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Liver disease cases are rapidly expanding worldwide, and transplantation remains the only effective cure for end-stage disease. There is an increasing demand for developing potential drug treatments, and regenerative therapies using in-vitro culture platforms. Human decellularized extracellular matrix (dECM) is an appealing alternative to conventional animal tissues as it contains human-specific proteins and can serve as scaffolding materials. Herein we exploit this with human donor tissue from discarded liver which was not suitable for transplant using a synergistic approach to combining biological and topographical cues in electrospun materials as an in-vitro culture platform. To realise this, we developed a methodology for incorporating human liver dECM into electrospun polycaprolactone (PCL) fibres with surface nanotopographies (230-580 nm). The hybrid scaffolds were fabricated using varying concentrations of dECM; their morphology, mechanical properties, hydrophilicity and stability were analysed. The scaffolds were validated using HepG2 and primary mouse hepatocytes, with subsequent results indicating that the modified scaffolds-maintained cell growth and influenced cell attachment, proliferation and hepatic-related gene expression. This work demonstrates a novel approach to harvesting the potential from decellularized human tissues in the form of innovative in-vitro culture platforms for liver., (© 2024. The Author(s).)

Left ventricular twist is influenced by the unique oriented structure of myocardial fibers. Replicating this intricate structural-functional relationship in an in vitro heart model remains challenging, mainly due to the difficulties in achieving a complex structure with synchrony between layers. This study introduces a novel approach through the utilization of bioprinting-assisted tissue assembly (BATA)-a synergistic integration of bioprinting and tissue assembly strategies. By flexibly manufacturing tissue modules and assembly platforms, BATA can create structures that traditional methods find difficult to achieve. This approach integrates engineered heart tissue (EHT) modules, each with intrinsic functional and structural characteristics, into a layered, multi-oriented tissue in a controlled manner. EHTs assembled in different orientations exhibit various contractile forces and electrical signal patterns. The BATA is capable of constructing complex myocardial fiber orientations within a chamber-like structure (MoCha). MoCha replicates the native cardiac architecture by exhibiting three layers and three alignment directions, and it reproduces the left ventricular twist by exhibiting synchronized contraction between layers and mimicking the native cardiac architecture. The potential of BATA extends to engineering tissues capable of constructing and functioning as complete organs on a large scale. This advancement holds the promise of realizing future organ-on-demand technology., (© 2024 The Authors. Advanced Materials published by Wiley‐VCH GmbH.)

Since cardiovascular diseases (CVDs) are globally one of the leading causes of death, of which myocardial infarction (MI) can cause irreversible damage and decrease survivors' quality of life, novel therapeutics are needed. Current approaches such as organ transplantation do not fully restore cardiac function or are limited. As a valuable strategy, tissue engineering seeks to obtain constructs that resemble myocardial tissue, vessels, and heart valves using cells, biomaterials as scaffolds, biochemical and physical stimuli. The latter can be induced using a bioreactor mimicking the heart's physiological environment. An extensive review of bioreactors providing perfusion, mechanical and electrical stimulation, as well as the combination of them is provided. An analysis of the stimulations' mechanisms and modes that best suit cardiac construct culture is developed. Finally, we provide insights into bioreactor configuration and culture assessment properties that need to be elucidated for its clinical translation., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cell encapsulation within three-dimensional hydrogels is a promising approach to mimic tissues. However, true biomimicry of the intricate microenvironment, biophysical and biochemical gradients, and the macroscale hierarchical spatial organizations of native tissues is an unmet challenge within tissue engineering. This review provides an overview of the macromolecular chemistries that have been applied toward the design of cell-friendly hydrogels, as well as their application toward controlling biophysical and biochemical bulk and gradient properties of the microenvironment. Furthermore, biofabrication technologies provide the opportunity to simultaneously replicate macroscale features of native tissues. Biofabrication strategies are reviewed in detail with a particular focus on the compatibility of these strategies with the current macromolecular toolkit described for hydrogel design and the challenges associated with their clinical translation. This review identifies that the convergence of the ever-expanding macromolecular toolkit and technological advancements within the field of biofabrication, along with an improved biological understanding, represents a promising strategy toward the successful tissue regeneration., (© 2023 The Authors. Macromolecular Bioscience published by Wiley‐VCH GmbH.)

Tissue engineering strategies have been extensively exploited to generate functional cardiac patches. To maintain cardiac functionality in vitro, bioreactors have been designed to provide perfusion and electrical stimulation, alone or combined. However, due to several design limitations the integration of optical systems to assess cardiac maturation level is still missing within these platforms. Here we present a bioreactor culture chamber that provides 3D cardiac constructs with a bidirectional interstitial perfusion and biomimetic electrical stimulation, allowing direct cellular optical monitoring and contractility test. The chamber design was optimized through finite element models to house an innovative scaffold anchoring system to hold and to release it for the evaluation of tissue maturation and functionality by contractility tests. Neonatal rat cardiac fibroblasts subjected to a combined perfusion and electrical stimulation showed positive cell viability over time. Neonatal rat cardiomyocytes were successfully monitored for the entire culture period to assess their functionality. The combination of perfusion and electrical stimulation enhanced patch maturation, as evidenced by the higher contractility, the enhanced beating properties and the increased level of cardiac protein expression. This new multifunctional bioreactor provides a relevant biomimetic environment allowing for independently culturing, real-time monitoring and testing up to 18 separated patches.

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., Competing Interests: The authors declare no conflict of interest.

With the increasing attention on cardiovascular disorders and the current inability of pre-clinical models to accurately predict human physiology, the need for advanced and reliable heart in vitro models is paramount. Microfabrication technologies provide potential solutions in the organs-on-chip systems: microengineered devices where cell cultures can be hosted and cultured to develop three-dimensional models or microtissues with high similarity to human physiology. We here described the fabrication and operation procedures for a beating heart-on-a-chip. The device features a culture region for a 3D cardiac microtissue and a system for applying tuned mechanical stimulation during culture to improve cardiac development. We additionally describe procedures for characterizing tissue maturation via immunofluorescence and functional evaluations of microtissue contractility., (© 2018 Elsevier Inc. All rights reserved.)

