Engineering Functional Vasculature in Decellularized Lungs Depends on Comprehensive Endothelial Cell Tropism.

Tissue engineering using decellularized whole lungs as matrix scaffolds began as a promise for creating autologous transplantable lungs for patients with end-stage lung disease and can also be used to study strategies for lung regeneration. Vascularization remains a critical component for all solid organ bioengineering, yet there has been limited success in generating functional re-endothelialization of most pulmonary vascular segments. We evaluated recellularization of the blood vessel conduits of acellular mouse scaffolds with highly proliferating, rat pulmonary microvascular endothelial progenitor cells (RMEPCs), pulmonary arterial endothelial cells (PAECs) or microvascular endothelial cells (MVECs). After 8 days of pulsatile perfusion, histological analysis showed that PAECs and MVECs possessed selective tropism for larger vessels or microvasculature, respectively. In contrast, RMEPCs lacked site preference and repopulated all vascular segments. RMEPC-derived endothelium exhibited thrombomodulin activity, expression of junctional genes, ability to synthesize endothelial signaling molecules, and formation of a restrictive barrier. The RMEPC phenotype described here could be useful for identifying endothelial progenitors suitable for efficient vascular organ and tissue engineering, regeneration and repair.

Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

Automated decellularization of intact, human-sized lungs for tissue engineering.

We developed an automated system that can be used to decellularize whole human-sized organs and have shown lung as an example. Lungs from 20 to 30 kg pigs were excised en bloc with the trachea and decellularized with our established protocol of deionized water, detergents, sodium chloride, and porcine pancreatic DNase. A software program was written to control a valve manifold assembly that we built for selection and timing of decellularization fluid perfusion through the airway and the vasculature. This system was interfaced with a prototypic bioreactor chamber that was connected to another program, from a commercial source, which controlled the volume and flow pressure of fluids. Lung matrix that was decellularized by the automated method was compared to a manual method previously used by us and others. Automation resulted in more consistent acellular matrix preparations as demonstrated by measuring levels of DNA, hydroxyproline (collagen), elastin, laminin, and glycosaminoglycans. It also proved highly beneficial in saving time as the decellularization procedure was reduced from days down to just 24 h. Developing a rapid, controllable, automated system for production of reproducible matrices in a closed system is a major step forward in whole-organ tissue engineering.

MSC therapy attenuates obliterative bronchiolitis after murine bone marrow transplant.

RATIONALE: Obliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease. OBJECTIVE: Administration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model. METHODS: Mice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT. MEASUREMENTS AND MAIN RESULTS: No ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206. CONCLUSIONS: These results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.

Evidence for early fibrosis and increased airway resistance in bone marrow transplant recipient mice deficient in MMP12.

Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality post-bone marrow transplantation (BMT) in humans. In our established murine IPS model in which lethally conditioned recipients are given allogeneic bone marrow and splenocytes, recruitment of host monocytes occurs early post-BMT, followed by donor T cells concomitant with development of severe lung dysfunction. Because matrix metalloproteinase 12 (MMP12) is important for macrophage infiltration and injury in other mouse models of lung disease such as emphysema, lethally conditioned MMP12(-/-) mice were used as allogeneic recipients to determine whether MMP12 plays a similar role in potentiating lung injury in IPS. Surprisingly, MMP12(-/-) mice developed IPS and exhibited an accelerated allogeneic T cell-dependent decrease in compliance compared with wild-type (WT) recipients. MMP12(-/-), but not WT, mice also had allogeneic T cell-dependent elevated lung resistance post-BMT. Recruitment of monocytes and T cells into the lungs was not altered on day 7 post-BMT, but the lungs of MMP12(-/-) recipients had increased collagen deposition, a feature normally not seen in our IPS model. MMP12(-/-) mice had a compensatory increase in MMP2 in the lungs post-BMT, as well as increased ß6-integrin compared with WT recipients, and only in the presence of allogeneic T cells. Levels of total transforming growth factor (TGF)-ß1 protein in the lungs were elevated compared with WT recipients, consistent with the profibrotic function of ß6-integrin as an activator of TGF-ß. These data indicate that host-derived MMP12 may be important in limiting development of IPS by allowing proper remodeling of extracellular matrix and effective repair of BMT-related injury.

Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.

We developed a decellularized murine lung matrix bioreactor system that could be used to evaluate the potential of stem cells to regenerate lung tissue. Lungs from 2-3-month-old C57BL/6 female mice were excised en bloc with the trachea and heart, and decellularized with sequential solutions of distilled water, detergents, NaCl, and porcine pancreatic DNase. The remaining matrix was cannulated and suspended in small airway growth medium, attached to a ventilator to simulate normal, murine breathing-induced stretch. After 7 days in an incubator, lung matrices were analyzed histologically. Scanning electron microscopy and histochemical staining demonstrated that the pulmonary matrix was intact and that the geographic placement of the proximal and distal airways, alveoli and vessels, and the basement membrane of these structures all remained intact. Decellularization was confirmed by the absence of nuclear 4',6-diamidino-2-phenylindole staining and negative polymerase chain reaction for genomic DNA. Collagen content was maintained at normal levels. Elastin, laminin, and glycosaminglycans were also present, although at lower levels compared to nondecellularized lungs. The decellularized lung matrix bioreactor was capable of supporting growth of fetal alveolar type II cells. Analysis of day 7 cryosections of fetal-cell-injected lung matrices showed pro-Sp-C, cytokeratin 18, and 4',6-diamidino-2-phenylindole-positive cells lining alveolar areas that appeared to be attached to the matrix. These data illustrate the potential of using decellularized lungs as a natural three-dimensional bioengineering matrix as well as provide a model for the study of lung regeneration from pulmonary stem cells.

A new murine model for bronchiolitis obliterans post-bone marrow transplant.

RATIONALE: Bronchiolitis obliterans (BO) is a major problem in lung transplantation and is also part of the spectrum of late-onset pulmonary complications that can occur after hematopoietic stem cell transplant. Better mouse models are needed to study the onset of this disease so that therapeutic interventions can be developed. OBJECTIVES: Our goal was to develop a BO mouse model. METHODS: Recipients were lethally conditioned and given a rescue dose of T-cell-depleted, allogeneic bone marrow (BM) supplemented with a sublethal dose of allogeneic T cells. MEASUREMENTS AND MAIN RESULTS: At 2 months post-BM transplant, the lungs had extensive perivascular and peribronchiolar inflammation consisting of CD4(+) T cells, CD8(+) T cells, B cells, macrophages, neutrophils, and fibroblasts. In contrast to the acute model, histology showed airway obstruction consistent with BO. Epithelial cells of airways in the early stages of occlusion exhibited changes in expression of cytokeratins. Although the lung had severe allogeneic BM transplant-mediated disease, there was only mild to moderate graft-versus-host disease in liver, colon, skin, and spleen. High wet/dry weight ratios and elevated hydroxyproline were seen, consistent with pulmonary edema and fibrosis. Mice with BO exhibited high airway resistance and low compliance. Increases in many inflammatory mediators in the lungs of mice that develop BO were seen early post-transplant and not later at the time of BO. CONCLUSIONS: This new mouse model will be useful for the study of BO associated with late post-hematopoietic stem cell transplant onset and chronic graft-versus-host disease, which also leads to poor outcome in the lung transplant setting.