A Beginner's Guide to the Characterization of Hydrogel Microarchitecture for Cellular Applications.

The extracellular matrix (ECM) is a three-dimensional, acellular scaffold of living tissues. Incorporating the ECM into cell culture models is a goal of cell biology studies and requires biocompatible materials that can mimic the ECM. Among such materials are hydrogels: polymeric networks that derive most of their mass from water. With the tuning of their properties, these polymer networks can resemble living tissues. The microarchitectural properties of hydrogels, such as porosity, pore size, fiber length, and surface topology can determine cell plasticity. The adequate characterization of these parameters requires reliable and reproducible methods. However, most methods were historically standardized using other biological specimens, such as 2D cell cultures, biopsies, or even animal models. Therefore, their translation comes with technical limitations when applied to hydrogel-based cell culture systems. In our current work, we have reviewed the most common techniques employed in the characterization of hydrogel microarchitectures. Our review provides a concise description of the underlying principles of each method and summarizes the collective data obtained from cell-free and cell-loaded hydrogels. The advantages and limitations of each technique are discussed, and comparisons are made. The information presented in our current work will be of interest to researchers who employ hydrogels as platforms for cell culture, 3D bioprinting, and other fields within hydrogel-based research.

Matrix Metalloproteases from Adipose Tissue-Derived Stromal Cells Are Spatiotemporally Regulated by Hydrogel Mechanics in a 3D Microenvironment.

Adipose tissue-derived stromal cells (ASCs) are of interest in tissue engineering and regenerative medicine (TERM) due to their easy acquisition, multipotency, and secretion of a host of factors that promote regeneration. Retention of ASCs in or around lesions is poor following direct administration. Therefore, for TERM applications, ASCs can be 'immobilized' via their incorporation into hydrogels such as gelatine methacryloyl (GelMA). Tweaking GelMA concentration is a common approach to approximate the mechanical properties found in organs or tissues that need repair. Distinct hydrogel mechanics influence the ability of a cell to spread, migrate, proliferate, and secrete trophic factors. Mesenchymal cells such as ASCs are potent remodellers of the extracellular matrix (ECM). Not only do ASCs deposit components, they also secrete matrix metalloproteases (MMPs) which degrade ECM. In this work, we investigated if GelMA polymer concentration influenced the expression of active MMPs by ASCs. In addition, MMPs' presence was interrogated with regard to ASCs morphology and changes in hydrogel ultrastructure. For this, immortalised ASCs were embedded in 5%, 10%, and 15% (w/v) GelMA hydrogels, photopolymerised and cultured for 14 d. Zymography in situ indicated that MMPs had a variable, hydrogel concentration-dependent influence on ASCs-secreted MMPs. In 5% GelMA, ASCs showed a high and sustained expression of MMPs, while, in 10% and 15% GelMA, such expression was almost null. ASCs morphology based on F-actin staining showed that increasing GelMA concentrations inhibit their spreading. Scanning electron microscopy (SEM) showed that hydrogel ultrastructure in terms of pore density, pore size, and percentage porosity were not consistently influenced by cells. Interestingly, changes in ultrastructural parameters were detected also in cell-free materials, albeit without a clear trend. We conclude that hydrogel concentration and its underlying mechanics influenced MMP expression by ASCs. The exact MMPs that respond to these mechanical cues should be defined in follow-up experiments.

Adipose Tissue-Derived Stromal Cells Alter the Mechanical Stability and Viscoelastic Properties of Gelatine Methacryloyl Hydrogels.

The extracellular matrix provides mechanical cues to cells within it, not just in terms of stiffness (elasticity) but also time-dependent responses to deformation (viscoelasticity). In this work, we determined the viscoelastic transformation of gelatine methacryloyl (GelMA) hydrogels caused by adipose tissue-derived stromal cells (ASCs) through mathematical modelling. GelMA-ASCs combination is of interest to model stem cell-driven repair and to understand cell-biomaterial interactions in 3D environments. Immortalised human ASCs were embedded in 5%, 10%, and 15% (w/v) GelMA hydrogels and evaluated for 14 d. GelMA had a concentration-dependent increase in stiffness, but cells decreased this stiffness over time, across concentrations. Viscoelastic changes in terms of stress relaxation increased progressively in 5% GelMA, while mathematical Maxwell analysis showed that the relative importance (Ri) of the fastest Maxwell elements increased proportionally. The 10% GelMA only showed differences at 7 d. In contrast, ASCs in 15% GelMA caused slower stress relaxation, increasing the Ri of the slowest Maxwell element. We conclude that GelMA concentration influenced the stiffness and number of Maxwell elements. ASCs changed the percentage stress relaxation and Ri of Maxwell elements transforming hydrogel viscoelasticity into a more fluid environment over time. Overall, 5% GelMA induced the most favourable ASC response.

