VA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding

The ability to replicate in vitro the native extracellular matrix (ECM) features and to control the three-dimensional (3D) cell organization plays a fundamental role in obtaining functional engineered bioconstructs. In tissue engineering (TE) applications, hydrogels have been successfully implied as biomatrices for 3D cell embedding, exhibiting high similarities to the natural ECM and holding easily tunable mechanical properties. In the present study, we characterized a promising photocrosslinking process to generate cell-laden methacrylate gelatin (GelMA) hydrogels in the presence of VA-086 photoinitiator using a ultraviolet LED source. We investigated the influence of prepolymer concentration and light irradiance on mechanical and biomimetic properties of resulting hydrogels. In details, the increasing of gelatin concentration resulted in enhanced rheological properties and shorter polymerization time. We then defined and validated a reliable photopolymerization protocol for cell embedding (1.5% VA-086, LED 2 mW/cm2) within GelMA hydrogels, which demonstrated to support bone marrow stromal cells viability when cultured up to 7 days. Moreover, we showed how different mechanical properties, derived from different crosslinking parameters, strongly influence cell behavior. In conclusion, this protocol can be considered a versatile tool to obtain biocompatible cell-laden hydrogels with properties easily adaptable for different TE applications

Fabrication of 3D cell-laden hydrogel microstructures through photo-mold patterning

Native tissues are characterized by spatially organized three-dimensional (3D) microscaled units which functionally define cells–cells and cells–extracellular matrix interactions. The ability to engineer biomimetic constructs mimicking these 3D microarchitectures is subject to the control over cell distribution and organization. In the present study we introduce a novel protocol to generate 3D cell laden hydrogel micropatterns with defined size and shape. The method, named photo-mold patterning (PMP), combines hydrogel micromolding within polydimethylsiloxane (PDMS) stamps and photopolymerization through a recently introduced biocompatible ultraviolet (UVA) activated photoinitiator (VA-086). Exploiting PDMS micromolds as geometrical constraints for two methacrylated prepolymers (polyethylene glycol diacrylate and gelatin methacrylate), micrometrically resolved structures were obtained within a 3 min exposure to a low cost and commercially available UVA LED. The PMP was validated both on a continuous cell line (human umbilical vein endothelial cells expressing green fluorescent protein, HUVEC GFP) and on primary human bone marrow stromal cells (BMSCs). HUVEC GFP and BMSCs were exposed to 1.5% w/v VA-086 and UVA light (1 W, 385 nm, distance from sample = 5 cm). Photocrosslinking conditions applied during the PMP did not negatively affect cells viability or specific metabolic activity. Quantitative analyses demonstrated the potentiality of PMP to uniformly embed viable cells within 3D microgels, creating biocompatible and favorable environments for cell proliferation and spreading during a seven days' culture. PMP can thus be considered as a promising and cost effective tool for designing spatially accurate in vitro models and, in perspective, functional constructs

In vitro mechanical stimulation to reproduce the pathological hallmarks of human cardiac fibrosis on a beating chip and predict the efficacy of drugs and advanced therapies

Cardiac fibrosis is one of the main causes of heart failure, significantly contributing to mortality. The discovery and development of effective therapies able to heal fibrotic pathological symptoms thus remain of paramount importance. Micro-physiological systems (MPS) are recently introduced as promising platforms able to accelerate this finding. Here a 3D in vitro model of human cardiac fibrosis, named uScar, is developed by imposing a cyclic mechanical stimulation to human atrial cardiac fibroblasts (AHCFs) cultured in a 3D beating heart-on-chip and exploited to screen drugs and advanced therapeutics. The sole provision of a cyclic 10% uniaxial strain at 1 Hz to the microtissues is sufficient to trigger fibrotic traits, inducing a consistent fibroblast-to-myofibroblast transition and an enhanced expression and production of extracellular matrix (ECM) proteins. Standard of care anti-fibrotic drugs (i.e., Pirfenidone and Tranilast) are confirmed to be efficient in preventing the onset of fibrotic traits in uScar. Conversely, the mechanical stimulation applied to the microtissues limit the ability of a miRNA therapy to directly reprogram fibroblasts into cardiomyocytes (CMs), despite its proved efficacy in 2D models. Such results demonstrate the importance of incorporating in vivo-like stimulations to generate more representative 3D in vitro models able to predict the efficacy of therapies in patients