Cardiovascular disease is a major cause of mortality worldwide, and current preclinical models including traditional animal models and 2D cell culture models have limitations in replicating human native heart physiology and response to drugs. Heart-on-a-chip (HoC) technology offers a promising solution by combining the advantages of cardiac tissue engineering and microfluidics to create in vitro 3D cardiac models, which can mimic key aspects of human microphysiological systems and provide controllable microenvironments. Herein, recent advances in HoC technologies are introduced, including engineered cardiac microtissue construction in vitro, microfluidic chip fabrication, microenvironmental stimulation, and real-time feedback systems. The development of cardiac tissue engineering methods is focused for 3D microtissue preparation, advanced strategies for HoC fabrication, and current applications of these platforms. Major challenges in HoC fabrication are discussed and the perspective on the potential for these platforms is provided to advance research and clinical applications., (© 2023 Wiley-VCH GmbH.)

In recent decades, microphysiological constructs and systems (MPCs and MPSs) have undergone significant development, ranging from self-organized organoids to high-throughput organ-on-a-chip platforms. Advances in biomaterials, bioinks, 3D bioprinting, micro/nanofabrication, and sensor technologies have contributed to diverse and innovative biofabrication tactics. MPCs and MPSs, particularly tissue chips relevant to absorption, distribution, metabolism, excretion, and toxicity, have demonstrated potential as precise, efficient, and economical alternatives to animal models for drug discovery and personalized medicine. However, current approaches mainly focus on the in vitro recapitulation of the human anatomical structure and physiological-biochemical indices at a single or a few simple levels. This review highlights the recent remarkable progress in MPC and MPS models and their applications. The challenges that must be addressed to assess the reliability, quantify the techniques, and utilize the fidelity of the models are also discussed., (© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.)

Engineering cardiac tissues and organ models remains a great challenge due to the hierarchical structure of the native myocardium. The need of integrating blood vessels brings additional complexity, limiting the available approaches that are suitable to produce integrated cardiovascular organoids. In this work we propose a novel hybrid strategy based on 3D bioprinting, to fabricate endothelialized myocardium. Enabled by the use of our composite bioink, endothelial cells directly bioprinted within microfibrous hydrogel scaffolds gradually migrated towards the peripheries of the microfibers to form a layer of confluent endothelium. Together with controlled anisotropy, this 3D endothelial bed was then seeded with cardiomyocytes to generate aligned myocardium capable of spontaneous and synchronous contraction. We further embedded the organoids into a specially designed microfluidic perfusion bioreactor to complete the endothelialized-myocardium-on-a-chip platform for cardiovascular toxicity evaluation. Finally, we demonstrated that such a technique could be translated to human cardiomyocytes derived from induced pluripotent stem cells to construct endothelialized human myocardium. We believe that our method for generation of endothelialized organoids fabricated through an innovative 3D bioprinting technology may find widespread applications in regenerative medicine, drug screening, and potentially disease modeling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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.

Cell adhesion plays a key role in cell behavior, in terms of migration, proliferation, differentiation and apoptosis. All of these events concur with tissue regeneration and remodeling mechanisms, integrating a complex network of intracellular signaling modules. Morphogenetic responses, which involve changes in cell shape, proliferation and differentiation, are thought to be controlled by both biochemical and biophysical cues. Indeed, the extracellular matrix not only displays adhesive ligands necessary for cell adhesion but also plays an essential biomechanical role - responsible, for instance, for the acquisition of the contractile phenotype. The substrate topography around the forming tissues and the associated mechanical stresses that are generated regulate cellular morphology, proliferation and differentiation. Thus, the ability to tailor topographical features around cells can be a crucial design parameter in tissue engineering applications, inducing cells to exhibit the required performances.In this work, we designed micropillared substrates using highly spaced arrays (interspacing equal to 25 µm) to evaluate the effects of topography on C2C12 myoblasts' adhesion and differentiation. Optical and fluorescence microscopy images were used to observe cell adhesion, together with Western blot analysis on vinculin and focal adhesion kinase (FAK) expression, a protein highly involved in adhesive processes. Differentiation marker (Myf5, myogenin and myosin heavy chain [MHC]) expression was also studied, in relation to the effect of different substrate topographies on the enhancement of a contractile phenotype. Our results demonstrated that microstructured surfaces may play a key role in the regeneration of functional tissues.

The development of in vitro models to screen the effect of different concentrations, combinations and temporal sequences of morpho-regulatory factors on stem/progenitor cells is crucial to investigate and possibly recapitulate developmental processes with adult cells. Here, we designed and validated a microfluidic platform to (i) allow cellular condensation, (ii) culture 3D micromasses of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) under continuous flow perfusion, and (ii) deliver defined concentrations of morphogens to specific culture units. Condensation of hBM-MSCs was obtained within 3 hours, generating micromasses in uniform sizes (56.2 ± 3.9 μm). As compared to traditional macromass pellet cultures, exposure to morphogens involved in the first phases of embryonic limb development (i.e. Wnt and FGF pathways) yielded more uniform cell response throughout the 3D structures of perfused micromasses (PMMs), and a 34-fold higher percentage of proliferating cells at day 7. The use of a logarithmic serial dilution generator allowed to identify an unexpected concentration of TGFβ3 (0.1 ng/ml) permissive to hBM-MSCs proliferation and inductive to chondrogenesis. This proof-of-principle study supports the described microfluidic system as a tool to investigate processes involved in mesenchymal progenitor cells differentiation, towards a 'developmental engineering' approach for skeletal tissue regeneration.

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.