Architecture and Composition Dictate Viscoelastic Properties of Organ-Derived Extracellular Matrix Hydrogels.

The proteins and polysaccharides of the extracellular matrix (ECM) provide architectural support as well as biochemical and biophysical instruction to cells. Decellularized, ECM hydrogels replicate in vivo functions. The ECM's elasticity and water retention renders it viscoelastic. In this study, we compared the viscoelastic properties of ECM hydrogels derived from the skin, lung and (cardiac) left ventricle and mathematically modelled these data with a generalized Maxwell model. ECM hydrogels from the skin, lung and cardiac left ventricle (LV) were subjected to a stress relaxation test under uniaxial low-load compression at a 20%/s strain rate and the viscoelasticity determined. Stress relaxation data were modelled according to Maxwell. Physical data were compared with protein and sulfated GAGs composition and ultrastructure SEM. We show that the skin-ECM relaxed faster and had a lower elastic modulus than the lung-ECM and the LV-ECM. The skin-ECM had two Maxwell elements, the lung-ECM and the LV-ECM had three. The skin-ECM had a higher number of sulfated GAGs, and a highly porous surface, while both the LV-ECM and the lung-ECM had homogenous surfaces with localized porous regions. Our results show that the elasticity of ECM hydrogels, but also their viscoelastic relaxation and gelling behavior, was organ dependent. Part of these physical features correlated with their biochemical composition and ultrastructure.

COL4A3 is degraded in allergic asthma and degradation predicts response to anti-IgE therapy.

BACKGROUND: Asthma is a heterogeneous syndrome substantiating the urgent requirement for endotype-specific biomarkers. Dysbalance of fibrosis and fibrolysis in asthmatic lung tissue leads to reduced levels of the inflammation-protective collagen 4 (COL4A3). OBJECTIVE: To delineate the degradation of COL4A3 in allergic airway inflammation and evaluate the resultant product as a biomarker for anti-IgE therapy response. METHODS: The serological COL4A3 degradation marker C4Ma3 (Nordic Bioscience, Denmark) and serum cytokines were measured in the ALLIANCE cohort (paediatric cases/controls: n=134/n=35; adult cases/controls: n=149/n=31). Exacerbation of allergic airway disease in mice was induced by sensitising to ovalbumin (OVA), challenge with OVA aerosol and instillation of poly(cytidylic-inosinic). Fulacimstat (chymase inhibitor; Bayer) was used to determine the role of mast cell chymase in COL4A3 degradation. Patients with cystic fibrosis (n=14) and cystic fibrosis with allergic bronchopulmonary aspergillosis (ABPA; n=9) as well as patients with severe allergic uncontrolled asthma (n=19) were tested for COL4A3 degradation. Omalizumab (anti-IgE) treatment was assessed using the Asthma Control Test. RESULTS: Serum levels of C4Ma3 were increased in asthma in adults and children alike and linked to a more severe, exacerbating allergic asthma phenotype. In an experimental asthma mouse model, C4Ma3 was dependent on mast cell chymase. Serum C4Ma3 was significantly elevated in cystic fibrosis plus ABPA and at baseline predicted the success of the anti-IgE therapy in allergic, uncontrolled asthmatics (diagnostic OR 31.5). CONCLUSION: C4Ma3 levels depend on lung mast cell chymase and are increased in a severe, exacerbating allergic asthma phenotype. C4Ma3 may serve as a novel biomarker to predict anti-IgE therapy response.

COL4A3 expression in asthmatic epithelium depends on intronic methylation and ZNF263 binding.

