Jenna L. Balestrini

Human iPS cell–derived alveolar epithelium repopulates lung extracellular matrix

The use of induced pluripotent stem cells (iPSCs) has been postulated to be the most effective strategy for developing patient-specific respiratory epithelial cells, which may be valuable for lung-related cell therapy and lung tissue engineering. We generated a relatively homogeneous population of alveolar epithelial type II (AETII) and type I (AETI) cells from human iPSCs that had phenotypic properties similar to those of mature human AETII and AETI cells. We used these cells to explore whether lung tissue can be regenerated in vitro. Consistent with an AETII phenotype, we found that up to 97% of cells were positive for surfactant protein C, 95% for mucin-1, 93% for surfactant protein B, and 89% for the epithelial marker CD54. Additionally, exposing induced AETII to a Wnt/β-catenin inhibitor (IWR-1) changed the iPSC-AETII-like phenotype to a predominantly AETI-like phenotype. We found that of induced AET1 cells, more than 90% were positive for type I markers, T1α, and caveolin-1. Acellular lung matrices were prepared from whole rat or human adult lungs treated with decellularization reagents, followed by seeding these matrices with alveolar cells derived from human iPSCs. Under appropriate culture conditions, these progenitor cells adhered to and proliferated within the 3D lung tissue scaffold and displayed markers of differentiated pulmonary epithelium.

Engineering Cellular Response Using Nanopatterned Bulk Metallic Glass

Nanopatterning of biomaterials is rapidly emerging as a tool to engineer cell function. Bulk metallic glasses (BMGs), a class of biocompatible materials, are uniquely suited to study nanopattern–cell interactions as they allow for versatile fabrication of nanopatterns through thermoplastic forming. Work presented here employs nanopatterned BMG substrates to explore detection of nanopattern feature sizes by various cell types, including cells that are associated with foreign body response, pathology, and tissue repair. Fibroblasts decreased in cell area as the nanopattern feature size increased, and fibroblasts could detect nanopatterns as small as 55 nm in size. Macrophages failed to detect nanopatterns of 150 nm or smaller in size, but responded to a feature size of 200 nm, resulting in larger and more elongated cell morphology. Endothelial cells responded to nanopatterns of 100 nm or larger in size by a significant decrease in cell size and elongation. On the basis of these observations, nondimensional analysis was employed to correlate cellular morphology and substrate nanotopography. Analysis of the molecular pathways that induce cytoskeletal remodeling, in conjunction with quantifying cell traction forces with nanoscale precision using a unique FIB-SEM technique, enabled the characterization of underlying biomechanical cues. Nanopatterns altered serum protein adsorption and effective substrate stiffness, leading to changes in focal adhesion density and compromised activation of Rho-A GTPase in fibroblasts. As a consequence, cells displayed restricted cell spreading and decreased collagen production. These observations suggest that topography on the nanoscale can be designed to engineer cellular responses to biomaterials.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Extracellular Matrix as a Driver for Lung Regeneration

Extracellular matrix has manifold roles in tissue mechanics, guidance of cellular behavior, developmental biology, and regenerative medicine. Over the past several decades, various pre-clinical and clinical studies have shown that many connective tissues may be replaced and/or regenerated using suitable extracellular matrix scaffolds. More recently, decellularization of lung tissue has shown that gentle removal of cells can leave behind a “footprint” within the matrix that may guide cellular adhesion, differentiation and homing following cellular repopulation. Fundamental issues like understanding matrix composition and micro-mechanics remain difficult to tackle, largely because of a lack of available assays and tools for systematically characterizing intact matrix from tissues and organs. This review will critically examine the role of engineered and native extracellular matrix in tissue and lung regeneration, and provide insights into directions for future research and translation.

Netrin‐1 Regulates Fibrocyte Accumulation in the Decellularized Fibrotic Sclerodermatous Lung Microenvironment and in Bleomycin‐Induced Pulmonary Fibrosis

Fibrocytes are collagen‐producing leukocytes that accumulate in patients with systemic sclerosis (SSc; scleroderma)–related interstitial lung disease (ILD) via unknown mechanisms that have been associated with altered expression of neuroimmune proteins. The extracellular matrix (ECM) influences cellular phenotypes. However, a relationship between the lung ECM and fibrocytes in SSc has not been explored. The aim of this study was to use a novel translational platform based on decellularized human lungs to determine whether the lung ECM of patients with scleroderma controls the development of fibrocytes from peripheral blood mononuclear cells.

Sterilization of lung matrices by supercritical carbon dioxide

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO2) that can achieve a sterility assurance level 10(-6) in decellularized lung matrix. The effects of ScCO2 treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO2 did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO2, indicating that ScCO2 produces a matrix that is stable during storage. The current studys results indicate that ScCO2 can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes.

Biaxial Stretch Improves Elastic Fiber Maturation, Collagen Arrangement, and Mechanical Properties in Engineered Arteries.