Bioreactors enabling direct perfusion of cell suspensions or culture media through the pores of 3D scaffolds have long been used in tissue engineering to improve cell seeding efficiency as well as uniformity of cell distribution and tissue development. A macro-scale U-shaped bioreactor for cell culture under perfusion (U-CUP) has been previously developed. In that system, the geometry of the perfusion chamber results in rather uniform flow through most of the scaffold volume, but not in the peripheral regions. Here, the design of the perfusion chamber has been optimized to provide a more homogenous perfusion flow through the scaffold. Then, the design of this macro-scale flow-optimized perfusion bioreactor (macro-Flopper) has been miniaturized to create a mini-scale device (mini-Flopper) compatible with medium-throughput assays. Computational fluid dynamic (CFD) modeling of the new chamber design, including a porous scaffold structure, revealed that Flopper bioreactors provide highly homogenous flow speed, pressure, and shear stress. Finally, a proof-of-principle of the functionality of the Flopper systems by engineering endothelialized stromal tissues using human adipose tissue-derived stromal vascular fraction (SVF) cells has been offered. Preliminary evidence showing that flow optimization improves cell maintenance in the engineered tissues will have to be confirmed in future studies. In summary, two bioreactor models with optimized perfusion flow and complementary sizes have been proposed that might be exploited to engineer homogenous tissues and, in the case of the mini-Flopper, for drug testing assays with a limited amount of biological material., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Cornea as the outermost layer of the eye is at risk of various genetic and environmental diseases that can damage the cornea and impair vision. Corneal transplantation is among the most applicable surgical procedures for repairing the defected tissue. However, the scarcity of healthy tissue donations as well as transplantation failure has remained as the biggest challenges in confront of corneal grafting. Therefore, alternative approaches based on stem-cell transplantation and classic regenerative medicine have been developed for corneal regeneration. In this review, the application and limitation of the recently-used advanced approaches for regeneration of cornea are discussed. Additionally, other emerging powerful techniques such as 5D printing as a new branch of scaffold-based technologies for construction of tissues other than the cornea are highlighted and suggested as alternatives for corneal reconstruction. The introduced novel techniques may have great potential for clinical applications in corneal repair including disease modeling, 3D pattern scheming, and personalized medicine., (© 2022 John Wiley & Sons Ltd.)

Gelatin methacryloyl (GelMA), a photocrosslinkable gelatin-based hydrogel, has been immensely used for diverse applications in tissue engineering and drug delivery. Apart from its excellent functionality and versatile mechanical properties, it is also suitable for a wide range of fabrication methodologies to generate tissue constructs of desired shapes and sizes. Despite its exceptional characteristics, it is predominantly limited by its weak mechanical strength, as some tissue types naturally possess high mechanical stiffness. The use of high GelMA concentrations yields high mechanical strength, but not without the compromise in its porosity, degradability, and three-dimensional (3D) cell attachment. Recently, GelMA has been blended with various natural and synthetic biomaterials to reinforce its physical properties to match with the tissue to be engineered. Among these, nanomaterials have been extensively used to form a composite with GelMA, as they increase its biological and physicochemical properties without affecting the unique characteristics of GelMA and also introduce electrical and magnetic properties. This review article presents the recent advances in the formation of hybrid GelMA nanocomposites using a variety of nanomaterials (carbon, metal, polymer, and mineral-based). We give an overview of each nanomaterial's characteristics followed by a discussion of the enhancement in GelMA's physical properties after its incorporation. Finally, we also highlight the use of each GelMA nanocomposite for different applications, such as cardiac, bone, and neural regeneration., (© 2021 Wiley Periodicals LLC.)

The mechanical environment of living cells is as critical as chemical signaling. Mechanical stimuli play a pivotal role in organogenesis and tissue homeostasis. Unbalances in mechanotransduction pathways often lead to diseases, such as cancer, cystic fibrosis, and neurodevelopmental disorders. Despite its inherent relevance, there is a lack of proper mechanoresponsive in vitro study systems. In this context, there is an urge to engineer innovative, robust, dynamic, and reliable organotypic technologies to better connect cellular processes to organ-level function and multi-tissue cross-talk. Mechanically active organoid-on-chip has the potential to surpass this challenge. These systems converge microfabrication, microfluidics, biophysics, and tissue engineering fields to emulate key features of living organisms, hence, reducing costs, time, and animal testing. In this review, we intended to present cutting-edge organ-on-chip platforms that integrate biomechanical stimuli as well as novel multicellular culture, such as organoids. We focused on its application in two main fields: precision medicine and drug development. Moreover, we also discussed the state of the art for the development of an engineered model to assess patient-derived tumor organoid metastatic potential. Finally, we highlighted the current drawbacks and emerging opportunities to match the industry needs. We envision the use of mechanoresponsive organotypic-on-chip microdevices as an indispensable tool for precision medicine, drug development, disease modeling, tissue engineering, and developmental biology., (© 2021 John Wiley & Sons Ltd.)

A sound understanding of developmental biology is part of the foundation of effective stem cell-derived tissue engineering. Here, the key concepts of cardiac development that are successfully applied in a bioinspired approach to growing engineered cardiac tissues, are reviewed. The native cardiac milieu is studied extensively from embryonic to adult phenotypes, as it provides a resource of factors, mechanisms, and protocols to consider when working toward establishing living tissues in vitro. It begins with the various cell types that constitute the cardiac tissue. It is discussed how myocytes interact with other cell types and their microenvironment and how they change over time from the embryonic to the adult states, with a view on how such changes affect the tissue function and may be used in engineered tissue models. Key embryonic signaling pathways that have been leveraged in the design of culture media and differentiation protocols are presented. The cellular microenvironment, from extracellular matrix chemical and physical properties, to the dynamic mechanical and electrical forces that are exerted on tissues is explored. It is shown that how such microenvironmental factors can inform the design of biomaterials, scaffolds, stimulation bioreactors, and maturation readouts, and suggest considerations for ongoing biomimetic advancement of engineered cardiac tissues and regeneration strategies for the future., (© 2021 Wiley-VCH GmbH.)

Many techniques for engineering and interrogating three-dimensional (3-D) muscle bundles from animal- or patient-derived myoblasts have recently been developed to overcome the limitations of existing in vitro and in vivo model systems. However, many approaches for engineering 3-D muscle bundles rely on specialized and time-consuming techniques, such as photolithography for fabrication and cryosectioning for histology. Cryosectioning also limits visualization to a single plane instead of the entire 3-D structure. To address these challenges, we first implemented a consumer-grade 3-D-printer to rapidly prototype multiple templates for engineering muscle bundles. We then employed our templates to engineer 3D muscle bundles and identify template geometries that promoted bundle survival over three weeks. Subsequently, we implemented tissue clearing, immunostaining, and confocal imaging to acquire z-stacks of intact muscle bundles labelled for myogenic markers. With this approach, we could select the imaging plane on-demand and visualize the intact 3-D structure of bundles. However, tissue clearing did cause some tissue degradation that should be considered. Together, these advances in muscle tissue engineering and imaging will accelerate the use of these 3-D tissue platforms for disease modeling and therapeutic discovery.

