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)

CD44 targeted delivery of siRNA by using HA-decorated nanotechnologies for KRAS silencing in cancer treatment

KRAS is a small GTPase that regulates cell proliferation and survival. In tumors, the KRAS gene is mutated, and leading to unregulated tumor growth. Despite the recognized importance of KRAS in cancer, attempts to develop small molecule inhibitors have proved unsuccessful. An alternative strategy is gene silencing and the use of small nucleic acid sequences (e.g. siRNA, shRNA), has been reported to successfully downregulate KRAS. In this study we developed ternary nanocomplexes to deliver an anti-KRAS siRNA to colorectal cancer cells, exploiting the interaction of hyaluronic acid (HA) with CD44 as a means to achieve selective targeting of CD44-positive cancer cells. Two different polycations, poly(hexamethylene biguanide) and chitosan, were complexed with siRNA and coated with HA. Physico-chemical properties and stability of nanoparticles were characterized, including size, surface charge, and degree of siRNA protection. We demonstrate nanoparticle internalization (flow cytometry), siRNA cytosolic release (confocal microscopy) and KRAS silencing (RT-qPCR) in CD44+/KRAS+ colorectal cancer cell line, HCT-116. Further we demonstrate that the uptake of HA-decorated nanoparticles in cancer cells is higher when co-cultured with fibroblasts

Colorectal tumor 3D in vitro models: advantages of biofabrication for the recapitulation of early stages of tumour development

The majority of cancer-related in vitro studies are conducted on cell monolayers or spheroids. Although this approach has led to key discoveries, it still has a poor outcome in recapitulating the different stages of tumor development. The advent of novel three-dimensional (3D) systems and technological methods for their fabrication is set to improve the field, offering a more physiologically relevant and high throughput in vitro system for the study of tumor development and treatment. Here we describe the fabrication of alginate-based 3D models that recapitulate the early stages of colorectal cancer, tracking two of the main biomarkers for tumor development: CD44 and HIF-1α. We optimized the fabrication process to obtain alginate micro-beads with controlled size and stiffness, mimicking the early stages of colorectal cancer. Human colorectal HCT-116 cancer cells were encapsulated with controlled initial number, and cell viability and protein expression of said 3D in vitro models was compared to that of current gold standards (cell monolayers and spheroids). Our results evidenced that encapsulated HCT-116 demonstrated a high viability, increase in stem-like cell populations (increased expression of CD44) and reduced hypoxic regions (lower HIF-1a expression) compared to spheroid cultures. In conclusion we show that our biofabricated system is a highly reproducible and easily accessible alternative to study cell behavior, allowing to better mimic the early stages of colorectal cancer in comparison to other in vitro models. The use of biofabricated in vitro models will improve the translatability of results, in particular when testing strategies for therapeutic intervention

Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask)

The impact of fabrication parameters and substrate stiffness in direct writing of living constructs.

Biomolecules and living cells can be printed in high-resolution patterns to fabricate living constructs for tissue engineering. To evaluate the impact of processing cells with rapid prototyping (RP) methods we modelled the printing phase of two RP systems that employ biomaterial inks containing living cells: a high-resolution inkjet system (BioJet) and a lower resolution nozzle based contact printing system (PAM(2) ). In the first fabrication method, we reasoned that cell damage occurs principally during drop collision on the printing surface, in the second we hypothesise that shear stresses act on cells during extrusion (within the printing nozzle). The two cases were modelled changing the printing conditions: biomaterial substrate stiffness and volumetric flow rate, respectively in BioJet and PAM(2) . Results show that during inkjet printing impact energies of about 10(-8) J are transmitted to cells, while extrusion energies of the order of 10(-11) J are exerted in direct printing. Viability tests of printed cells can be related to those numerical simulations, suggesting a threshold energy of 10(-9) J to avoid permanent cell damage. To obtain well-defined living constructs, a combination of these methods is proposed for the fabrication of scaffolds with controlled 3D architecture and spatial distribution of biomolecules and cell

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

Riboflavin and collagen: New crosslinking methods to tailor the stiffness of hydrogels

Fabricating materials with tailored mechanical properties is a challenge and crucial for their successful application in a variety of fields such as tissue engineering. Here collagen and riboflavin were used to create hydrogels with controlled mechanical properties mimicking those of soft tissues (e.g. liver). Collagen-based hydrogels were obtained using a two-step gelation method. Firstly a physical gelation step (i.e. modulation of temperature and pH) was used to fix a specific shape; then photo-initiated cross-links were formed to increase the stiffness. Specifically the chemical cross-linking step was initiated with UV (ultra-violet) radiation to obtain riboflavin derivatised radical polymerization of collagen chains. Cylindrical shaped samples with controlled dimensions were fabricated, and then tested using compressive loading. We show that the compressive elastic modulus of collagen-based hydrogels can be tuned between 0.9 and 3.6 kPa by changing collagen concentration, irradiation with UV in the presence of riboflavin and freeze-drying

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