Tissue-engineered blood vessels (TEVs) are typically produced using the pulsatile, uniaxial circumferential stretch to mechanically condition and strengthen the arterial grafts. Despite improvements in the mechanical integrity of TEVs after uniaxial conditioning, these tissues fail to achieve critical properties of native arteries such as matrix content, collagen fiber orientation, and mechanical strength. As a result, uniaxially loaded TEVs can result in mechanical failure, thrombus, or stenosis on implantation. In planar tissue equivalents such as artificial skin, biaxial loading has been shown to improve matrix production and mechanical properties. To date however, multiaxial loading has not been examined as a means to improve mechanical and biochemical properties of TEVs during culture. Therefore, we developed a novel bioreactor that utilizes both circumferential and axial stretch that more closely simulates loading conditions in native arteries, and we examined the suture strength, matrix production, fiber orientation, and cell proliferation. After 3 months of biaxial loading, TEVs developed a formation of mature elastic fibers that consisted of elastin cores and microfibril sheaths. Furthermore, the distinctive features of collagen undulation and crimp in the biaxial TEVs were absent in both uniaxial and static TEVs. Relative to the uniaxially loaded TEVs, tissues that underwent biaxial loading remodeled and realigned collagen fibers toward a more physiologic, native-like organization. The biaxial TEVs also showed increased mechanical strength (suture retention load of 303 ± 14.53 g, with a wall thickness of 0.76 ± 0.028 mm) and increased compliance. The increase in compliance was due to combinatorial effects of mature elastic fibers, undulated collagen fibers, and collagen matrix orientation. In conclusion, biaxial stretching is a potential means to regenerate TEVs with improved matrix production, collagen organization, and mechanical properties.

Biomimetic Culture Reactor for Whole-Lung Engineering.

Abstract Decellularized organs are now established as promising scaffolds for whole-organ regeneration. For this work to reach therapeutic practice, techniques and apparatus are necessary for doing human-scale clinically applicable organ cultures. We have designed and constructed a bioreactor system capable of accommodating whole human or porcine lungs, and we describe in this study relevant technical details, means of assembly and operation, and validation. The reactor has an artificial diaphragm that mimics the conditions found in the chest cavity in vivo, driving hydraulically regulated negative pressure ventilation and custom-built pulsatile perfusion apparatus capable of driving pressure-regulated or volume-regulated vascular flow. Both forms of mechanical actuation can be tuned to match specific physiologic profiles. The organ is sealed in an elastic artificial pleura that mounts to a support architecture. This pleura reduces the fluid volume required for organ culture, maintains the organs position during mechanical conditioning, and creates a sterile barrier allowing disassembly and maintenance outside of a biosafety cabinet. The combination of fluid suspension, negative-pressure ventilation, and physiologic perfusion allows the described system to provide a biomimetic mechanical environment not found in existing technologies and especially suited to whole-organ regeneration. In this study, we explain the design and operation of this apparatus and present data validating intended functions.

New Functional Tools for Antithrombogenic Activity Assessment of Live Surface Glycocalyx

Objective—It is widely accepted that the presence of a glycosaminoglycan-rich glycocalyx is essential for endothelialized vasculature health; in fact, a damaged or impaired glycocalyx has been demonstrated in many vascular diseases. Currently, there are no methods that characterize glycocalyx functionality, thus limiting investigators’ ability to assess the role of the glycocalyx in vascular health. Approach and Results—We have developed novel, easy-to-use, in vitro assays that directly quantify live endothelialized surface’s functional heparin weights and their anticoagulant capacity to inactivate Factor Xa and thrombin. Using our assays, we characterized 2 commonly used vascular models: native rat aorta and cultured human umbilical vein endothelial cell monolayer. We determined heparin contents to be ≈10 000 ng/cm2 on the native aorta and ≈10-fold lower on cultured human umbilical vein endothelial cells. Interestingly, human umbilical vein endothelial cells demonstrated a 5-fold lower anticoagulation capacity in inactivating both Factor Xa and thrombin relative to native aortas. We verified the validity and accuracy of the novel assays developed in this work using liquid chromatography–mass spectrometry analysis. Conclusions—Our assays are of high relevance in the vascular community because they can be used to establish the antithrombogenic capacity of many different types of surfaces such as vascular grafts and transplants. This work will also advance the capacity for glycocalyx-targeting therapeutics development to treat damaged vasculatures.

Extracellular matrix in lung development, homeostasis and disease

The lungs unique extracellular matrix (ECM), while providing structural support for cells, is critical in the regulation of developmental organogenesis, homeostasis and injury-repair responses. The ECM, via biochemical or biomechanical cues, regulates diverse cell functions, fate and phenotype. The composition and function of lung ECM become markedly deranged in pathological tissue remodeling. ECM-based therapeutics and bioengineering approaches represent promising novel strategies for regeneration/repair of the lung and treatment of chronic lung diseases. In this review, we assess the current state of lung ECM biology, including fundamental advances in ECM composition, dynamics, topography, and biomechanics; the role of the ECM in normal and aberrant lung development, adult lung diseases and autoimmunity; and ECM in the regulation of the stem cell niche. We identify opportunities to advance the field of lung ECM biology and provide a set recommendations for research priorities to advance knowledge that would inform novel approaches to the pathogenesis, diagnosis, and treatment of chronic lung diseases.