Gelatin methacrylate (GelMA) is a photo-cross-linkable biopolymer. A combination of GelMA with biodegradable polyesters such as PCL (poly‐ε‐caprolactone) and their triblock derivatives improve the mechanical properties of GelMA. PCL-PEG-PCL (PCEC) was synthesized using ring-opening polymerization of ε-caprolactone in the presence of polyethylene glycol (PEG). The GelMA- PCEC was fabricated using freeze-drying and 3D printing and their porosity, mechanical properties, and swelling behavior were investigated. Human dental pulp stem cells were cultured on the scaffolds for a period of 14 days and cell adhesion was evaluated by scanning electron microscopy. Cell viability was analyzed by MTT and osteogenic differentiation was evaluated by Alizarin red S. Results showed that the 3D-printed scaffold had higher water absorption rate, retaining its structure up to a strain of 0.2 %, and a higher Young's modulus compared to the freeze-dried scaffold. In terms of cell viability, the 3D-printed scaffold outperformed the freeze-dried scaffold with a percentage of 86 % and 63 % viability respectively. Moreover, the 3D-printed scaffold exhibited better osteodifferentiation with calcium deposition. Overall, these findings suggest that the 3D-printed scaffold may have advantages over the freeze-dried scaffold in tissue engineering applications that require high water absorption, elasticity, and cell viability. The fabricated scaffolds provided suitable cell proliferation. [ABSTRACT FROM AUTHOR]

A major challenge in myocardial tissue engineering is replicating the heart's highly complex three-dimensional (3D) anisotropic structure. Heart-on-a-chip (HOC) is an emerging technology for constructing myocardial tissue in vitro in recent years, but most existing HOC systems face difficulties in constructing 3D myocardial tissue aligned with multiple cell layers. Electrospun nanofibers are commonly used as scaffolds for cell growth in myocardial tissue engineering, which can structurally simulate the extracellular matrix to induce the aligned growth of myocardial cells. Here, we developed an HOC that integrates multi-layered aligned polycaprolactone (PCL) nanofiber scaffolds inside microfluidic chips, and constructed 3D thick and aligned tissue with a layered seeding approach. By culturing human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) on chip, the myocardial tissue on the two layered nanofibers reached a thickness of ~53 μm compared with ~19 μm for single-layered nanofibers. The obtained myocardial tissue presented well-aligned structures, with densely distributed α-actinin. By the third day post seeding, the hiPSC-CMs contract highly synchronously, with a contraction frequency of 18 times/min. The HOC with multi-layered biomimetic scaffolds provided a dynamic in vitro culture environment for hiPSC-CMs. Together with the layered cell-seeding process, the designed HOC promoted the formation of thick, well-aligned myocardial tissue. [ABSTRACT FROM AUTHOR]

Atherosclerosis is the primary cause of cardiovascular disease, resulting in mortality, elevated healthcare costs, diminished productivity, and reduced quality of life for individuals and their communities. This is exacerbated by the limited understanding of its underlying causes and limitations in current therapeutic interventions, highlighting the need for sophisticated models of atherosclerosis. This review critically evaluates the computational and biological models of atherosclerosis, focusing on the study of hemodynamics in atherosclerotic coronary arteries. Computational models account for the geometrical complexities and hemodynamics of the blood vessels and stenoses, but they fail to capture the complex biological processes involved in atherosclerosis. Different in vitro and in vivo biological models can capture aspects of the biological complexity of healthy and stenosed vessels, but rarely mimic the human anatomy and physiological hemodynamics, and require significantly more time, cost, and resources. Therefore, emerging strategies are examined that integrate computational and biological models, and the potential of advances in imaging, biofabrication, and machine learning is explored in developing more effective models of atherosclerosis. [ABSTRACT FROM AUTHOR]

Organ-on-chip (OOC) technology has gained importance for biomedical studies and drug development. This technology involves microfluidic devices that mimic the structure and function of specific human organs or tissues. OOCs are a promising alternative to traditional cell-based models and animals, as they provide a more representative experimental model of human physiology. By creating a microenvironment that closely resembles in vivo conditions, OOC platforms enable the study of intricate interactions between different cells as well as a better understanding of the underlying mechanisms pertaining to diseases. OOCs can be integrated with other technologies, such as sensors and imaging systems to monitor real-time responses and gather extensive data on tissue behavior. Despite these advances, OOCs for many organs are in their initial stages of development, with several challenges yet to be overcome. These include improving the complexity and maturity of these cellular models, enhancing their reproducibility, standardization, and scaling them up for high-throughput uses. Nonetheless, OOCs hold great promise in advancing biomedical research, drug discovery, and personalized medicine, benefiting human health and well-being. Here, we review several recent OOCs that attempt to overcome some of these challenges. These OOCs with unique applications can be engineered to model organ systems such as the stomach, cornea, blood vessels, and mouth, allowing for analyses and investigations under more realistic conditions. With this, these models can lead to the discovery of potential therapeutic interventions. In this review, we express the significance of the relationship between mucosal tissues and vasculature in organ-on-chip (OOC) systems. This interconnection mirrors the intricate physiological interactions observed in the human body, making it crucial for achieving accurate and meaningful representations of biological processes within OOC models. Vasculature delivers essential nutrients and oxygen to mucosal tissues, ensuring their proper function and survival. This exchange is critical for maintaining the health and integrity of mucosal barriers. This review will discuss the OOCs used to represent the mucosal architecture and vasculature, and it can encourage us to think of ways in which the integration of both can better mimic the complexities of biological systems and gain deeper insights into various physiological and pathological processes. This will help to facilitate the development of more accurate predictive models, which are invaluable for advancing our understanding of disease mechanisms and developing novel therapeutic interventions. [ABSTRACT FROM AUTHOR]

