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.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

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 (DDP). New methods enabling to perform more representative preclinical 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 1 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 preclinical 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.

Correction to: Current strategies of mechanical stimulation for maturation of cardiac microtissues.

[This corrects the article DOI: 10.1007/s12551-021-00841-6.].

Photo and Soft Lithography for Organ-on-Chip Applications.

Organs-on-Chip devices are generally fabricated by means of photo- and soft lithographic techniques. Photolithography is a process that involves the transfer of a pattern onto a substrate by a selective exposure to light. In particular, in this chapter two different photolithography methods will be described: liquid and dry photolithography. In liquid photolithography, a silicon wafer is spin-coated with liquid photoresist and exposed to UV light in order to be patterned. In dry photolithography, the silicon wafer is laminated with resist dry film before being patterned through UV light. In both cases, the UV light can be collimated on top of the wafer either through photomasks or by direct laser exposure. The obtained patterned wafer is then used as a mold for the soft lithographic process (i.e., replica molding) to produce polymer-based microdevices.

Electromechanical Stimulation of 3D Cardiac Microtissues in a Heart-on-Chip Model.

Modeling human cardiac tissues in vitro is essential to elucidate the biological mechanisms related to the heart physiopathology, possibly paving the way for new treatments. Organs-on-chips have emerged as innovative tools able to recreate tissue-specific microenvironments, guiding the development of miniaturized models and offering the opportunity to directly analyze functional readouts. Here we describe the fabrication and operational procedures for the development of a heart-on-chip model, reproducing cardiac biomimetic microenvironment. The device provides 3D cardiac microtissue with a synchronized electromechanical stimulation to support the tissue development. We additionally describe procedures for characterizing tissue evolution and functionality through immunofluorescence, real time qPCR, calcium imaging and microtissue contractility investigations.

Mechanical Induction of Osteoarthritis Traits in a Cartilage-on-a-Chip Model.

The present lack of effective therapies for osteoarthritis, the most diffused musculoskeletal disease, correlates with the absence of representative in vitro disease models. Microfabrication techniques and soft lithography allow the development of organs and tissues on chip with increased mimicry of human pathophysiology. Exploitation of polydimethylsiloxane elasticity, furthermore, permits to incorporate finely controlled mechanical actuators which are of the utmost importance in a faithful representation of the intrinsically active environment of musculoskeletal districts, to increase our comprehension of the disease onset and to successfully predict the response to pharmacological therapies. Here, we portray the fabrication and operational processes for the development of a cartilage-on-a-chip model. Additionally, we describe the methodologies to induce a phenotype reminiscent of osteoarthritis solely through hyperphysiological cyclic compression. The techniques to assess achievement of such features through immunofluorescence and gene expression are also detailed.

Current strategies of mechanical stimulation for maturation of cardiac microtissues.

The most advanced in vitro cardiac models are today based on the use of induced pluripotent stem cells (iPSCs); however, the maturation of cardiomyocytes (CMs) has not yet been fully achieved. Therefore, there is a rising need to move towards models capable of promoting an adult-like cardiomyocytes phenotype. Many strategies have been applied such as co-culture of cardiomyocytes, with fibroblasts and endothelial cells, or conditioning them through biochemical factors and physical stimulations. Here, we focus on mechanical stimulation as it aims to mimic the different mechanical forces that heart receives during its development and the post-natal period. We describe the current strategies and the mechanical properties necessary to promote a positive response in cardiac tissues from different cell sources, distinguishing between passive stimulation, which includes stiffness, topography and static stress and active stimulation, encompassing cyclic strain, compression or perfusion. We also highlight how mechanical stimulation is applied in disease modelling.

A dynamic microscale mid-throughput fibrosis model to investigate the effects of different ratios of cardiomyocytes and fibroblasts.

Cardiac fibrosis is a maladaptive remodeling of the myocardium hallmarked by contraction impairment and excessive extracellular matrix deposition (ECM). The disease progression, nevertheless, remains poorly understood and present treatments are not capable of controlling the scarring process. This is partly due to the absence of physiologically relevant, easily operable, and low-cost in vitro models, which are of the utmost importance to uncover pathological mechanisms and highlight possible targets for anti-fibrotic therapies. In classic models, fibrotic features are usually obtained using substrates with scar mimicking stiffness and/or supplementation of morphogens such as transforming growth factor ß1 (TGF-ß1). Qualities such as the interplay between activated fibroblasts (FBs) and cardiomyocytes (CMs), or the mechanically active, three-dimensional (3D) environment, are, however, neglected or obtained at the expense of the number of experimental replicates achievable. To overcome these shortcomings, we engineered a micro-physiological system (MPS) where multiple 3D cardiac micro-tissues can be subjected to cyclical stretching simultaneously. Up to six different biologically independent samples are incorporated in a single device, increasing the experimental throughput and paving the way for higher yielding drug screening campaigns. The newly developed MPS was used to co-culture different ratios of neonatal rat CMs and FBs, investigating the role of CMs in the modulation of fibrosis traits, without the addition of morphogens, and in soft substrates. The expression of contractile stress fibers and of degradative enzymes, as well as the deposition of fibronectin and type I collagen were superior in microtissues with a low amount of CMs. Moreover, high CM-based microconstructs simulating a ratio similar to that of healthy tissues, even if subjected to both cyclic stretch and TGF-ß1, did not show any of the investigated fibrotic signs, indicating a CM fibrosis modulating effect. Overall, this in vitro fibrosis model could help to uncover new pathological aspects studying, with mid-throughput and in a mechanically active, physiologically relevant environment, the crosstalk between the most abundant cell types involved in fibrosis.

