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)

Fibronectin localization and fibrillization are affected by the presence of serum in culture media

In vitro models of fibrotic phenomena are often based on the fibroblast-myofibroblast transition as the contraction-triggering cellular event. There are, however, multiple sources of concern regarding the appropriateness of such models; a first and widely investigated issue is the often inappropriate nature of the interactions between mesenchymal cells and surrounding/underlying matrix/substrate. A second set of problems concerns the composition of the fluid phase, which includes both dispersed/dissolved paracrine messengers and matrix elements. In this study, we have focused on the effects that serum may generate. We have observed that A) serum causes high variability in the expression of typical markers of myofibroblast differentiation (ED-A fibronectin and α-Smooth Muscle Actin) upon treatment with TGF-β1; this is probably due to intrinsic variability of cytokine concentrations in different batches of serum. B) the fibrillization of endogenous fibronectin is partially hampered and its localization changed from ventral (on the substrate) to dorsal (upper surface); the latter morphology appears to be largely overlooked in literature, even though it may have a significant role in terms of mechanotransductive signaling. This quite dramatic change possibly occurs as a result of competition with serum proteins, although our data seem to rule out a direct role of serum fibronectin

Rapid Prototyping Composite and Complex Scaffolds with PAM(2).

To create composite synthetic scaffolds with the same degree of complexity and multilevel organization as biological tissue, we need to integrate multilevel biomaterial processing in rapid prototyping systems. The scaffolds then encompass the entire range of properties, which characterize biological tissue. A multilevel microfabrication system, PAM(2), has been developed to address this gap in material processing. It is equipped with different modules, each covering a range of material properties and spatial resolutions. Together, the modules in PAM(2) can be used to realize complex and composite scaffolds for tissue engineering, bringing us a step closer to real clinical applications. This chapter describes the PAM(2) system and discusses some of the practical issues associated with scaffold microfabrication and biomaterial processing

Strain rate viscoelastic analysis of soft and highly hydrated biomaterials.

Measuring the viscoelastic behavior of highly hydrated biological materials is challenging because of their intrinsic softness and labile nature. In these materials, it is difficult to avoid prestress and therefore to establish precise initial stress and strain conditions for lumped parameter estimation using creep or stress-relaxation (SR) tests. We describe a method ([Image: see text] or epsilon dot method) for deriving the viscoelastic parameters of soft hydrated biomaterials which avoids prestress and can be used to rapidly test degradable samples. Standard mechanical tests are first performed compressing samples using different strain rates. The dataset obtained is then analyzed to mathematically derive the material's viscoelastic parameters. In this work a stable elastomer, polydimethylsiloxane, and a labile hydrogel, gelatin, were first tested using the[Image: see text], in parallel SR was used to compare lumped parameter estimation. After demonstrating that the elastic parameters are equivalent and that the estimation of short-time constants is more precise using the proposed method, the viscoelastic behavior of porcine liver was investigated using this approach. The results show that the constitutive parameters of hepatic tissue can be quickly quantified without the application of any prestress and before the onset of time-dependent degradation phenomena. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 3352–3360, 201