Cardiac Meets Skeletal: What's New in Microfluidic Models for Muscle Tissue Engineering

In the last few years microfluidics and microfabrication technique principles have been extensively exploited for biomedical applications. In this framework, organs-on-a-chip represent promising tools to reproduce key features of functional tissue units within microscale culture chambers. These systems offer the possibility to investigate the effects of biochemical, mechanical, and electrical stimulations, which are usually applied to enhance the functionality of the engineered tissues. Since the functionality of muscle tissues relies on the 3D organization and on the perfect coupling between electrochemical stimulation and mechanical contraction, great efforts have been devoted to generate biomimetic skeletal and cardiac systems to allow high-throughput pathophysiological studies and drug screening. This review critically analyzes microfluidic platforms that were designed for skeletal and cardiac muscle tissue engineering. Our aim is to highlight which specific features of the engineered systems promoted a typical reorganization of the engineered construct and to discuss how promising design solutions exploited for skeletal muscle models could be applied to improve cardiac tissue models and vice versa

Versatile electrical stimulator for providing cardiac-like electrical impulses in vitro

In the perspective of reliable methods alternative to in vivo animal testing for cardiac tissue engineering (CTE) research, the versatile electrical stimulator ELETTRA has been developed. ELETTRA delivers controlled and stable cardiac-like electrical impulses, and it can be coupled to already existing bioreactors for providing in vitro combined biomimetic culture conditions. Designed to be cost-effective and easy to use, this device could contribute to the reduction and replacement of in vivo animal experiments in CTE

Versatile electrical stimulator for cardiac tissue engineering—Investigation of charge-balanced monophasic and biphasic electrical stimulations

The application of biomimetic physical stimuli replicating the in vivo dynamic microenvironment is crucial for the in vitro development of functional cardiac tissues. In particular, pulsed electrical stimulation (ES) has been shown to improve the functional properties of in vitro cultured cardiomyocytes. However, commercially available electrical stimulators are expensive and cumbersome devices while customized solutions often allow limited parameter tunability, constraining the investigation of different ES protocols. The goal of this study was to develop a versatile compact electrical stimulator (ELETTRA) for biomimetic cardiac tissue engineering approaches, designed for delivering controlled parallelizable ES at a competitive cost. ELETTRA is based on an open-source micro-controller running custom software and is combinable with different cell/tissue culture set-ups, allowing simultaneously testing different ES patterns on multiple samples. In particular, customized culture chambers were appositely designed and manufactured for investigating the influence of monophasic and biphasic pulsed ES on cardiac cell monolayers. Finite element analysis was performed for characterizing the spatial distributions of the electrical field and the current density within the culture chamber. Performance tests confirmed the accuracy, compliance, and reliability of the ES parameters delivered by ELETTRA. Biological tests were performed on neonatal rat cardiac cells, electrically stimulated for 4 days, by comparing, for the first time, the monophasic waveform (electric field = 5 V/cm) to biphasic waveforms by matching either the absolute value of the electric field variation (biphasic ES at ±2.5 V/cm) or the total delivered charge (biphasic ES at ±5 V/cm). Findings suggested that monophasic ES at 5 V/cm and, particularly, charge-balanced biphasic ES at ±5 V/cm were effective in enhancing electrical functionality of stimulated cardiac cells and in promoting synchronous contraction

Surface-Patterned Electrode Bioreactor for Electrical Stimulation

We present a microscale cell culture system with an interdigitated microarray of excimer-laser-ablated indium tin oxide electrodes for electrical stimulation of cultured cells. The system has been characterized in a range of geometeries and stimulation regimes via electrochemical impedance spectroscopy and used to culture primary cardiomyocytes and human adipose derived stem cells. Over 6 days of culture with electrical stimulation (2 ms duration, 1 Hz, 180 μm wide electrodes with 200 μm spacing), both cell types exhibited enhanced proliferation, elongation and alignment, and adipose derived stem cells exhibited higher numbers of Connexin-43-composed gap junctions

Modeling methodology for defining a priori the hydrodynamics of a dynamic suspension bioreactor. Application to human induced pluripotent stem cell culture

