The field of lung regenerative medicine is still in its infancy, and it largely overlooks the potential role of biomaterial-based microenvironments in promoting lung regeneration in emphysema in COPD. In this study, we aimed at understanding the interactions between primary human lung mesenchymal cells (MCs) and an elastin-like (EL) scaffold that mimics the extracellular matrix, which may be used to trigger regenerative pathways in host MCs in vivo. As a first step, we have evaluated the cytocompatibility and the morphology of the MCs when seeded inside the scaffold in 3D, in comparison to regular 2D cultures on plastic surfaces. To this end, the MCs were embedded in the EL scaffolds homogeneously, and the cytocompatibility was assessed in terms of cell viability (LIVE/DEAD) and cell metabolic activity (resazurin reduction) at 0, 1, 3 and 7 days of culture. The MCs remained alive similarly to the 2D control, showing excellent cell viability (approx. 98%). The metabolic activity showed a slower increase in 3D than in 2D, with stable levels after 1 day. This is considered more physiological, given that cells expand fast in 2D, which does not reflect the in vivo situation. A similar conclusion could be drawn from the morphological evaluation of the cells by actin (Phalloidin iFluor-555) and nuclei (DAPI) staining. The MCs seeded in 3D were found spread anisotropically in branches, which suggests a more physiological phenotype than the cells grown in 2D. Altogether, this work sets the ground for further investigations on the interaction of EL scaffolds with lung cells, as a first step to study the potential induction of the regeneration of emphysema in COPD.

Rationale: Mesenchymal stromal cells (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate different cell populations involved in the advance of the disease, such as macrophages. These immune cells possess a broad spectrum of inflammatory responses. Previous data have shown that the MSC secretome influences macrophage phenotype and functional capacities. Furthermore, preconditioning MSC with physiomimetic cues from the extracellular matrix (ECM) have shown to improve their repairing actions upon transplantation which could be exploited to boost their terapeutic efficacy. Aim: To assess the macrophage activity exerted by the secretome from physiomimetically-cultured lung MSC (LMSC). Methods: LMSC from human donors were cultured on in-house developed devices that enable lung-mimetic strain. Medium from LMSC cultured in either lung ECM scaffolds and in lung ECM hydrogels whilst subjected to cyclic stretch, and on tissue culture plates. Human monocytes were differentiated to macrophages by adding PMA and polarized to M1 and M2 phenotypes by adding LPS or IL-4 plus IL-10, respectively. M0, M1 and M2 macrophages were exposed to the medium of LMSC from the different culture conditions and analysed for typical surface markers by flow cytometry and their secretome content. Results: Secretome of LMSC subjected to stretch in lung scaffolds elicited changes in the gene expression of IL-10 compared to the static conditions. On the other hand, hydrogel conditions induced changes in IL-6 secreted by macrophages. Conclusion: Mechanical features of the lung ECM orchestrate key on LMSC, hence providing new insights into preconditioning of MSC for therapy.

Mesenchymal stromal cell (MSC)-based therapies for inflammatory diseases rely mainly on the paracrine ability to modulate the activity of macrophages. Despite recent advances, there is scarce information regarding changes of the secretome content attributed to physiomimetic cultures and, especially, how secretome content influence on macrophage activity for therapy. hLMSCs from human donors were cultured on devices developed in house that enabled lung-mimetic strain. hLMSC secretome was analyzed for typical cytokines, chemokines and growth factors. RNA was analyzed for the gene expression of CTGF and CYR61. Human monocytes were differentiated to macrophages and assessed for their phagocytic capacity and for M1/M2 subtypes by the analysis of typical cell surface markers in the presence of hLMSC secretome. CTGF and CYR61 displayed a marked reduction when cultured in lung-derived hydrogels (L-Hydrogels). The secretome showed that lung-derived scaffolds had a distinct secretion while there was a large overlap between L-Hydrogel and the conventionally (2D) cultured samples. Additionally, secretome from L-Scaffold showed an HGF increase, while IL-6 and TNF-α decreased in lung-mimetic environments. Similarly, phagocytosis decreased in a lung-mimetic environment. L-Scaffold showed a decrease of M1 population while stretch upregulated M2b subpopulations. In summary, mechanical features of the lung ECM and stretch orchestrate anti-inflammatory and immunosuppressive outcomes of hLMSCs.

Healthy regeneration of tissue relies on a well-orchestrated release of growth factors. Herein, we show the use of synthetic glycosaminoglycans for controlled binding and release of growth factors to induce a desired cellular response. First, we screened glycosaminoglycans with growth factors of interest to determine kon (association rate constant), koff (dissociation rate constant), and Kd (equilibrium rate constant). As proof-of-concept, we functionalized an elastin-like recombinamer (ELR) hydrogel with a synthetic glycosaminoglycan and immobilized fibroblast growth factor 2 (FGF2), demonstrating that human umbilical vein endothelial cells cultured on top of ELR hydrogel differentiated into tube-like structures. Taking this concept further, we developed a tunable macroporous ELR cryogel material, containing a synthetic glycosaminoglycan and FGF2 that showed increased blood vessel formation and reduced immune response compared to control when implanted in a subcutaneous mouse model. These results demonstrated the possibility for specific release of desired growth factors in/from a modular 3D scaffold in vitro and in vivo.