Hydrogels have emerged as versatile biomaterials with remarkable applications in biomedicine and tissue engineering. Here, we present an overview of recent and ongoing research in Italy, focusing on extracellular matrix-derived, natural, and synthetic hydrogels specifically applied to biomedicine and tissue engineering. The analyzed studies highlight the versatile nature and wide range of applicability of hydrogel-based studies. Attention is also given to the integration of hydrogels within bioreactor systems, specialized devices used in biological studies to culture cells under controlled conditions, enhancing their potential for regenerative medicine, drug discovery, and drug delivery. Despite the abundance of literature on this subject, a comprehensive overview of Italian contributions to the field of hydrogels-based biomedical research is still missing and is thus our focus for this review. Consolidating a diverse range of studies, the Italian scientific community presents a complete landscape for hydrogel use, shaping the future directions of biomaterials research. This review aspires to serve as a guide and map for Italian researchers interested in the development and use of hydrogels in biomedicine. [ABSTRACT FROM AUTHOR]

Electrical stimulation (ES) has been described as a promising tool for bone tissue engineering, being known to promote vital cellular processes such as cell proliferation, migration, and differentiation. Despite the high variability of applied protocol parameters, direct coupled electric fields have been successfully applied to promote osteogenic and osteoinductive processes in vitro and in vivo. Our work aims to study the viability, proliferation, and osteogenic differentiation of human bone marrow-derived mesenchymal stem/stromal cells when subjected to five different ES protocols. The protocols were specifically selected to understand the biological effects of different parts of the generated waveform for typical direct-coupled stimuli. In vitro culture studies evidenced variations in cell responses with different electric field magnitudes (numerically predicted) and exposure protocols, mainly regarding tissue mineralization (calcium contents) and osteogenic marker gene expression while maintaining high cell viability and regular morphology. Overall, our results highlight the importance of numerical guided experiments to optimize ES parameters towards improved in vitro osteogenesis protocols. [ABSTRACT FROM AUTHOR]

The intricate nature of native cartilage, characterized by zonal variations in oxygen levels and ECM composition, poses a challenge for existing hydrogelbased tissue models. Consequently, these 3D models often present simplified renditions of the native tissue, failing to fully capture its heterogenous nature. The combined effects of hydrogel components, network properties, and structural designs on cellular responses are often overlooked. In this work, we aim to establish more physiological cartilage models through biofabrication of photopolymerizable allylated-gelatin (GelAGE) and Thiolated Heparin (HepSH) constructs with tailorable matrix stiffness and customized architectures. This involves systematically studying how the native glycosaminoglycan Heparin together with hydrogel stiffness, and oxygen availability within 3D structures influence chondrogenic differentiation and regional heterogeneity. A comprehensive library of 3D hydrogel constructs was successfully developed, encompassing GelAGE-HepSH hydrogels with three distinct stiffness levels: 12, 55 and 121 kPa, and three unique geometries: spheres, discs, and square lattices. In soft GelAGE-HepSH hydrogels, the localization of differentiating cells was observed to be irregular, while stiff hydrogels restricted the overall secretion of ECM components. The medium-stiff hydrogels were found to be most applicable, supporting both uniform tissue formation and maintained shape fidelity. Three different 3D architectures were explored, where biofabrication of smaller GelAGE-HepSH spheres without oxygen gradients induced homogenous, hyaline cartilage tissue formation. Conversely, fabrication of larger constructs (discs and lattices) with oxygen gradients could be utilized to design heterogenous cartilage tissue models. Similarly, temporal oxygen gradients were observed to drive interconnected deposition of glycosaminoglycans (GAGs). Control samples of GelAGE without HepSH did not exhibit any notable changes in chondrogenesis as a function of stiffness, architectures, or oxygen concentrations. Overall, the incorporation of HepSH within GelAGE hydrogels was observed to serve as an amplifier for the biological effects from both stiffness and oxygen cues. In conclusion, fabrication of GelAGE-HepSH constructs designed to impose limitations on oxygen availability induce more zone-specific cartilage tissue alignment. This systematic study of matrix components, network stiffness, and oxygen levels in 3D biofabricated structures contributes to the development of more physiologically relevant cartilage models while further enhancing our overall understanding of cartilage tissue engineering. [ABSTRACT FROM AUTHOR]

Three dimensional printing has emerged as a widely acceptable strategy for the fabrication of mammalian cell laden constructs with complex microenvironments for tissue engineering and regenerative medicine. More recently 3D printed living materials containing microorganisms have been developed and matured into living biofilms. The potential for engineered 3D biofilms as in vitro models for biomedical applications, such as antimicrobial susceptibility testing, and environmental applications, such as bioleaching, bioremediation, and wastewater purification, is extensive but the need for an in‐depth understanding of the structure–function relationship between the complex construct and the microorganism response still exists. This review discusses 3D printing fabrication methods for engineered biofilms with specific structural features. Next, it highlights the importance of bioink compositions and 3D bioarchitecture design. Finally, a brief overview of current and potential applications of 3D printed biofilms in environmental and biomedical fields is discussed. [ABSTRACT FROM AUTHOR]

Emergent cell behaviors that drive tissue morphogenesis are the integrated product of instructions from gene regulatory networks, mechanics and signals from the local tissue microenvironment. How these discrete inputs intersect to coordinate diverse morphogenic events is a critical area of interest. Organ-on-chip technology has revolutionized the ability to construct and manipulate miniaturized human tissues with organotypic three-dimensional architectures in vitro. Applications of organ-on-chip platforms have increasingly transitioned from proof-of-concept tissue engineering to discovery biology, furthering our understanding of molecular and mechanical mechanisms that operate across biological scales to orchestrate tissue morphogenesis. Here, we provide the biological framework to harness organ-on-chip systems to study tissue morphogenesis, and we highlight recent examples where organ-on-chips and associated microphysiological systems have enabled new mechanistic insight in diverse morphogenic settings. We further highlight the use of organ-on-chip platforms as emerging test beds for cell and developmental biology. [ABSTRACT FROM AUTHOR]