Three-dimensional dynamic suspension is becoming an effective cell culture method for a wide range of bioprocesses, with an increasing number of bioreactors proposed for this purpose. The complex hydrodynamics establishing within these devices affects bioprocess outcomes and efficiency, and usually expensive in vitro trial-and-error experiments are needed to properly set the working parameters. Here we propose a methodology to define a priori the hydrodynamic working parameters of a dynamic suspension bioreactor, selected as a test case because of the complex hydrodynamics characterizing its operating condition. A combination of computational and analytical approaches was applied to generate operational guideline graphs for defining a priori specific working parameters. In detail, 43 simulations were performed under pulsed flow regime to characterize advective transport within the device depending on different operative conditions, i.e., culture medium flow rate and its duty cycle, cultured particle diameter, and initial particle suspension volume. The operational guideline graphs were then used to set specific hydrodynamic working parameters for an in vitro proof-of-principle test, where human induced pluripotent stem cell (hiPSC) aggregates were cultured for 24 h within the bioreactor. The in vitro findings showed that, under the selected pulsed flow regime, sedimentation was avoided, hiPSC aggregate circularity and viability were preserved, and culture heterogeneity was reduced, thus confirming the appropriateness of the a priori method. This methodology has the potential to be adaptable to other dynamic suspension devices to support experimental studies by providing in silico-based a priori knowledge, useful to limit costs and to optimize culture bioprocesses

Bizonal cardiac engineered tissues with differential maturation features in a mid-throughput multimodal bioreactor

Functional three-dimensional (3D) engineered cardiac tissue (ECT) models are essential for effective drug screening and biological studies. Application of physiological cues mimicking those typical of the native myocardium is known to promote the cardiac maturation and functionality in vitro. Commercially available bioreactors can apply one physical force type at a time and often in a restricted loading range. To overcome these limitations, a millimetric-scalemicroscope-integrated bioreactor was developed to deliver multiple biophysical stimuli to ECTs. In this study, we showed that the single application of auxotonic loading (passive) generated a bizonal ECT with a unique cardiac maturation pattern. Throughout the statically cultured constructs and in the ECT region exposed to high passive loading, cardiomyocytes predominantly displayed a round morphology and poor contractility ability. The ECT region with a low passive mechanical stimulation instead showed both rat- and human-origin cardiac cell maturation and organization, as well as increased ECT functionality

Control of angiogenesis and host response by modulating the cell adhesion properties of an Elastin-Like Recombinamer-based hydrogel

Producción CientíficaThe control of the in vivo vascularization of engineered tissue substitutes is essential in order to obtain either a rapid induction or a complete inhibition of the process (e.g. in muscles and hyaline-cartilage, respectively). Among the several polymers available, Elastin-Like Recombinamers (ELR)-based hydrogel stands out as a promising material for tissue engineering thanks to its viscoelastic properties, non-toxicity, and non-immunogenicity. In this study, we hypothesized that varying the cell adhesion properties of ELR-hydrogels could modulate the high angiogenic potential of adipose tissue-derived stromal vascular fraction (SVF) cells, predominantly composed of endothelial/mural and mesenchymal cells. Human SVF cells, embedded in RGD-REDV-bioactivated or unmodified ELR-hydrogels, were implanted in rat subcutaneous pockets either immediately or upon 5-day-culture in perfusion-bioreactors. Perfusion-based culture enhanced the endothelial cell cord-like-organization and the release of pro-angiogenic factors in functionalized constructs. While in vivo vascularization and host cell infiltration within the bioactivated gels were highly enhanced, the two processes were strongly inhibited in non-functionalized SVF-based hydrogels up to 28 days. ELR-based hydrogels showed a great potential to determine the successful integration of engineered substitutes thanks to their capacity to finely control the angiogenic/inflammation process at the recipient site, even in presence of SVF cells.Ministerio de Economía, Industria y Competitividad (Project MAT-2010-15982, MAT2010- 15310, PRI-PIBAR-2011-1403 and MAT2012-38043-C02-01)Junta de Castilla y León (programa de apoyo a proyectos de investigación – Ref.VA152A12-2, VA244U13 and VA155A12-2

Bioreactor Platform for Biomimetic Culture and in situ Monitoring of the Mechanical Response of in vitro Engineered Models of Cardiac Tissue