Lab-on-Chip for testing myelotoxic effect of drugs and chemicals

In the last 20 years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods has approved the colony-forming unit granulocytes–macrophages test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime

Predicting human cardiac QT alterations and pro-arrhythmic effects of compounds with a 3D beating heart-on-chip platform

Determining the potential cardiotoxicity and pro-arrhythmic effects of drug candidates remains one of the most relevant issues in the drug development pipeline. New methods enabling to perform more representative pre-clinical in vitro studies by exploiting induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are under investigation to increase the translational power of the outcomes. Here we present a pharmacological campaign conducted to evaluate the drug-induced QT alterations and arrhythmic events on uHeart, a 3D miniaturized in-vitro model of human myocardium encompassing iPSC-CM and dermal fibroblasts embedded in fibrin. uHeart was mechanically trained resulting in synchronously beating cardiac microtissues in one week, characterized by a clear field potential (FP) signal that was recorded by means of an integrated electrical system. A drug screening protocol compliant with the new International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines was established and uHeart was employed for testing the effect of 11 compounds acting on single or multiple cardiac ion channels and well-known to elicit QT prolongation or arrhythmic events in clinics. The alterations of uHeart's electrophysiological parameters such as the beating period, the FP duration, the FP amplitude and the detection of arrhythmic events prior and after drug administration at incremental doses were effectively analyzed through a custom developed algorithm. Results demonstrated the ability of uHeart to successfully anticipate clinical outcome and to predict the QT prolongation with a sensitivity of 83.3%, a specificity of 100% and an accuracy of 91.6%. Cardiotoxic concentrations of drugs were notably detected in the range of the clinical highest blood drug concentration (Cmax), qualifying uHeart as a fit-to-purpose pre-clinical tool for cardiotoxicity studies

Chondrocyte Hypertrophy in Osteoarthritis: Mechanistic Studies and Models for the Identification of New Therapeutic Strategies

Articular cartilage shows limited self-healing ability owing to its low cellularity and avascularity. Untreated cartilage defects display an increased propensity to degenerate, leading to osteoarthritis (OA). During OA progression, articular chondrocytes are subjected to significant alterations in gene expression and phenotype, including a shift towards a hypertrophic-like state (with the expression of collagen type X, matrix metalloproteinases-13, and alkaline phosphatase) analogous to what eventuates during endochondral ossification. Present OA management strategies focus, however, exclusively on cartilage inflammation and degradation. A better understanding of the hypertrophic chondrocyte phenotype in OA might give new insights into its pathogenesis, suggesting potential disease-modifying therapeutic approaches. Recent developments in the field of cellular/molecular biology and tissue engineering proceeded in the direction of contrasting the onset of this hypertrophic phenotype, but knowledge gaps in the cause–effect of these processes are still present. In this review we will highlight the possible advantages and drawbacks of using this approach as a therapeutic strategy while focusing on the experimental models necessary for a better understanding of the phenomenon. Specifically, we will discuss in brief the cellular signaling pathways associated with the onset of a hypertrophic phenotype in chondrocytes during the progression of OA and will analyze in depth the advantages and disadvantages of various models that have been used to mimic it. Afterwards, we will present the strategies developed and proposed to impede chondrocyte hypertrophy and cartilage matrix mineralization/calcification. Finally, we will examine the future perspectives of OA therapeutic strategies

Beating organs-on-chip as advanced tools in drug screening: Engineered in vitro models of human organs and diseases