Cardiac regeneration is a critical endeavor in the treatment of heart diseases, aimed at repairing and enhancing the structure and function of damaged myocardium. This review offers a comprehensive overview of current advancements and strategies in cardiac regeneration, with a specific focus on regenerative medicine and tissue engineering-based approaches. Stem cell-based therapies, which involve the utilization of adult stem cells and pluripotent stem cells hold immense potential for replenishing lost cardiomyocytes and facilitating cardiac tissue repair and regeneration. Tissue engineering also plays a prominent role employing synthetic or natural biomaterials, engineering cardiac patches and grafts with suitable properties, and fabricating upscale bioreactors to create functional constructs for cardiac recovery. These constructs can be transplanted into the heart to provide mechanical support and facilitate tissue healing. Additionally, the production of organoids and chips that accurately replicate the structure and function of the whole organ is an area of extensive research. Despite significant progress, several challenges persist in the field of cardiac regeneration. These include enhancing cell survival and engraftment, achieving proper vascularization, and ensuring the long-term functionality of engineered constructs. Overcoming these obstacles and offering effective therapies to restore cardiac function could improve the quality of life for individuals with heart diseases. [ABSTRACT FROM AUTHOR]

Heart regeneration after myocardial infarction (MI) using human stem cell-derived cardiomyocytes (CMs) is rapidly accelerating with large animal and human clinical trials. However, vascularization methods to support the engraftment, survival, and development of implanted CMs in the ischemic environment of the infarcted heart remain a key and timely challenge. To this end, we developed a dual remuscularization-revascularization therapy that is evaluated in a rat model of ischemia-reperfusion MI. This study details the differentiation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for engineering cardiac tissue containing patterned engineered vessels 400 μm in diameter. Vascularized engineered human myocardial tissues (vEHMs) are cultured in static conditions or perfused in vitro prior to implantation and evaluated after two weeks. Immunohistochemical staining indicates improved engraftment of hiPSC-CMs in in vitro-perfused vEHMs with greater expression of SMA+ vessels and evidence of inosculation. Three-dimensional vascular reconstructions reveal less tortuous and larger intra-implant vessels, as well as an improved branching hierarchy in in vitro-perfused vEHMs relative to non-perfused controls. Exploratory RNA sequencing of explanted vEHMs supports the hypothesis that co-revascularization impacts hiPSC-CM development in vivo. Our approach provides a strong foundation to enhance vEHM integration, develop hierarchical vascular perfusion, and maximize hiPSC-CM engraftment for future regenerative therapy. [ABSTRACT FROM AUTHOR]

Approximately half of an adult human's body weight is made up of muscles. Thus, restoring the functionality and aesthetics of lost muscle tissue is critical. The body is usually able to repair minor muscle injuries. However, when volumetric muscle loss occurs due to tumour extraction, for instance, the body will form fibrous tissue instead. Gelatin methacryloyl (GelMA) hydrogels have been applied for drug delivery, tissue adhesive, and various tissue engineering applications due to their tuneable mechanical properties. Here, we have synthesised GelMA from different gelatin sources (i.e., porcine, bovine, and fish) with varying bloom numbers, which refers to the gel strength, and investigated for the influence of the source of gelatin and the bloom number on biological activities and mechanical properties. The results indicated that the source of the gelatin and variable bloom numbers have an impact on GelMA hydrogel properties. Furthermore, our findings established that the bovine-derived gelatin methacryloyl (B-GelMA) has better mechanical properties than the other varieties composed of porcine and fish with 60 kPa, 40 kPa, and 10 kPa in bovine, porcine, and fish, respectively. Additionally, it showed a noticeably greater swelling ratio (SR) ~1100% and a reduced rate of degradation, improving the stability of hydrogels and giving cells adequate time to divide and proliferate to compensate for muscle loss. Furthermore, the bloom number of gelatin was also proven to influence the mechanical properties of GelMA. Interestingly, although GelMA made of fish had the lowest mechanical strength and gel stability, it demonstrated excellent biological properties. Overall, the results emphasise the importance of gelatin source and bloom number, allowing GelMA hydrogels to have a wide range of mechanical and excellent biological properties and making them suitable for various muscle tissue regeneration applications. [ABSTRACT FROM AUTHOR]

Organ-on-a-chip (OOC) is an emerging interdisciplinary technology that reconstitutes the structure, function, and physiology of human tissues as an alternative to conventional preclinical models for drug screening. Over the last decade, substantial progress has been made in mimicking tissue- and organ-level functions on chips through technical advances in biomaterials, stem cell engineering, microengineering, and microfluidic technologies. Structural and engineering constituents, as well as biological components, are critical factors to be considered to reconstitute the tissue function and microenvironment on chips. In this review, we highlight critical engineering technologies for reconstructing the tissue microarchitecture and dynamic spatiotemporal microenvironment in OOCs. We review the technological advances in the field of OOCs for a range of applications, including systemic analysis tools that can be integrated with OOCs, multiorgan-on-chips, and large-scale manufacturing. We then discuss the challenges and future directions for the development of advanced end-user-friendly OOC systems for a wide range of applications. [ABSTRACT FROM AUTHOR]

Advancements and developments in the 3D bioprinting have been promising and have met the needs of organ transplantation. Current improvements in tissue engineering constructs have enhanced their applications in regenerative medicines and other medical fields. The synergistic effects of 3D bioprinting have brought technologies such as tissue engineering, microfluidics, integrated tissue organ printing, in vivo bioprinted tissue implants, artificial intelligence and machine learning approaches together. These have greatly impacted interventions in medical fields, such as medical implants, multi-organ-on-chip models, prosthetics, drug testing tissue constructs and much more. This technological leap has offered promising personalized solutions for patients with chronic diseases, and neurodegenerative disorders, and who have been in severe accidents. This review discussed the various standing printing methods, such as inkjet, extrusion, laser-assisted, digital light processing, and stereolithographic 3D bioprinter models, adopted for tissue constructs. Additionally, the properties of natural, synthetic, cell-laden, dECM-based, short peptides, nanocomposite and bioactive bioinks are briefly discussed. Sequels of several tissue-laden constructs such as skin, bone and cartilage, liver, kidney, smooth muscles, cardiac and neural tissues are briefly analyzed. Challenges, future perspectives and the impact of microfluidics in resolving the limitations in the field, along with 3D bioprinting, are discussed. Certainly, a technology gap still exists in the scaling up, industrialization and commercialization of this technology for the benefit of stakeholders. [ABSTRACT FROM AUTHOR]

