline-induced pulmonary hypertension (MCT-PHT). Decellularization of control and MCT-PHT Sprague-Dawley rat lungs was accomplished by treating the lungs with a combination of Triton X-100, sodium deoxycholate, NaCl, and DNase. The resulting acellular matrices were characterized by DNA quantification, Western blotting, immunohistochemistry, and proteomic analyses revealing that decellularization was able to remove cells while leaving the extracellular matrix (ECM) components and lung ultrastructure intact. Decellularization significantly reduced DNA content (*30-fold in MCT-PHT lungs and *50-fold in the control lungs) and enriched ECM components ( > 60-fold in both the control and MCT-PHT lungs) while depleting cellular proteins. MicroCT visualization of MCT-PHT rat lungs indicated that the vasculature was narrowed as a result of MCT treatment, and this characteristic was unchanged by decellularization. Mean arterial vessel diameter of representative decellularized MCT-PHT and control scaffolds was estimated to be 0.152 -0.134 mm and 0.247 -0.160 mm, respectively. Decellularized MCT-PHT lung scaffolds supported attachment and survival of rat adipose-derived stem cells (rASCs), seeded into the airspace or the vasculature, for at least 2 weeks. The cells seeded in MCT-PHT lung scaffolds proliferated and underwent apoptosis similar to control scaffolds; however, the initial percentage of apoptotic cells was slightly higher in MCT-PHT lungs (2.79 -2.03% vs. 1.05 -1.02% of airway-seeded rASCs, and 4.47 -1.21% vs. 2.66 -0.10% of vascular seeded rASCs). The ECM of cell-seeded scaffolds showed no signs of degradation by the cells after 14 days in culture. These data suggest that diseased hypertensive lungs can be efficiently decellularized similar to control lungs and have the potential to be recellularized with mesenchymal stem cells with the ultimate goal of generating healthy, functional pulmonary tissue.

Revascularization of decellularized lung scaffolds: principles and progress

“…Hence, rather than/in lieu of optimizing the decellularization techniques, replenishment of essential, cell-instructive matricellular ECM components might be an alternative approach for countering ECM loss and improving functional recellularization (52). The general maintenance of the morphology of the lung vascular ECM network following decellularization has been reported, especially for the macrovasculature (10,21,74,83,93,119). As detailed earlier, in intact lung tissue one can utilize histological stains such as Verhoeff's van Gieson, which stains elastic fibers, to identify large diameter arteries from large-diameter veins and airways by the presence of a thick internal elastic lamina in the arteries and its low abundance in the veins and airways (61).…”

“…Unlike the vessels with diameter greater than 100 m, the structure of small diameter arterioli and venules is similar and difficult to distinguish without highly specific histological techniques (61). 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).…”

“…However, the physiological impact of changes in the precise composition and micromechanical properties of the decellularized scaffolds on the functionality of recellularized engineered organs remains poorly understood and will require further mechanistic studies looking at survival, proliferation, differentiation, and functionality of the cells seeded into the various DLS (113). Decellularized rodent lungs can be perfused throughout the entire pulmonary vascular tree, albeit leakage is observed at all levels of the vasculature, as shown with vascular casting visualized with micro-CT imaging (80,93) or dye perfusion visualized with real-time imaging [Evans blue dye (21) or Trypan blue dye (58)]. Current analysis methods such as Western blotting, high-pressure liquid chromatography, and mass spectroscopy do not distinguish between the ECM/BM of airways and that of the vascular compartments but rather provide global assessments of the lung ECM protein content and localization.…”

“…For this purpose, MSCs (9), primary somatic cells (120), or ESCs (69) were delivered either via the airway or vascular network and left in situ under static conditions in the absence of perfusion for no more than 24 h. Thereafter the tissue was sectioned and further cultured in vitro to assess anatomical reconstitution of the two physiological compartments. The whole DLS was seeded via tracheal instillation or vascular perfusion, each driven by gravity (27) or by hand (93). Instillation by gravity allows for control of perfusion pressure and flow rate into the lung, whereas instillation by hand (referred to in Table 1 as "manually") lacks such control and therefore could lead to high variability in cell distribution throughout the lungs.…”

Coming to terms with tissue engineering and regenerative medicine in the lung

“…MSCs within lungs have also been identified that have the ability to express markers of ATI and ATII cells (73,95,214). In this regard, a particularly relevant finding is that MSCs may be less finicky toward the scaffold milieu in terms of attachment (19,21,170,180,213,217). However, what is not yet established is whether MSCs fully redifferentiate into specific lung cell types within a scaffold and recapitulate the functions of the native cell type.…”