BACKGROUND: Reduction of COL4A3, one of the six isoforms of collagen 4, in asthmatic airways results in increased inflammation and angiogenesis, implicating it as a central part of asthma pathogenesis. However, to date, the path underlying these diminished COL4A3 levels has been elusive. This study investigated a possible mechanism underlying the reduction of COL4A3 expression. METHODS: Bronchial biopsies of 76 patients with asthma and 83 controls were subjected to RNA-sequencing and DNA methylation bead arrays to identify expression and methylation changes. The binding of ZNF263 was analysed by chromatin-immunoprecipitation sequencing coupled with quantitative (q)PCR. Effects of ZNF263 silencing, using small interfering RNA, on the COL4A3 expression were studied using qPCR. RESULTS: COL4A3 expression was significantly reduced in bronchial biopsies compared to healthy controls, whereas DNA methylation levels at cg11797365 were increased. COL4A3 expression levels were significantly low in asthmatics without inhaled corticosteroid (ICS) use, whereas the expression was not statistically different between asthmatics using ICS and controls. Methylation levels at cg11797365 in vitro were increased upon consecutive rhinovirus infections. CONCLUSION: Our data indicate an epigenetic modification as a contributing factor for the loss of COL4A3 expression in asthmatic airway epithelium.

An immune regulatory 3D-printed alginate-pectin construct for immunoisolation of insulin producing ß-cells.

Different bioinks have been used to produce cell-laden alginate-based hydrogel constructs for cell replacement therapy but some of these approaches suffer from issues with print quality, long-term mechanical instability, and bioincompatibility. In this study, new alginate-based bioinks were developed to produce cell-laden grid-shaped hydrogel constructs with stable integrity and immunomodulating capacity. Integrity and printability were improved by including the co-block-polymer Pluronic F127 in alginate solutions. To reduce inflammatory responses, pectin with a low degree of methylation was included and tested for inhibition of Toll-Like Receptor 2/1 (TLR2/1) dimerization and activation and tissue responses under the skin of mice. The viscoelastic properties of alginate-Pluronic constructs were unaffected by pectin incorporation. The tested pectin protected printed insulin-producing MIN6 cells from inflammatory stress as evidenced by higher numbers of surviving cells within the pectin-containing construct following exposure to a cocktail of the pro-inflammatory cytokines namely, IL-1ß, IFN-γ, and TNF-α. The results suggested that the cell-laden construct bioprinted with pectin-alginate-Pluronic bioink reduced tissue responses via inhibiting TLR2/1 and support insulin-producing ß-cell survival under inflammatory stress. Our study provides a potential novel strategy to improve long-term survival of pancreatic islet grafts for Type 1 Diabetes (T1D) treatment.

The Pericytic Phenotype of Adipose Tissue-Derived Stromal Cells Is Promoted by NOTCH2.

Long-term diabetes leads to macrovascular and microvascular complication. In diabetic retinopathy (DR), persistent hyperglycemia causes permanent loss of retinal pericytes and aberrant proliferation of microvascular endothelial cells (ECs). Adipose tissue-derived stromal cells (ASCs) may serve to functionally replace retinal pericytes and normalize retinal microvasculature during disease progression. We hypothesized that Notch signaling in ASC underlies regulation and stabilization of dysfunctional retinal microvascular networks such as in DR. ASC prominently and constitutively expressed NOTCH2. Genetic knockdown of NOTCH2 in ASC (SH-NOTCH2) disturbed the formation of vascular networks of human umbilical cord vein endothelial cells both on monolayers of ASC and in organotypical three-dimensional cocultures with ASC. On ASC SH-NOTCH2, cell surface platelet-derived growth factor receptor beta was downregulated which disrupted their migration toward the chemoattractant platelet-derived growth factor beta subunits (PDGF-BB) as well as to conditioned media from EC and bovine retinal EC. This chemoattractant is secreted by pro-angiogenic EC in newly formed microvascular networks to attract pericytes. Intravitreal injected ASC SH-NOTCH2 in oxygen-induced retinopathy mouse eyes did not engraft in the preexisting retinal microvasculature. However, the in vivo pro-angiogenic capacity of ASC SH-NOTCH2 did not differ from controls. In this respect, multifocal electroretinography displayed similar b-wave amplitudes in the avascular zones when either wild type ASC or SH-NOTCH2 ASC were injected. In conclusion, our results indicate that NOTCH2 is essential to support in vitro vasculogenesis via juxtacrine interactions. In contrast, ongoing in vivo angiogenesis is influenced by paracrine signaling of ASC, irrespective of Notch signaling. Stem Cells 2018;36:240-251.

Tumstatin regulates the angiogenic and inflammatory potential of airway smooth muscle extracellular matrix.

