Decellularized lung tissue has been recognized as a potential platform to engineer whole lung organs suitable for transplantation or for modeling a variety of lung diseases. However many technical hurdles remain before this potential may be fully realized. Inability to efficiently re-endothelialize the pulmonary vasculature with a functional endothelium appears to be the primary cause of failure of recellularized lung scaffolds in early transplant studies. This dissertation research aims to enhance the re-endothelialization of decellularized rodent lung scaffolds with rat lung microvascular endothelial cells. This was achieved by adjusting the posture of the lung to a supine position during cell seeding through the pulmonary artery. The supine position allowed for significantly more homogeneous seeding and better cell retention in the apex regions of all lobes than the traditional upright position, especially in the right upper and left lobes. Additionally, the supine position allowed for greater cell retention within large diameter vessels (proximal – 100 µm to 5,000 µm) than the upright position, with little to no difference in the small diameter distal vessels. Endothelial cell adhesion in the proximal regions of the pulmonary vasculature in the decellularized lung was dependent on the binding of endothelial cell integrins, specifically α1β1, α2β1 and α5β1 integrins to, respectively, collagen type-I, type-IV and fibronectin in the residual ECM. Following in vitro maturation of the seeded constructs under perfusion culture, the seeded endothelial cells spread along the vascular wall, leading to a partial re-establishment of endothelial barrier function as inferred from a custom-designed leakage assay. The results of this dissertation research suggest that attention to cellular distribution within the whole organ is of paramount importance for restoring proper vascular function. / Bioengineering

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13634号 / 論医博第2325号 / 新制||医||1074(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 尾野 亘, 教授 伊達 洋至, 教授 安達 泰治 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Chronic obstructive pulmonary disease (COPD) including emphysema is a devastating condition, increasing in prevalence in the US and worldwide. There remains no cure for COPD, rather only symptomatic treatments. Due to unique challenges of the lung, translation of therapies for acute lung injury to target chronic lung diseases like COPD has not been successful. We have been investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. During the course of this work we have developed and characterized a lug derived ECM hydrogel that exhibits “injectability,” allowing cells or dugs to be delivered in a liquid and encapsulated at body temperature. The hydrogel self assembles in <5 minutes and achieves mechanical stiffness similar to other soft tissue ECM hydrogels. The hydrogel can support 3D cell growth and encapsulated cell viability. Encapsulated hMSCs can also still be activated by simulated inflammatory environments. Naïve mouse macrophages exposed to the fully formed gel were not significantly induced to express markers for pro or anti-inflammatory polarized phenotypes, but increased expression for several secreted inflammatory mediators was observed. We also investigated a novel approach for preparing and solubilizing the isolated ECM proteins, using digestion time as a variable for controlling hydrogel density (interconnectivity), mechanical stiffness, component protein size distribution, and cell behavior on fully formed gels. The potential future impact for the presented research includes optimization for future animal studies, expansion to additional applications, and the development of new derivative materials.

In vivo, adipose tissue demonstrates only a limited capacity for self-repair, and the long-term treatment of subcutaneous defects remains an unresolved clinical problem. With the goal of regenerating healthy tissues, many tissue-engineering strategies have pointed to the potential of implementing three-dimensional (3-D), cell-seeded scaffolds for soft tissue augmentation and wound healing. In particular, microcarriers have shown promise as both cell expansion substrates and injectable cell-delivery vehicles for these applications. However, limited research has investigated the engineering of tissue-specific microcarriers, designed to closely mimic the native extracellular matrix (ECM) composition. In this work, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via non-cytotoxic protocols. Characterization by microscopy confirmed the efficacy of the fabrication protocols in producing stable beads, as well as the production of a microporous surface topography. The mean bead diameter was 934 ± 51 μm, while the porosity was measured to be 29 ± 4 % using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT-based microcarriers were effectively stabilized with the non-cytotoxic photochemical crosslinking agent rose bengal, with only low levels of protein release measured within a simulated physiological environment. In cell-based studies, the DAT-based microcarriers successfully supported the proliferation and adipogenic differentiation of human adipose-derived stem cells (hASCs) in a dynamic spinner flask system, with a more favorable response observed in terms of adhesion, proliferation, and adipogenesis on the DAT-based microcarriers relative to gelatin control beads. More specifically, dynamically-cultured hASCs on DAT-based microcarriers demonstrated greater lipid loading, as well as higher glycerol-3-phosphate dehydrogenase (GPDH) activity, a key enzyme involved in triacylglycerol biosynthesis, at 7 days and 14 days in culture in an inductive medium. Overall, the results indicated that the DAT-based microcarriers provided a uniquely supportive environment for adipogenesis. Established microcarrier sterility and injectability further support the broad potential of these tissue-specific microcarriers as a novel, adipogenic, clinically-translatable strategy for soft tissue engineering. / Thesis (Master, Chemical Engineering) -- Queen's University, 2010-12-01 14:28:14.628

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