A tri-layer decellularized, dehydrated human amniotic membrane scaffold supports the cellular functions of human tenocytes in vitro.

Differences in scaffold design have the potential to influence cell-scaffold interactions. This study sought to determine whether a tri-layer design influences the cellular function of human tenocytes in vitro. The single-layer decellularized, dehydrated human amniotic membrane (DDHAM) and the tri-layer DDHAM (DDHAM-3L) similarly supported tenocyte function as evidenced by improved cell growth and migration, reduced dedifferentiation, and an attenuated inflammatory response. The tri-layer design provides a mechanically more robust scaffold without altering biological activity.

Modulation of Synthetic Tracheal Grafts with Extracellular Matrix Coatings.

Synthetic scaffolds for the repair of long-segment tracheal defects are hindered by insufficient biocompatibility and poor graft epithelialization. In this study, we determined if extracellular matrix (ECM) coatings improved the biocompatibility and epithelialization of synthetic tracheal grafts (syn-TG). Porcine and human ECM substrates (pECM and hECM) were created through the decellularization and lyophilization of lung tissue. Four concentrations of pECM and hECM coatings on syn-TG were characterized for their effects on scaffold morphologies and on in vitro cell viability and growth. Uncoated and ECM-coated syn-TG were subsequently evaluated in vivo through the orthotopic implantation of segmental grafts or patches. These studies demonstrated that ECM coatings were not cytotoxic and, enhanced the in vitro cell viability and growth on syn-TG in a dose-dependent manner. Mass spectrometry demonstrated that fibrillin, collagen, laminin, and nephronectin were the predominant ECM components transferred onto scaffolds. The in vivo results exhibited similar robust epithelialization of uncoated and coated syn-TG patches; however, the epithelialization remained poor with either uncoated or coated scaffolds in the segmental replacement models. Overall, these findings demonstrated that ECM coatings improve the seeded cell biocompatibility of synthetic scaffolds in vitro; however, they do not improve graft epithelialization in vivo.

Development of alginate and gelatin-based pleural and tracheal sealants.

Pleural and tracheal injuries remain significant problems, and an easy to use, effective pleural or tracheal sealant would be a significant advance. The major challenges are requirements for adherence, high strength and elasticity, dynamic durability, appropriate biodegradability, and lack of cell or systemic toxicity. We designed and evaluated two sealant materials comprised respectively of alginate methacrylate and of gelatin methacryloyl, each functionalized by conjugation with dopamine HCl. Both compounds are cross-linked into easily applied as pre-formed hydrogel patches or as in situ hydrogels formed at the wound site utilizing FDA-approved photo-initiators and oxidants. Material testing demonstrates appropriate adhesiveness, tensile strength, burst pressure, and elasticity with no significant cell toxicity in vitro assessments. Air-leak was absent after sealant application to experimentally-induced injuries in ex-vivo rat lung and tracheal models and in ex vivo pig lungs. Sustained repair of experimentally-induced pleural injury was observed for up to one month in vivo rat models and for up to 2 weeks in vivo rat tracheal injury models without obvious air leak or obvious toxicities. The alginate-based sealant worked best in a pre-formed hydrogel patch whereas the gelatin-based sealant worked best in an in situ formed hydrogel at the wound site thus providing two potential approaches. These studies provide a platform for further pre-clinical and potential clinical investigations. STATEMENT OF SIGNIFICANCE: Pneumothorax and pleural effusions resulting from trauma and a range of lung diseases and critical illnesses can result in lung collapse that can be immediately life-threatening or result in chronic leaking (bronchopleural fistula) that is currently difficult to manage. This leads to significantly increased morbidity, mortality, hospital stays, health care costs, and other complications. We have developed sealants originating from alginate and gelatin biomaterials, each functionalized by methacryloylation and by dopamine conjugation to have desired mechanical characteristics for use in pleural and tracheal injuries. The sealants are easily applied, non-cytotoxic, and perform well in vitro and in vivo model systems of lung and tracheal injuries. These initial proof of concept investigations provide a platform for further studies.

Comparative immunogenicity of decellularized wild type and alpha 1,3 galactosyltransferase knockout pig lungs.

Decellularized pig lungs recellularized with human lung cells offer a novel approach for organ transplantation. However, the potential immunogenicity of decellularized pig lungs following exposure to human tissues has not been assessed. We found that exposure of native lungs from wildtype and transgenic pigs lacking alpha (1,3)-galactosyltransferase (α-gal KO) to sera from normal healthy human volunteers demonstrated similar robust IgM and IgG immunoreactivity, comparably decreased in decellularized lungs. Similar results were observed with sera from patients who had previously undergone transcutaneous porcine aortic valve replacement (TAVR) or from patients with increased circulating anti-α-gal IgE antibodies (α-gal syndrome). Depleting anti-α-gal antibodies from the sera demonstrated both specificity of α-gal immunoreactivity and also residual immunoreactivity similar between wildtype and α-gal KO pig lungs. Exposure of human monocytes and macrophages to native wildtype lungs demonstrated greater induction of M2 phenotype than native α-gal KO pig lungs, which was less marked with decellularized lungs of either type. Overall, these results demonstrate that native wildtype and α-gal KO pig lungs provoke similar immune responses that are comparably decreased following decellularization. This provides a further platform for potential use of decellularized pig lungs in tissue engineering approaches and subsequent transplantation schemes but no obvious overall immunologic advantage of utilizing lungs obtained from α-gal KO pigs.