In the past two decades, relevant advances have been made in the generation of engineered cardiac constructs to be used as functional in vitro models for cardiac research or drug testing, and with the ultimate but still challenging goal of repairing the damaged myocardium. To support cardiac tissue generation and maturation in vitro, the application of biomimetic physical stimuli within dedicated bioreactors is crucial. In particular, cardiac-like mechanical stimulation has been demonstrated to promote development and maturation of cardiac tissue models. Here, we developed an automated bioreactor platform for tunable cyclic stretch and in situ monitoring of the mechanical response of in vitro engineered cardiac tissues. To demonstrate the bioreactor platform performance and to investigate the effects of cyclic stretch on construct maturation and contractility, we developed 3D annular cardiac tissue models based on neonatal rat cardiac cells embedded in fibrin hydrogel. The constructs were statically pre-cultured for 5 days and then exposed to 4 days of uniaxial cyclic stretch (sinusoidal waveform, 10% strain, 1 Hz) within the bioreactor. Explanatory biological tests showed that cyclic stretch promoted cardiomyocyte alignment, maintenance, and maturation, with enhanced expression of typical mature cardiac markers compared to static controls. Moreover, in situ monitoring showed increasing passive force of the constructs along the dynamic culture. Finally, only the stretched constructs were responsive to external electrical pacing with synchronous and regular contractile activity, further confirming that cyclic stretching was instrumental for their functional maturation. This study shows that the proposed bioreactor platform is a reliable device for cyclic stretch culture and in situ monitoring of the passive mechanical response of the cultured constructs. The innovative feature of acquiring passive force measurements in situ and along the culture allows monitoring the construct maturation trend without interrupting the culture, making the proposed device a powerful tool for in vitro investigation and ultimately production of functional engineered cardiac constructs

Spontaneous In Vivo Chondrogenesis of Bone Marrow-Derived Mesenchymal Progenitor Cells by Blocking Vascular Endothelial Growth Factor Signaling

Chondrogenic differentiation of bone marrow-derived mesenchymal stromal/stem cells (MSCs) can be induced by presenting morphogenetic factors or soluble signals but typically suffers from limited efficiency, reproducibility across primary batches, and maintenance of phenotypic stability. Considering the avascular and hypoxic milieu of articular cartilage, we hypothesized that sole inhibition of angiogenesis can provide physiological cues to direct in vivo differentiation of uncommitted MSCs to stable cartilage formation. Human MSCs were retrovirally transduced to express a decoy soluble vascular endothelial growth factor (VEGF) receptor-2 (sFlk1), which efficiently sequesters endogenous VEGF in vivo, seeded on collagen sponges and immediately implanted ectopically in nude mice. Although naïve cells formed vascularized fibrous tissue, sFlk1-MSCs abolished vascular ingrowth into engineered constructs, which efficiently and reproducibly developed into hyaline cartilage. The generated cartilage was phenotypically stable and showed no sign of hypertrophic evolution up to 12 weeks. In vitro analyses indicated that spontaneous chondrogenic differentiation by blockade of angiogenesis was related to the generation of a hypoxic environment, in turn activating the transforming growth factor-β pathway. These findings suggest that VEGF blockade is a robust strategy to enhance cartilage repair by endogenous or grafted mesenchymal progenitors. This article outlines the general paradigm of controlling the fate of implanted stem/progenitor cells by engineering their ability to establish specific microenvironmental conditions rather than directly providing individual morphogenic cues.; Chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs) is typically targeted by morphogen delivery, which is often associated with limited efficiency, stability, and robustness. This article proposes a strategy to engineer MSCs with the capacity to establish specific microenvironmental conditions, supporting their own targeted differentiation program. Sole blockade of angiogenesis mediated by transduction for sFlk-1, without delivery of additional morphogens, is sufficient for inducing MSC chondrogenic differentiation. The findings represent a relevant step forward in the field because the method allowed reducing interdonor variability in MSC differentiation efficiency and, importantly, onset of a stable, nonhypertrophic chondrocyte phenotype

Vascular Endothelial Growth Factor Sequestration Enhances In Vivo Cartilage Formation

Autologous chondrocyte transplantation for cartilage repair still has unsatisfactory clinical outcomes because of inter-donor variability and poor cartilage quality formation. Re-differentiation of monolayer-expanded human chondrocytes is not easy in the absence of potent morphogens. The Vascular Endothelial Growth Factor (VEGF) plays a master role in angiogenesis and in negatively regulating cartilage growth by stimulating vascular invasion and ossification. Therefore, we hypothesized that its sole microenvironmental blockade by either VEGF sequestration by soluble VEGF receptor-2 (Flk-1) or by antiangiogenic hyperbranched peptides could improve chondrogenesis of expanded human nasal chondrocytes (NC) freshly seeded on collagen scaffolds. Chondrogenesis of several NC donors was assessed either in vitro or ectopically in nude mice. VEGF blockade appeared not to affect NC in vitro differentiation, whereas it efficiently inhibited blood vessel ingrowth in vivo. After 8 weeks, in vivo glycosaminoglycan deposition was approximately two-fold higher when antiangiogenic approaches were used, as compared to the control group. Our data indicates that the inhibition of VEGF signaling, independently of the specific implementation mode, has profound effects on in vivo NC chondrogenesis, even in the absence of chondroinductive signals during prior culture or at the implantation site