Elsevier

Biomaterials

Volume 34, Issue 13, April 2013, Pages 3256-3269
Biomaterials

The effect of age and emphysematous and fibrotic injury on the re-cellularization of de-cellularized lungs

https://doi.org/10.1016/j.biomaterials.2013.01.028Get rights and content

Abstract

Use of de-cellularized cadaveric lungs as 3-dimensional scaffolds for ex vivo lung tissue generation offers a new potential therapeutic approach for clinical lung transplantation. However, it is likely that some of the available cadaveric human lungs may be from older donors or from donors with previously existing structural lung diseases such as emphysema or pulmonary fibrosis. It is not known whether these lungs will be suitable for either de-cellularization or re-cellularization. To investigate this, we assessed the effects of advanced age, representative emphysematous and fibrotic injuries, and the combination of advanced age and emphysematous injury and found significant differences both in histologic appearance and in the retention of extracellular matrix (ECM) and other proteins, as assessed by immunohistochemistry and mass spectrometry, between the different conditions. However, despite these differences, binding, retention and growth of bone marrow-derived mesenchymal stromal cells (MSCs) over a 1-month period following intratracheal inoculation were similar between the different experimental conditions. In contrast, significant differences occurred in the growth of C10 mouse lung epithelial cells between the different conditions. Therefore, age, lung injury, and the cell type used for re-cellularization may significantly impact the usefulness of de-cellularized whole lungs for ex vivo lung tissue regeneration.

Introduction

Approximately 1000–1500 lung transplants per year are performed in the United States, but a significant shortage of suitable donor lungs and the drawbacks of lung transplantation, including lifelong immunosuppression and an approximate 50% 5-year mortality, demonstrate a critical need for new approaches [1]. Although transplantation of cadaveric lungs has not yet been feasible to date, use of de-cellularized whole lung as scaffolds for ex vivo lung bioengineering has recently been investigated as an alternative approach that could potentially allow use of cadaveric lungs seeded with autologous stem or progenitor cells obtained from the eventual transplant recipient [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13].

However, some of the donor lungs that might be utilized for de-cellularization and ex vivo bioengineering may originate from aged donors, donors with pre-existing structural lung diseases, or a combination of both age and lung disease. At present it is unknown how these factors might affect either de-cellularization or subsequent re-cellularization. To assess these questions, we comparatively assessed architecture and ECM content in de-cellularized mouse lungs from young (8–12 weeks) vs. old (15–18 months) mice, lungs from young mice after induction of either emphysematous lung injury following intratracheal inoculation with elastase or of fibrotic injury following intratracheal instillation of bleomycin, or in young mice injured with elastase and allowed to age. We then further assessed growth of two different cell types, murine bone marrow-derived mesenchymal stromal cells (MSCs) and C10 mouse lung epithelial cells following intratracheal inoculation into the different de-cellularized lungs.

Section snippets

Mice

Adult C57BL/6J male mice aged 8–12 weeks (young mice) or 15–18 months (old mice) (Jackson Laboratories), were maintained at UVM in accordance with institutional and American Association for Accreditation of Laboratory Animal Care (AAALAC) standards and review.

Lung de-cellularization

Mice were euthanized by lethal intraperitoneal injection of sodium pentobarbital in accordance with accepted AAALAC standards. After opening the chest, the trachea was cannulated with a blunted 18 gauge Luer-lock syringe. The thymus was

Statistical analyses

Heat maps for the natural log of unique peptide hits for each positively identified protein in the mass spectrometric analyses of lungs de-cellularized under each experimental condition were generated using the ‘pheatmap’ package for ‘R’ statistical software version 2.15.1. Two group comparisons were done using the raw peptide counts (i.e. non-log transformed) using the non-parametric exact permutation test with p < 0.05 considered statistically significant [18]. This non-parametric equivalent

Comparison of de-cellularized lungs obtained from young, old, elastase, and bleomycin-treated mice

As we and others have previously demonstrated, architecture is largely preserved in de-cellularized lungs obtained from young healthy mice, as assessed by H&E, Masson's Trichrome (collagen), and Von Gieson's (elastin) staining (Fig. 1A, [3], [4], [5], [6], [7], [8], [9], [10], [11], [12]). There is an overall decrease in Alcian Blue staining for glycosaminoglycans (GAGS, Fig. 1A and B panels G, H, I, likely reflecting loss of cell associated GAGs [3], [4], [5], [6], [7], [8], [9], [10], [11],

Discussion

Lung transplantation is currently limited by a shortage of available donor lungs and by immune reactions to the transplanted lungs resulting in both acute and chronic rejection with significant morbidity and mortality. To date, use of cadaveric lungs has not been feasible. However, use of de-cellularized whole lung scaffolds may provide a viable option for clinical lung transplantation [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]. As demonstrated by us and others,

Conclusion

These results suggest that de-cellularized lungs obtained from aged lungs may be appropriate for ex vivo lung bioengineering approaches utilizing de-cellularization and re-cellularization strategies. However, a wider range of cell types, including pulmonary vascular endothelial cells and different stem and progenitor cell populations that might be utilized for re-cellularization, need to be tested. Our data suggests that fibrotic lungs support prolonged growth of inoculated cells but whether

Disclosure statement

No competing financial interests exist.

Acknowledgments

The authors gratefully acknowledge the staffs of the Offices of Animal Care Management at the University of Vermont, Bruce Bunnell and Christine Finck for critical reads of the manuscript and Tyler Bittner and Ian Johnson for valuable contributions to the experimental studies. Studies were supported by NIH ARRA RC4HL106625 (D.J.W.), NHLBI R21HL094611 (D.J.W.), UVM Lung Biology Training grant T32 HL076122 from the NHLBI, and UVM Environmental Pathology Training grant T32 ES007122 from the NIEHS.

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