Functionalized Enzyme-Responsive Biomaterials to Model Tissue Stiffening in vitro.

The mechanical properties of the cellular microenvironment play a crucial role in modulating cell function, and many pathophysiological processes are accompanied by variations in extracellular matrix (ECM) stiffness. Lysyl oxidase (LOx) is one of the enzymes involved in several ECM-stiffening processes. Here, we engineered poly(ethylene glycol) (PEG)-based hydrogels with controlled mechanical properties in the range typical of soft tissues. These hydrogels were functionalized featuring free primary amines, which allows an additional chemical LOx-responsive behavior with increase in crosslinks and hydrogel elastic modulus, mimicking biological ECM-stiffening mechanisms. Hydrogels with elastic moduli in the range of 0.5-4 kPa were obtained after a first photopolymerization step. The increase in elastic modulus of the functionalized and enzyme-responsive hydrogels was also characterized after the second-step enzymatic reaction, recording an increase in hydrogel stiffness up to 0.5 kPa after incubation with LOx. Finally, hydrogel precursors containing HepG2 (bioinks) were used to form three-dimensional (3D) in vitro models to mimic hepatic tissue and test PEG-based hydrogel biocompatibility. Hepatic functional markers were measured up to 7 days of culture, suggesting further use of such 3D models to study cell mechanobiology and response to dynamic variation of hydrogels stiffness. The results show that the functionalized hydrogels presented in this work match the mechanical properties of soft tissues, allow dynamic variations of hydrogel stiffness, and can be used to mimic changes in the microenvironment properties of soft tissues typical of inflammation and pathological changes at early stages (e.g., fibrosis, cancer).

* An Injectable Recombinant Collagen I Peptide-Based Macroporous Microcarrier Allows Superior Expansion of C2C12 and Human Bone Marrow-Derived Mesenchymal Stromal Cells and Supports Deposition of Mineralized Matrix.

The development of scaffold formulations based on extracellular matrix (ECM)-inspired synthetic materials constitutes an important resource for the advance of cell-based therapies in bone tissue engineering approaches, where both cell and scaffold implantation are often needed. Culturing cells on porous microcarriers (MCs) allows cell expansion in a three-dimensional microenvironment and constitutes a possible solution for minimally invasive cell and scaffold simultaneous delivery, but the reduced pore dimension and pore interconnection diameter of several commercially available MCs limits de facto cell ingrowth, and ultimately their suitability for in vivo cell delivery. In this study we investigated the potential of a new macroporous MC based on a collagen I-based recombinant peptide (Cellnest™) for C2C12 cells and human bone marrow-derived mesenchymal stromal cells (hBMSCs) expansion and we analyzed the influence of dehydrothermal (DHT), hexamethylene diisocyanate (HMDIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) cross-linking strategies on cell vitality, proliferation, and hBMSCs differentiation. We established a double emulsification protocol for the manufacturing of MCs characterized by external pores of 20-40 µm diameter, 73% porosity, and 20 ± 3 µm pore interconnection diameter supporting cell ingrowth and proliferation into the MC. MCs cross-linked with DHT and HMDIC supported higher cell proliferation comparing to a commercially available equivalent over the course of 7 days and resulted in higher cell yield by day 28. Moreover, while hBMSCs expansion on Cellnest-MCs did not lead to a significant upregulation of the early markers of osteogenic differentiation Col1a1 and Runx2, their differentiation potential into the osteogenic lineage was preserved when cultured in differentiation medium, as confirmed by mineralized ECM deposition. We believe that Cellnest-MCs will help in reaching clinically relevant cell quantities and ultimately help in accelerating the translation of cell-based therapies for bone tissue engineering in the clinical practice.

Antioxidant effect of a fermented powder of Lady Joy bean in primary rat hepatocytes.

The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H2O2-induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H2O2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H2O2-induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H2O2.

A novel dual-flow bioreactor simulates increased fluorescein permeability in epithelial tissue barriers.

Permeability studies across epithelial barriers are of primary importance in drug delivery as well as in toxicology. However, traditional in vitro models do not adequately mimic the dynamic environment of physiological barriers. Here, we describe a novel two-chamber modular bioreactor for dynamic in vitro studies of epithelial cells. The fluid dynamic environment of the bioreactor was characterized using computational fluid dynamic models and measurements of pressure gradients for different combinations of flow rates in the apical and basal chambers. Cell culture experiments were then performed with fully differentiated Caco-2 cells as a model of the intestinal epithelium, comparing the effect of media flow applied in the bioreactor with traditional static transwells. The flow increases barrier integrity and tight junction expression of Caco-2 cells with respect to the static controls. Fluorescein permeability increased threefold in the dynamic system, indicating that the stimulus induced by flow increases transport across the barrier, closely mimicking the in vivo situation. The results are of interest for studying the influence of mechanical stimuli on cells, and underline the importance of developing more physiologically relevant in vitro tissue models. The bioreactor can be used to study drug delivery, chemical, or nanomaterial toxicity and to engineer barrier tissues.