Biomimetic materials have emerged as attractive and competitive alternatives for tissue engineering (TE) and regenerative medicine. In contrast to conventional biomaterials or synthetic materials, biomimetic scaffolds based on natural biomaterial can offer cells a broad spectrum of biochemical and biophysical cues that mimic the in vivo extracellular matrix (ECM). Additionally, such materials have mechanical adaptability, microstructure interconnectivity, and inherent bioactivity, making them ideal for the design of living implants for specific applications in TE and regenerative medicine. This paper provides an overview for recent progress of biomimetic natural biomaterials (BNBMs), including advances in their preparation, functionality, potential applications and future challenges. We highlight recent advances in the fabrication of BNBMs and outline general strategies for functionalizing and tailoring the BNBMs with various biological and physicochemical characteristics of native ECM. Moreover, we offer an overview of recent key advances in the functionalization and applications of versatile BNBMs for TE applications. Finally, we conclude by offering our perspective on open challenges and future developments in this rapidly-evolving field. [ABSTRACT FROM AUTHOR]

(1) Background: Synovial tissue plays a fundamental role in inflammatory processes. Therefore, understanding the mechanisms regulating healthy and diseased synovium functions, as in rheumatic diseases, is crucial to discovering more effective therapies to minimize or prevent pathological progress. The present study aimed at developing a bioartificial synovial tissue as an in vitro model for drug screening or personalized medicine applications using 3D bioprinting technology. (2) Methods: The volumetric extrusion technique has been used to fabricate cell-laden scaffolds. Gelatin Methacryloyl (GelMA), widely applied in regenerative medicine and tissue engineering, was selected as a bioink and combined with an immortalized cell line of fibroblast-like synoviocytes (K4IM). (3) Results: Three different GelMA formulations, 7.5–10–12.5% w/v, were tested for the fabrication of the scaffold with the desired morphology and internal architecture. GelMA 10% w/v was chosen and combined with K4IM cells to fabricate scaffolds that showed high cell viability and negligible cytotoxicity for up to 14 days tested by Live & Dead and lactate dehydrogenase assays. (4) Conclusions: We successfully 3D bioprinted synoviocytes-laden scaffolds as a proof-of-concept (PoC) towards the fabrication of a 3D synovial membrane model suitable for in vitro studies. However, further research is needed to reproduce the complexity of the synovial microenvironment to better mimic the physiological condition. [ABSTRACT FROM AUTHOR]

Nutrient supply via a functional vasculature is essential during regenerative processes, tissue growth, and homeostasis. 3D bioprinting offers the opportunity to engineer vascularized constructs by combining cells and biocompatible materials in specifically designed fashions. However, the complexity of microvascular dynamic networks can hardly be recapitulated yet, even by sophisticated 3D manufacturing. Ideally, the natural organizational competences of endothelial cells will be harnessed such that engineered vascular networks self‐assemble to form complex, controllable microvascular patterns. Here, a bioengineering approach is presented to control microvascular structure formation and to steer cellular self‐assembly of endothelial and supporting cells within a multi‐material stereolithographic 3D bioprinting concept. Bioengineered vascularized constructs are generated by controlled cell deposition in an enzymatically degradable or a non‐degradable material. In vitro, the microvascular structures are regulated in distribution, network orientation, vessel length and branching behavior and developed lumen, signs of vascular stabilization and an interconnected vascular network including anastomosis. This novel biofabrication approach demonstrates the capability to control microvascular network formation by using cellular and spatial cues allowing the generation of distinctly yet precisely vascularized constructs. Such novel approach of controlled microvascular formation may play a fundamental role in the development of vascularized implants or in vitro screening models. [ABSTRACT FROM AUTHOR]

Development in microfluidic technology has contributed to increased understanding in neural tissue engineering through the in vitro observation of cell-on-chip (CoC) systems. This has been further helped by the integration with the broader MEMS (micro mechanical and electromechanical systems) technology that offers external devices such as detectors or biosensors to show the characteristics of the observed object. An on-chip microsystem microfluidic platform for dopamine detection is presented here. The microfluidic platform integrates electrochemical detection with finger pumping and a valve system as means to control the fluid flow. This microenvironment offers a quicker result in observing the phenomena related to the neural cell activities with a relatively small specimen volume of 50-100 µL, eases the handling of movement, and consequently reduces the cost of consumable items. The microfluidic platform presented here showed that the pump module that also serves as a mixing point was able to deliver at maximum of 121.36 µL with 2-3 strokes of normal finger pressure priming. A series of valves aids in the termination or isolation of fluid flow in a specific zone for further processing. Ultimately, the microfluidic platform is also equipped with a portable electrochemical detection module that allows us to measure the dopamine concentration up to 1 mM. This development showed that the on-chip testing of dopamine could be conducted easier and be more portable to handle. [ABSTRACT FROM AUTHOR]

Tissue engineers have made great strides in the past decade thanks to the advent of three-dimensional (3D) bioprinting technology, which has allowed them to create highly customized biological structures with precise geometric design ability, allowing us to close the gap between manufactured and natural tissues. In this work, we first survey the state-of-the-art methods, cells, and materials for 3D bioprinting. The modern uses of this method in tissue engineering are then briefly discussed. Following this, the main benefits of 3D bioprinting in tissue engineering are outlined in depth, including the ability to rapidly prototype the individualized structure and the ability to engineer with a highly controllable microenvironment. Finally, we offer some predictions for the future of 3D bioprinting in the field of tissue engineering. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases are the leading cause of global mortality. Over the past two decades, researchers have tried to provide novel solutions for end-stage heart failure to address cardiac transplantation hurdles such as donor organ shortage, chronic rejection, and life-long immunosuppression. Cardiac decellularized extracellular matrix (dECM) has been widely explored as a promising approach in tissue-regenerative medicine because of its remarkable similarity to the original tissue. Optimized decellularization protocols combining physical, chemical, and enzymatic agents have been developed to obtain the perfect balance between cell removal, ECM composition, and function maintenance. However, proper assessment of decellularized tissue composition is still needed before clinical translation. Recellularizing the acellular scaffold with organ-specific cells and evaluating the extent of cardiomyocyte repopulation is also challenging. This review aims to discuss the existing literature on decellularized cardiac scaffolds, especially on the advantages and methods of preparation, pointing out areas for improvement. Finally, an overview of the state of research regarding the application of cardiac dECM and future challenges in bioengineering a human heart suitable for transplantation is provided. [ABSTRACT FROM AUTHOR]