The extracellular matrix (ECM) creates the microenvironment of the tissue; an altered ECM in the asthmatic airway may be central in airway inflammation and remodelling. Tumstatin is a collagen IV-derived matrikine reduced in the asthmatic airway wall that reverses airway inflammation and remodelling in small and large animal models of asthma. This study hypothesized that the mechanisms underlying the broad asthma-resolving effects of tumstatin were due to autocrine remodelling of the ECM. Neutrophils and endothelial cells were seeded on decellularized ECM of non-asthmatic (NA) or asthmatic (A) airway smooth muscle (ASM) cells previously exposed to tumstatin in the presence or absence of a broad matrix metalloproteinase inhibitor, Marimastat. Gene expression in NA and A ASM induced by tumstatin was assessed using RT-PCR arrays. The presence of tumstatin during ECM deposition affected neutrophil and endothelial cell properties on both NA and A ASM-derived matrices and this was only partly due to MMP activity. Gene expression patterns in response to tumstatin in NA and A ASM cells were different. Tumstatin may foster an anti-inflammatory and anti-angiogenic microenvironment by modifying ASM-derived ECM. Further work is required to examine whether restoring tumstatin levels in the asthmatic airway represents a potential novel therapeutic approach.

Doxycycline reduces the migration of tuberous sclerosis complex-2 null cells - effects on RhoA-GTPase and focal adhesion kinase.

Lymphangioleiomyomatosis (LAM) is associated with dysfunction of the tuberous sclerosis complex (TSC) leading to enhanced cell proliferation and migration. This study aims to examine whether doxycycline, a tetracycline antibiotic, can inhibit the enhanced migration of TSC2-deficient cells, identify signalling pathways through which doxycycline works and to assess the effectiveness of combining doxycycline with rapamycin (mammalian target of rapamycin complex 1 inhibitor) in controlling cell migration, proliferation and wound closure. TSC2-positive and TSC2-negative mouse embryonic fibroblasts (MEF), 323-TSC2-positive and 323-TSC2-null MEF and Eker rat uterine leiomyoma (ELT3) cells were treated with doxycycline or rapamycin alone, or in combination. Migration, wound closure and proliferation were assessed using a transwell migration assay, time-lapse microscopy and manual cell counts respectively. RhoA-GTPase activity, phosphorylation of p70S6 kinase (p70S6K) and focal adhesion kinase (FAK) in TSC2-negative MEF treated with doxycycline were examined using ELISA and immunoblotting techniques. The enhanced migration of TSC2-null cells was reduced by doxycycline at concentrations as low as 20 pM, while the rate of wound closure was reduced at 2-59 µM. Doxycycline decreased RhoA-GTPase activity and phosphorylation of FAK in these cells but had no effect on the phosphorylation of p70S6K, ERK1/2 or AKT. Combining doxycycline with rapamycin significantly reduced the rate of wound closure at lower concentrations than achieved with either drug alone. This study shows that doxycycline inhibits TSC2-null cell migration. Thus doxycycline has potential as an anti-migratory agent in the treatment of diseases with TSC2 dysfunction.

Lamstatin--a novel inhibitor of lymphangiogenesis derived from collagen IV.

The lymphatic system is essential for the maintenance of tissue homeostasis and immunity. Its dysfunction in disease (such as lymphangioleiomyomatosis) can lead to chylous effusions, oedema or dissemination of malignant cells. Collagen IV has six α chains, of which some of the non-collagenous-1 domains have endogenous anti-angiogenic properties, however, little is known about specific endogenous anti-lymphangiogenic characteristics. In this study we sought to investigate the expression levels of collagen IV non-collagenous-1 domains in lung tissue of patients with and without lymphangioleiomyomatosis to explore the hypothesis that a member of the collagen IV family, specifically the non-collagenous domain-1 of α5, which we named lamstatin, has anti-lymphangiogenic properties. Levels of lamstatin detected by immunohistochemistry were decreased in lungs of lymphangioleiomyomatosis patients. We produced recombinant lamstatin in an E.coli expression system and synthesized a 17-amino acid peptide from a theoretically identified, active region (CP17) and tested their effects in vitro and in vivo. Recombinant lamstatin and CP17 inhibited proliferation, migration and cord formation of human microvascular lung lymphatic endothelial cells, in vitro. Furthermore, lamstatin and CP17 decreased complexity and dysplasia of the tumour-associated lymphatic network in a lung adenocarcinoma xenograft mouse model. In this study we identified a novel, direct inhibitor of lymphangiogenesis, derived from collagen IV. This may prove useful for exploring new avenues of treatment for lymphangioleiomyomatosis and metastasis via the lymphatic system in general.