Heparan sulfates from bat and human lung and their binding to the spike protein of SARS-CoV-2 virus.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has resulted in a pandemic and continues to spread at an unprecedented rate around the world. Although a vaccine has recently been approved, there are currently few effective therapeutics to fight its associated disease in humans, COVID-19. SARS-CoV-2 and the related severe acute respiratory syndrome (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV) result from zoonotic respiratory viruses that have bats as the primary host and an as yet unknown secondary host. While each of these viruses has different protein-based cell-surface receptors, each rely on the glycosaminoglycan, heparan sulfate as a co-receptor. In this study we compare, for the first time, differences and similarities in the structure of heparan sulfate in human and bat lungs. Furthermore, we show that the spike glycoprotein of COVID-19 binds 3.5 times stronger to human lung heparan sulfate than bat lung heparan sulfate.

Porcine Lung-Derived Extracellular Matrix Hydrogel Properties Are Dependent on Pepsin Digestion Time.

Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM) in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.

Functional role of glycosaminoglycans in decellularized lung extracellular matrix.

Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGFß1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2, HGF, and TGFß1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. STATEMENT OF SIGNIFICANCE: Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGs) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives.

Lung bioengineering: advances and challenges in lung decellularization and recellularization.

PURPOSE OF REVIEW: Bioengineering the lung based on its natural extracellular matrix (ECM) offers novel opportunities to overcome the shortage of donors, to reduce chronic allograft rejections, and to improve the median survival rate of transplanted patients. During the last decade, lung tissue engineering has advanced rapidly to combine scaffolds, cells, and biologically active molecules into functional tissues to restore or improve the lung's main function, gas exchange. This review will inspect the current progress in lung bioengineering using decellularized and recellularized lung scaffolds and highlight future challenges in the field. RECENT FINDINGS: Lung decellularization and recellularization protocols have provided researchers with tools to progress toward functional lung tissue engineering. However, there is continuous evolution and refinement particularly for optimization of lung recellularization. These further the possibility of developing a transplantable bioartificial lung. SUMMARY: Bioengineering the lung using recellularized scaffolds could offer a curative option for patients with end-stage organ failure but its accomplishment remains unclear in the short-term. However, the state-of-the-art of techniques described in this review will increase our knowledge of the lung ECM and of chemical and mechanical cues which drive cell repopulation to improve the advances in lung regeneration and lung tissue engineering.

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 °C and can be used immediately following neutralization or stored at 4 °C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

Electrospun Decellularized Lung Matrix Scaffold for Airway Smooth Muscle Culture.

Chronic respiratory disease affects many people worldwide with little known about the intricate mechanisms driving the pathology, making it difficult to develop novel therapies. Improving the understanding of airway smooth muscle and extracellular matrix (ECM) interactions is key to developing treatments for this leading cause of death. With currently no relevant or controllable in vivo or in vitro models to investigate cell-ECM interactions in the small airways, the development of a biomimetic in vitro model with cell attachment, signaling, and organization is needed. The goal of this study was to create a biologically and structurally relevant in vitro model of small airway smooth muscle. In order to achieve this goal, a scaffold was engineered from synthetic poly-l-lactic acid (PLLA) and decellularized pig lung ECM (PLECM). PLECM scaffolds have improved physical characteristics over synthetic scaffolds, by exhibiting a significant decrease in the elastic modulus and an increase in hydrophilicity. Histological staining and SDS-PAGE showed that essential proteins or protein fragments found in natural ECM were present after processing. Human bronchial smooth muscle cells (HBSMCs) seeded onto PLECM 3D scaffolds formed confluent layers and maintained a contractile phenotype, as demonstrated by the organized arrangement of actin filaments within the cell and expected contractile protein expression of calponin 1. HBSMCs cultured on electrospun PLECM scaffold also increased alpha-1 type 1 collagen compared to those cultured on PLLA scaffolds. In summary, this research demonstrates that a PLLA/PLECM composite electrospun mat is a promising tool to produce an in vitro model of the airway with the potential for a better understanding of bronchiole smooth muscle behavior in diseased or normal states.

Development and characterization of a naturally derived lung extracellular matrix hydrogel.

The complexity and rapid clearance mechanisms of lung tissue make it difficult to develop effective treatments for many chronic pathologies. We are investigating lung derived extracellular matrix (ECM) hydrogels as a novel approach for delivery of cellular therapies to the pulmonary system. The main objectives of this study include effective decellularization of porcine lung tissue, development of a hydrogel from the porcine ECM, and characterization of the material's composition, mechanical properties, and ability to support cellular growth. Our evaluation of the decellularized tissue indicated successful removal of cellular material and immunogenic remnants in the ECM. The self-assembly of the lung ECM hydrogel was rapid, reaching maximum modulus values within 3 min at 37°C. Rheological characterization showed the lung ECM hydrogel to have a concentration dependent storage modulus between 15 and 60 Pa. The purpose of this study was to evaluate our novel ECM derived hydrogel and measure its ability to support 3D culture of MSCs in vitro and in vivo delivery of MSCs. Our in vitro experiments using human mesenchymal stem cells demonstrated our novel ECM hydrogel's ability to enhance cellular attachment and viability. Our in vivo experiments demonstrated that rat MSC delivery in pre-gel solution significantly increased cell retention in the lung over 24 h in an emphysema rat model. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1922-1935, 2016.