Modeling In Vitro Osteoarthritis Phenotypes in a Vascularized Bone Model Based on a Bone-Marrow Derived Mesenchymal Cell Line and Endothelial Cells.

The subchondral bone and its associated vasculature play an important role in the onset of osteoarthritis (OA). Integration of different aspects of the OA environment into multi-cellular and complex human, in vitro models is therefore needed to properly represent the pathology. In this study, we exploited a mesenchymal stromal cell line/endothelial cell co-culture to produce an in vitro human model of vascularized osteogenic tissue. A cocktail of inflammatory cytokines, or conditioned medium from mechanically-induced OA engineered microcartilage, was administered to this vascularized bone model to mimic the inflamed OA environment, hypothesizing that these treatments could induce the onset of specific pathological traits. Exposure to the inflammatory factors led to increased network formation by endothelial cells, reminiscent of the abnormal angiogenesis found in OA subchondral bone, demineralization of the constructs, and increased collagen production, signs of OA related bone sclerosis. Furthermore, inflammation led to augmented expression of osteogenic (alkaline phosphatase (ALP) and osteocalcin (OCN)) and angiogenic (vascular endothelial growth factor (VEGF)) genes. The treatment, with a conditioned medium from the mechanically-induced OA engineered microcartilage, also caused increased demineralization and expression of ALP, OCN, ADAMTS5, and VEGF; however, changes in network formation by endothelial cells were not observed in this second case, suggesting a possible different mechanism of action in inducing OA-like phenotypes. We propose that this vascularized bone model could represent a first step for the in vitro study of bone changes under OA mimicking conditions and possibly serve as a tool in testing anti-OA drugs.

Engineered nasal cartilage for the repair of osteoarthritic knee cartilage defects.

Osteoarthritis (OA) is the most prevalent joint disorder, causing pain and disability predominantly in the aging population but also affecting young individuals. Current treatments are limited to use of anti-inflammatory drugs to alleviate symptoms or degenerated joint replacement by a prosthetic implant at the end stage of the disease. We hypothesized that degenerative cartilage defects can be treated using nasal chondrocyte­based tissue-engineered cartilage (N-TEC). We demonstrate that N-TEC maintained cartilaginous properties when exposed in vitro to inflammatory stimuli found in osteoarthritic joints and favorably altered the inflammatory profile of cells from osteoarthritic joints. These effects were at least partially mediated by down-regulation of the WNT (wingless/integrated) signaling pathway through sFRP1 (secreted frizzled-related protein-1). We further report that N-TEC survive and engraft in vivo in ectopic mouse models reproducing a human osteochondral OA tissue environment, as well as in sheep articular cartilage defects that mimic degenerative settings. Last, we tested the safety of autologous N-TEC for the treatment of osteoarthritic cartilage defects in the knees of two patients with advanced OA (Kellgren and Lawrence grades 3 and 4) who were otherwise considered for unicondylar knee arthroplasty. No adverse reactions were recorded, and patients reported reduced pain as well as improved joint function and life quality 14 months after surgery. Together, our findings indicate that N-TEC can directly contribute to cartilage repair in osteoarthritic joints. A suitably powered clinical trial is now required to assess its efficacy in the treatment of patients with OA.

Micro-electrode channel guide (µECG) technology: an online method for continuous electrical recording in a human beating heart-on-chip.

Cardiac toxicity still represents a common adverse outcome causing drug attrition and post-marketing withdrawal. The development of relevantin vitromodels resembling the human heart recently opened the path towards a more accurate detection of drug-induced human cardiac toxicity early in the drug development process. Organs-on-chip have been proposed as promising tools to recapitulatein vitrothe key aspects of thein vivocardiac physiology and to provide a means to directly analyze functional readouts. In this scenario, a new device capable of continuous monitoring of electrophysiological signals from functionalin vitrohuman hearts-on-chip is here presented. The development of cardiac microtissues was achieved through a recently published method to control the mechanical environment, while the introduction of a technology consisting in micro-electrode coaxial guides allowed to conduct direct and non-destructive electrophysiology studies. The generated human cardiac microtissues exhibited synchronous spontaneous beating, as demonstrated by multi-point and continuous acquisition of cardiac field potential, and expression of relevant genes encoding for cardiac ion-channels. A proof-of-concept pharmacological validation on three drugs proved the proposed model to potentially be a powerful tool to evaluate functional cardiac toxicity.