Introduction: Chronic obstructive pulmonary disease (COPD) is a worldwide health problem, where the tissue is gradually degraded, leading to emphysema and loss of tissue integrity [1]. Aims and Objectives: Our goal is to regenerate the damaged tissue by inserting a biomaterial and activate regeneration in the border zone between destroyed areas and remains of healthy tissue in COPD. Elastin-like Recombinamer (ELR) is a biomaterial that has proved to have excellent biocompatibility properties [2]. Methods: The ELR is modified in that one part has an alkyne modification and the second part an azide modification. The ELR is in a liquid state at 4°C and solidifies at 37°C. When the two parts are mixed in subzero temperatures, the alkyne reacts with the azide and creates a covalent bond at the same time as ice crystals are formed. The ELR structures around the ice crystals resulting in the formation of a macroporous material, a cryogel. This material has been further tested in vivo using a subcutaneous mouse model for 8 weeks, enabling assessment of the formation and integration of blood vessels. Results and Discussion: We successfully formed a cryogel that closely mimic alveolar morphology. Histological evaluation showed blood vessel formation in the cryogels as well as an integration into the skin over time. Conclusion and Further Studies: These data demonstrate that an ELR-based cryogel is a promising synthetic scaffold for lung tissue engineering. We further aim to develop this model by replacing resected lung tissue with the ELR cryogel in vivo to regain functional lung tissue. References [1] O.Hallgren et al. Respiratory Research 2010 11:55 [2] A. Ibanez-Fonseca et al. J Tissue Eng Regen Med. 2018;12:e1450–e1460.

It is known that the cell environment such as biomechanical properties and extracellular matrix (ECM) composition dictate cell behaviour including migration, proliferation, and differentiation. Important constituents of the microenvironment, including ECM molecules such as proteoglycans and glycosaminoglycans (GAGs), determine events in both embryogenesis and repair of the adult lung. Mesenchymal stromal/stem cells (MSC) have been shown to have immunomodulatory properties and may be potent actors regulating tissue remodelling and regenerative cell responses upon lung injury. Using MSC in cell-based therapy holds promise for treatment of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, so far clinical trials with MSCs in COPD have not had a significant impact on disease amelioration nor on IPF, where low cell survival rate and pulmonary retention time are major hurdles to overcome. Research shows that the microenvironment has a profound impact on transplanted MSCs. In our studies on acellular lung tissue slices (lung scaffolds) from IPF patients versus healthy individuals, we see a profound effect on cellular activity, where healthy cells cultured in diseased lung scaffolds adapt and produce proteins further promoting a diseased environment, whereas cells on healthy scaffolds sustain a healthy proteomic profile. Therefore, modulating the environmental context for cell-based therapy may be a potent way to improve treatment using MSCs. In this review, we will describe the importance of the microenvironment for cell-based therapy in chronic lung diseases, how MSC-ECM interactions can affect therapeutic output and describe current progress in the field of cell-based therapy.

Five novel xylosides tagged with the fluorescent probe Pacific Blue™ were synthesized and found to act as substrates for β4GalT7, a bottleneck enzyme in the biosynthetic pathways leading to glycosaminoglycans. By confocal microscopy of A549 cells, we showed that the xylosides were taken up by the cells, but did not enter the Golgi apparatus where most of the glycosaminoglycan biosynthesis occurs. Instead, after a possible double galactosylation by β4GalT7 and β3GalT6, the biosynthesis was terminated. We hypothesize this is due to the charge of the fluorescent probe, which is required for fluorescent ability and stability under physiological conditions.

We present a xylosylated naphthoxyloside carrying a terminal azide functionality that can be used for conjugation using click chemistry. We show that this naphthoxyloside serves as a substrate for β4GalT7 and induces the formation of soluble glycosaminoglycan (GAG) chains with physiologically relevant lengths and sulfation patterns. Finally, we demonstrate its usefulness by conjugation to the Alexa Fluor 647 and TAMRA fluorophores and coupling to a surface plasmon resonance chip for interaction studies with the hepatocyte growth factor known to interact with the GAG heparan sulfate.

Lung mesenchymal stromal cells (lung-MSCs) show a great potential in the regeneration of lung tissue. However, one of the main limitations is their expansion in 2D, which is very far from their physiological niche. This may induce phenotypic changes that lead to misleading results in their characterization and the potential loss of their regenerative profile. Therefore, we hypothesize that if a specific subpopulation of lung-MSCs is cultured in a 3D niche mimicking some of the features of the native tissue, the cells will keep their phenotype to a higher extent. Lung-MSCs were sorted from healthy human lung biopsies. Further sorting allowed to isolate subpopulations of CD13− and CD13+ (N-aminopeptidase), a subset currently being studied in our laboratory. Elastin-like recombinamer cryogels with a controlled pore size of 200-500 µm (similar to lung alveoli) and hydrogels where used as 3D niches. The metabolic activity of the cells was measured up to 3 weeks as a signature of cell growth. Cells were imaged inside the scaffolds by confocal microscopy upon actin (Phalloidin-iFluor 555) and nuclei (DAPI) staining. The metabolic activity of lung-MSCs in the 3D scaffolds increased over time, indicating cell growth and expansion, with significant differences (p < 0.001) in comparison to 2D cell culture. Cell morphology showed great differences between the groups. Overall, these results validate the 3D expansion of CD13-sorted lung-MSCs. The differences found in terms of metabolic activity and morphology in comparison with the 2D culture suggest different cell behaviors. Further studies should focus in the phenotypic characterization of the cells through gene expression and protein profiling, which might have important implications in lung-MSC culture for regenerative purposes.