Characterising the elastic and viscoelastic interaction between the cell and its matrix in 3D: because it takes two to salsa dance

The extracellular matrix (ECM) is a three-dimensional, acellular component of all organs and tissues. The ECM has elastic and viscoelastic properties, quantified through the elastic modulus (i.e. stiffness) and stress relaxation, respectively, that guide cell fate. Stiffness and stress relaxation drive cellular plasticity in homeostasis and disease. Therefore, to represent the mechanics of the ECM in vitro it is necessary to employ models that recapitulate these properties. Among said models are hydrogels: polymeric networks whose mass mainly consists of water. Importantly, hydrogel viscoelasticity remains an understudied property, notably in cell-loaded materials. Hence, this thesis investigated the elastic and viscoelastic properties of diverse cell-free and cell-loaded hydrogels. The hydrogels evaluated in this thesis include organ-derived ECM, gelatine methacryloyl, agarose, human-derived platelet-poor plasma, alginate and pluronic. These hydrogels have tissue engineering and regenerative medicine (TERM) potential. Particular emphasis was placed on investigating and mathematically modelling the elastic and viscoelastic fate of cell-loaded hydrogels. Our data show that increasing polymer concentration tailored hydrogel elasticity and viscoelasticity. Hydrogel architecture, composition and the bonds forming the polymer network dictated hydrogel elasticity and viscoelasticity. Also, cells altered hydrogel stiffness and stress relaxation in a polymer type, hydrogel concentration and time-dependent manner. A generalised Maxwell model of viscoelasticity further revealed cell-induced changes in hydrogel time-dependent mechanics. Overall, this thesis furthers our understanding of cell-matrix biology in vitro. The data presented here also have implications for the TERM field and areas of hydrogel-based research for cellular applications

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 (R(i)) 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 R(i) 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 R(i) of Maxwell elements transforming hydrogel viscoelasticity into a more fluid environment over time. Overall, 5% GelMA induced the most favourable ASC response

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

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

Viscoelastic properties of plasma-agarose hydrogels dictate favorable fibroblast responses for skin tissue engineering applications

Dermal wound healing relies on the properties of the extracellular matrix (ECM). Thus, hydrogels that replicate skin ECM have reached clinical application. After a dermal injury, a transient, biodegradable fibrin clot is instrumental in wound healing. Human plasma, and its main constituent, fibrin would make a suitable biomaterial for improving wound healing and processed as hydrogels albeit with limited mechanical strength. To overcome this, plasma-agarose (PA) composite hydrogels have been developed and used to prepare diverse bioengineered tissues. To date, little is known about the influence of variable agarose concentrations on the viscoelastic properties of PA hydrogels and their correlation to cell biology. This study reports the characterization of the viscoelastic properties of different concentrations of agarose in PA hydrogels: 0 %, 0.5 %, 1 %, 1.5 %, and 2 % (w/v), and their influence on the cell number and mitochondrial activity of human dermal fibroblasts. Results show that agarose addition increased the stiffness, relaxation time constants 1 (τ1) and 2 (τ2), and fiber diameter, whereas the porosity decreased. Changes in cell metabolism occurred at the early stages of culturing and correlated to the displacement of fast (τ1) and intermediate (τ2) Maxwell elements. Fibroblasts seeded in low PA concentrations spread faster during 14 d than cells cultured in higher agarose concentrations. Collectively, these results confirm that PA viscoelasticity and hydrogel architecture strongly influenced cell behavior. Therefore, viscoelasticity is a key parameter in the design of PA-based implants

From Macro to Micro: Comparison of Imaging Techniques to Detect Vascular Network Formation in Left Ventricle Decellularized Extracellular Matrix Hydrogels

Background: Angiogenesis is a crucial process in physiological maintenance and tissue regeneration. To understand the contribution of angiogenesis, it is essential to replicate this process in an environment that reproduces the biochemical and physical properties which are largely governed by the extracellular matrix (ECM). We investigated vascularization in cardiac left ventricular ECM hydrogels to mimic post-myocardial repair. We set out to assess and compare different destructive and non-destructive methods, optical as well as non-optical, to visualize angiogenesis and associated matrix remodeling in myocardial ECM hydrogels. Methods: A total of 100,000, 300,000, and 600,000 Human Pulmonary Microvascular Endothelial Cells (HPMEC) were seeded in left ventricular cardiac ECM hydrogel in 48-well plates. After 1, 7, and 14 days of culture, the HPMEC were imaged by inverted fluorescence microscopy and 3D confocal laser scanning microscopy (Zeiss Cell Discoverer 7). In addition, cell-seeded ECM hydrogels were scanned by optical coherence tomography (OCT). Fixed and paraffin-embedded gels were thin-sectioned and assessed for ECM components via H&E, picrosirius red histochemical staining, and immunostaining for collagen type I. ImageJ-based densitometry was used to quantify vascular-like networks and GraphPad was used for statistical analyses. Results: Qualitative analyses were realized through fluoromicrographs obtained by the confocal laser scanning microscope which allowed us to visualize the extensive vascular-like networks that readily appeared at all seeding densities. Quantification of networks was only possible using fluoromicrographs from inverted microscopy. These showed that, after three days, the number of master junctions was seeding density-dependent. The resolution of optical coherence tomography was too low to distinguish between signals caused by the ECM and cells or networks, yet it did show that gels, irrespective of cells, were heterogeneous. Interestingly, (immuno)histochemistry could clearly distinguish between the cast cardiac-derived matrix and newly deposited ECM in the hydrogels. The H&E staining corroborated the presence of vascular-like network structures, albeit that sectioning inevitably led to the loss of 3D structure. Conclusions: Except for OCT, all methods had complementary merit and generated qualitative and quantitative data that allowed us to understand vascular network formation in organ-derived ECM hydrogels

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

An immune regulatory 3D-printed alginate-pectin construct for immunoisolation of insulin producing β-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-1β, 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 β-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

Viscoelastic properties of plasma-agarose hydrogels dictate favorable fibroblast responses for skin tissue engineering applications

Dermal wound healing relies on the properties of the extracellular matrix (ECM). Thus, hydrogels that replicate skin ECM have reached clinical application. After a dermal injury, a transient, biodegradable fibrin clot is instrumental in wound healing. Human plasma, and its main constituent, fibrin would make a suitable biomaterial for improving wound healing and processed as hydrogels albeit with limited mechanical strength. To overcome this, plasma-agarose (PA) composite hydrogels have been developed and used to prepare diverse bioengineered tissues. To date, little is known about the influence of variable agarose concentrations on the viscoelastic properties of PA hydrogels and their correlation to cell biology. This study reports the characterization of the viscoelastic properties of different concentrations of agarose in PA hydrogels: 0 %, 0.5 %, 1 %, 1.5 %, and 2 % (w/v), and their influence on the cell number and mitochondrial activity of human dermal fibroblasts. Results show that agarose addition increased the stiffness, relaxation time constants 1 (τ1) and 2 (τ2), and fiber diameter, whereas the porosity decreased. Changes in cell metabolism occurred at the early stages of culturing and correlated to the displacement of fast (τ1) and intermediate (τ2) Maxwell elements. Fibroblasts seeded in low PA concentrations spread faster during 14 d than cells cultured in higher agarose concentrations. Collectively, these results confirm that PA viscoelasticity and hydrogel architecture strongly influenced cell behavior. Therefore, viscoelasticity is a key parameter in the design of PA-based implants