Intervertebral Disc-on-a-Chip as Advanced In Vitro Model for Mechanobiology Research and Drug Testing: A Review and Perspective.

Discogenic back pain is one of the most diffused musculoskeletal pathologies and a hurdle to a good quality of life for millions of people. Existing therapeutic options are exclusively directed at reducing symptoms, not at targeting the underlying, still poorly understood, degenerative processes. Common intervertebral disc (IVD) disease models still do not fully replicate the course of degenerative IVD disease. Advanced disease models that incorporate mechanical loading are needed to investigate pathological causes and processes, as well as to identify therapeutic targets. Organs-on-chip (OoC) are microfluidic-based devices that aim at recapitulating tissue functions in vitro by introducing key features of the tissue microenvironment (e.g., 3D architecture, soluble signals and mechanical conditioning). In this review we analyze and depict existing OoC platforms used to investigate pathological alterations of IVD cells/tissues and discuss their benefits and limitations. Starting from the consideration that mechanobiology plays a pivotal role in both IVD homeostasis and degeneration, we then focus on OoC settings enabling to recapitulate physiological or aberrant mechanical loading, in conjunction with other relevant features (such as inflammation). Finally, we propose our view on design criteria for IVD-on-a-chip systems, offering a future perspective to model IVD mechanobiology.

Blockage of bone morphogenetic protein signalling counteracts hypertrophy in a human osteoarthritic micro-cartilage model.

Bone morphogenetic protein (BMP) signalling plays a significant role during embryonic cartilage development and has been associated with osteoarthritis (OA) pathogenesis, being in both cases involved in triggering hypertrophy. Inspired by recent findings that BMP inhibition counteracts hypertrophic differentiation of human mesenchymal progenitors, we hypothesized that selective inhibition of BMP signalling would mitigate hypertrophic features in OA cartilage. First, a 3D in vitro OA micro-cartilage model was established using minimally expanded OA chondrocytes that was reproducibly able to capture OA-like hypertrophic features. BMP signalling was then restricted by means of two BMP receptor type I inhibitors, resulting in reduction of OA hypertrophic traits while maintaining synthesis of cartilage extracellular matrix. Our findings open potential pharmacological strategies for counteracting cartilage hypertrophy in OA and support the broader perspective that key signalling pathways known from developmental processes can guide the understanding, and possibly the mitigation, of adult pathological features.

Integrating Biosensors in Organs-on-Chip Devices: A Perspective on Current Strategies to Monitor Microphysiological Systems.

Organs-on-chip (OoC), often referred to as microphysiological systems (MPS), are advanced in vitro tools able to replicate essential functions of human organs. Owing to their unprecedented ability to recapitulate key features of the native cellular environments, they represent promising tools for tissue engineering and drug screening applications. The achievement of proper functionalities within OoC is crucial; to this purpose, several parameters (e.g., chemical, physical) need to be assessed. Currently, most approaches rely on off-chip analysis and imaging techniques. However, the urgent demand for continuous, noninvasive, and real-time monitoring of tissue constructs requires the direct integration of biosensors. In this review, we focus on recent strategies to miniaturize and embed biosensing systems into organs-on-chip platforms. Biosensors for monitoring biological models with metabolic activities, models with tissue barrier functions, as well as models with electromechanical properties will be described and critically evaluated. In addition, multisensor integration within multiorgan platforms will be further reviewed and discussed.

Hyperphysiological compression of articular cartilage induces an osteoarthritic phenotype in a cartilage-on-a-chip model.

Owing to population aging, the social impact of osteoarthritis (OA)-the most common musculoskeletal disease-is expected to increase dramatically. Yet, therapy is still limited to palliative treatments or surgical intervention, and disease-modifying OA (DMOA) drugs are scarce, mainly because of the absence of relevant preclinical OA models. Therefore, in vitro models that can reliably predict the efficacy of DMOA drugs are needed. Here, we show, using a newly developed microphysiological cartilage-on-a-chip model that enables the application of strain-controlled compression to three-dimensional articular cartilage microtissue, that a 30% confined compression recapitulates the mechanical factors involved in OA pathogenesis and is sufficient to induce OA traits. Such hyperphysiological compression triggers a shift in cartilage homeostasis towards catabolism and inflammation, hypertrophy, and the acquisition of a gene expression profile akin to those seen in clinical osteoarthritic tissue. The cartilage on-a-chip model may enable the screening of DMOA candidates.