Drug development continuously faces challenges in efficiently predicting toxicity and verifying efficacy of new compounds during the early pre-clinical phases [1]. Organs-on-Chips (OOCs) have recently emerged as innovative in vitro tools holding the potential to improve prediction over human drug responses earlier in the timeline, thus reducing failures and costs of the clinical trials [2]. Here we present new beating OOCs providing cells with a 3D environment and mimicking the mechanical stimulation that tissues sense in vivo. This results in improved functionalities or induction of pathological changes. Our technology, named uBeat, relies on specific geometrical structures that modulate the mechanical deformation exerted on 3D microtissues, to achieve different magnitudes of either uniaxial strain or confined compression. Based on uBeat, we developed two platforms: i) uHeart, a spontaneously beating heart-on-chip integrating real-time measurement of cardiac electrophysiological signals [3] and ii) uKnee, the first in vitro 3D model of human osteoarthritic (OA) cartilage on chip [4]. We also exploited our models for drug screening purposes, by testing the effect of well-known compounds. uHeart provides 3D cardiac microtissues with a physiological cyclic uniaxial strain (~10%, 1Hz) and integrates electrodes to specifically measure the field potential (FP) signals online. Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) and human dermal fibroblast (h-DFs) were embedded in fibrin hydrogel in a 3:1 ratio and cultured for 7 days within uHeart. The cardiac model developed a synchronous beating and the electrical activity was recorded through paired electrodes specifically inserted. We preliminary calibrated the system by assessing the pro-arrhythmic effect of known compounds (i.e. Verapamil, Sotalol and Terfenadine) on uHeart electrical activity. Aspirin and DMSO were used as negative control and vehicle, respectively. As expected, Terfenadine and Sotalol prolonged the repolarization time of cardiac microtissues at 100-1000 nM and 10-60 µM, respectively. In contrast, Verapamil decreased the FP duration and both Aspirin (up to 100 µM) and DMSO (up to 0.5% w/v) did not alter the beating properties. uKnee provides 3D cartilage-like constructs with either physiological (10%, 1Hz), or hyper-physiological compression (30%,1 Hz). The latter is able to elicit OA pathogenesis by mechanical factors. Primary human articular chondrocytes were embedded in a poly(ethylene-glycol)-based hydrogel and cultured in uKnee under static chondrogenic conditions for 2 weeks. Deposition of a cartilage-like matrix was demonstrated by immunofluorescence staining (i.e. Aggrecan, Collagen II) and a stable cartilage phenotype was evidenced by the increased expression of specific genes (i.e. ACAN, PRG4, ATX, FRZB and GREM1). After maturation, constructs were subjected to additional 7 days of confined compression at both intensity levels. Hyper-physiological compression induced OA-like traits and significantly enhanced catabolic and inflammatory response, as evidenced by MMP13 and IL8 gene expression. Responses to 4 drugs already used in the clinic (i.e. Rapamacyn, Celecoxib, IL-1Ra and dexamethasone) were assessed and resulted consistent with data from animal studies, probing the potential of uKnee as anti-OA drugs screening platform. Our new technology (uBeat) allows to develop beating OOC as powerful and reliable pre-clinical tools for efficient in vitro drug screening and disease modelling

Learn, simplify and implement: developmental re-engineering strategies for cartilage repair

The limited self-healing capacity of cartilage in adult individuals, and its tendency to deteriorate once structurally damaged, makes the search for therapeutic strategies following cartilage-related traumas relevant and urgent. To date, autologous cell-based therapies represent the most advanced treatments, but their clinical success is still hampered by the long-term tendency to form fibrous as opposed to hyaline cartilage tissue. Would the efficiency and robustness of therapies be enhanced if cartilage regeneration approaches were based on the attempt to recapitulate processes occurring during cartilage development ("developmental engineering")? And from this perspective, shouldn't cartilage repair strategies be inspired by development, but adapted to be effective in a context (an injured joint in an adult individual) that is different from the embryo ("developmental re-engineering")? Here, starting from mesenchymal stem/stromal cells (MSCs) as an adult cell source possibly resembling features of the embryonic mesenchyme, we propose a developmental re-engineering roadmap based on the following three steps: (i) learn from embryonic cartilage development which are the key pathways involved in MSC differentiation towards stable cartilage, (ii) simplify the complex developmental events by approximation to essential molecular pathways, possibly by using in vitro high-throughput models and, finally, (iii) implement the outcomes at the site of the injury by establishing an appropriate interface between the delivered signals and the recipient environment (e.g., by controlling inflammation and angiogenesis). The proposed re-design of developmental machinery by establishing artificial developmental events may offer a chance for regeneration to those tissues, like cartilage, with limited capacity to recover from injuries