Challenges Toward the Identification of Predictive Markers for Human Mesenchymal Stromal Cells Chondrogenic Potential.

Human bone marrow derived mesenchymal stromal cells (BMSCs) represent a putative cell source candidate for tissue engineering-based strategies to repair cartilage and bone. However, traditional isolation of BMSCs by adhesion to plastic leads to very heterogeneous cell populations, accounting for high variability of chondrogenic differentiation outcome, both across donors and across clonally derived strains. Identification of putative surface markers able to select BMSC subpopulations with higher chondrogenic capacity (CC) and reduced variance in chondrogenic differentiation could aid the development of BMSC-based cartilage and bone regeneration approaches. With the goal to identify predictive markers for chondrogenic BMSC populations, we assessed the gene expression profile of single cell-derived clones with high and low CC. While a clustering between high and low CC clones was observed for one donor, donor-to-donor variability hampered the possibility to achieve conclusive results when different donors were considered. Nevertheless, increased NCAM1/CD56 expression correlated in clones derived from one donor with higher CC, the same trend was observed for three additional donors (even if no significance was achieved). Enriching multiclonal BMSCs for CD56+ expression led to an increase in CC, though still highly affected by donor-to-donor variability. Our study finally suggests that definition of predictive marker(s) for BMSCs chondrogenesis is challenged by the large donor heterogeneity of these cells, and by the high complexity and plasticity of the BMSCs system. Multiple pathways and external parameters may be indeed involved in determining the chondrogenic potential of BMSCs, making the identification of putative markers still an open issue. Stem Cells Translational Medicine 2019;8:194&11.

Developmentally inspired programming of adult human mesenchymal stromal cells toward stable chondrogenesis.

It is generally accepted that adult human bone marrow-derived mesenchymal stromal cells (hMSCs) are default committed toward osteogenesis. Even when induced to chondrogenesis, hMSCs typically form hypertrophic cartilage that undergoes endochondral ossification. Because embryonic mesenchyme is obviously competent to generate phenotypically stable cartilage, it is questioned whether there is a correspondence between mesenchymal progenitor compartments during development and in adulthood. Here we tested whether forcing specific early events of articular cartilage development can program hMSC fate toward stable chondrogenesis. Inspired by recent findings that spatial restriction of bone morphogenetic protein (BMP) signaling guides embryonic progenitors toward articular cartilage formation, we hypothesized that selective inhibition of BMP drives the phenotypic stability of hMSC-derived chondrocytes. Two BMP type I receptor-biased kinase inhibitors were screened in a microfluidic platform for their time- and dose-dependent effect on hMSC chondrogenesis. The different receptor selectivity profile of tested compounds allowed demonstration that transient blockade of both ALK2 and ALK3 receptors, while permissive to hMSC cartilage formation, is necessary and sufficient to maintain a stable chondrocyte phenotype. Remarkably, even upon compound removal, hMSCs were no longer competent to undergo hypertrophy in vitro and endochondral ossification in vivo, indicating the onset of a constitutive change. Our findings demonstrate that adult hMSCs effectively share properties of embryonic mesenchyme in the formation of transient but also of stable cartilage. This opens potential pharmacological strategies to articular cartilage regeneration and more broadly indicates the relevance of developmentally inspired protocols to control the fate of adult progenitor cell systems.

A three-dimensional in vitro dynamic micro-tissue model of cardiac scar formation.

In vitro cardiac models able to mimic the fibrotic process are paramount to develop an effective anti-fibrosis therapy that can regulate fibroblast behaviour upon myocardial injury. In previously developed in vitro models, typical fibrosis features were induced by using scar-like stiffness substrates and/or potent morphogen supplementation in monolayer cultures. In our model, we aimed to mimic in vitro a fibrosis-like environment by applying cyclic stretching of cardiac fibroblasts embedded in three-dimensional fibrin-hydrogels alone. Using a microfluidic device capable of delivering controlled cyclic mechanical stretching (10% strain at 1 Hz), some of the main fibrosis hallmarks were successfully reproduced in 7 days. Cyclic strain indeed increased cell proliferation, extracellular matrix (ECM) deposition (e.g. type-I-collagen, fibronectin) and its stiffness, forming a scar-like tissue with superior quality compared to the supplementation of TGFß1 alone. Taken together, the observed findings resemble some of the key steps in the formation of a scar: (i) early fibroblast proliferation, (ii) later phenotype switch into myofibroblasts, (iii) ECM deposition and (iv) stiffening. This in vitro scar-on-a-chip model represents a big step forward to investigate the early mechanisms possibly leading later to fibrosis without any possible confounding supplementation of exogenous potent morphogens.