Cardiovascular diseases (CVDs) are known as the major cause of death worldwide. In spite of tremendous advancements in medical therapy, the gold standard for CVD treatment is still transplantation. Tissue engineering, on the other hand, has emerged as a pioneering field of study with promising results in tissue regeneration using cells, biological cues, and scaffolds. 3D bioprinting is a rapidly growing technique in tissue engineering because of its ability to create complex scaffold structures, encapsulate cells, and perform these tasks with precision. More recently, 3D bioprinting has made its debut in cardiac tissue engineering, and scientists are investigating this technique for development of new strategies for cardiac tissue regeneration. In this review, the fundamentals of cardiac tissue biology, available 3D bioprinting techniques and bioinks, and cells implemented for cardiac regeneration are briefly summarized and presented. Afterward, the pioneering and state‐of‐the‐art works that have utilized 3D bioprinting for cardiac tissue engineering are thoroughly reviewed. Finally, regulatory pathways and their contemporary limitations and challenges for clinical translation are discussed. [ABSTRACT FROM AUTHOR]

Ex vivo modelling systems for cardiovascular research are becoming increasingly important in reducing lab animal use and boosting personalized medicine approaches. Integrating multiple cell types in complex setups adds a higher level of significance to the models, simulating the intricate intercellular communication of the microenvironment in vivo. Cardiac fibrosis represents a key pathogenetic step in multiple cardiovascular diseases, such as ischemic and diabetic cardiomyopathies. Indeed, allowing inter-cellular interactions between cardiac stromal cells, endothelial cells, cardiomyocytes, and/or immune cells in dedicated systems could make ex vivo models of cardiac fibrosis even more relevant. Moreover, culture systems with 3D architectures further enrich the physiological significance of such in vitro models. In this review, we provide a summary of the multicellular 3D models for the study of cardiac fibrosis described in the literature, such as spontaneous microtissues, bioprinted constructs, engineered tissues, and organs-on-chip, discussing their advantages and limitations. Important discoveries on the physiopathology of cardiac fibrosis, as well as the screening of novel potential therapeutic molecules, have been reported thanks to these systems. Future developments will certainly increase their translational impact for understanding and modulating mechanisms of cardiac fibrosis even further. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is an extremely complex disease, as it combines both biological-chemical and mechanical aspects, and it also involves the entire joint consisting of various types of tissues, including cartilage and bone. This paper describes the methods of conducting cell cultures aimed at searching for the mechanical causes of OA development, therapeutic solutions, and methods of preventing the disease. It presents the systems for the cultivation of cartilage cells depending on the level of their structural complexity, and taking into account the most common solutions aimed at recreating the most important factors contributing to the development of OA, that is mechanical loads. In-vitro systems used in tissue engineering to investigate the phenomena associated with OA were specified depending on the complexity and purposefulness of conducting cell cultures. [ABSTRACT FROM AUTHOR]

At present, there is an increasing need to mimic the in vivo micro-environment in the culture of cells and tissues in micro-tissue engineering. Concave microwells are becoming increasingly popular since they can provide a micro-environment that is closer to the in vivo environment compared to traditional microwells, which can facilitate the culture of cells and tissues. Here, we will summarize the fabrication methods of concave microwells, as well as their applications in micro-tissue engineering. The fabrication methods of concave microwells include traditional methods, such as lithography and etching, thermal reflow of photoresist, laser ablation, precision-computerized numerical control (CNC) milling, and emerging technologies, such as surface tension methods, the deformation of soft membranes, 3D printing, the molding of microbeads, air bubbles, and frozen droplets. The fabrication of concave microwells is transferring from professional microfabrication labs to common biochemical labs to facilitate their applications and provide convenience for users. Concave microwells have mostly been used in organ-on-a-chip models, including the formation and culture of 3D cell aggregates (spheroids, organoids, and embryoids). Researchers have also used microwells to study the influence of substrate topology on cellular behaviors. We will briefly review their applications in different aspects of micro-tissue engineering and discuss the further applications of concave microwells. We believe that building multiorgan-on-a-chip by 3D cell aggregates of different cell lines will be a popular application of concave microwells, while integrating physiologically relevant molecular analyses with the 3D culture platform will be another popular application in the near future. Furthermore, 3D cell aggregates from these biosystems will find more applications in drug screening and xenogeneic implantation. [ABSTRACT FROM AUTHOR]

Capacitively Coupled (CCoupled) electric fields are used to stimulate cell cultures in Tissue Engineering. Knowing the electric field (E-Field) magnitude in the culture medium is fundamental to establish a relationship between stimulus strength and cellular effects. We analysed eight CCoupled studies and sought to corroborate the reported estimates of the E-Field in the culture medium. First, we reviewed the basic physics underlying CCoupled stimulation and delineated three approaches to estimate the E-field. Using these approaches, we found that the reported values were overestimated in five studies, four of which were based on incorrect assumptions. In all studies, insufficient information was provided to reproduce the setup exactly. Creating electrical models of the experimental setup should improve the accuracy of the E-field estimates and enhance reproducibility. For this purpose, we developed a free open-source tool, the E-field Calculator for CCoupled systems, which is available for download from an internet hosting platform. [ABSTRACT FROM AUTHOR]

Endothelial cell dysfunction plays a central role in many pathologies, rendering it crucial to understand the underlying mechanism for potential therapeutics. Tissue engineering offers opportunities for in vitro studies of endothelial dysfunction in pathological mimicry environments. Here, we begin by analyzing hydrogel biomaterials as a platform for understanding the roles of the extracellular matrix and hypoxia in vascular formation. We next examine how three-dimensional bioprinting has been applied to recapitulate healthy and diseased tissue constructs in a highly controllable and patient-specific manner. Similarly, studies have utilized organs-on-a-chip technology to understand endothelial dysfunction's contribution to pathologies in tissue-specific cellular components under well-controlled physicochemical cues. Finally, we consider studies using the in vitro construction of multicellular blood vessels, termed tissue-engineered blood vessels, and the spontaneous assembly of microvascular networks in organoids to delineate pathological endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Bioengineering a solid organ for organ replacement is a growing endeavor in regenerative medicine. Our approach – recellularization of a decellularized cadaveric organ scaffold with human cells – is currently the most promising approach to building a complex solid vascularized organ to be utilized in vivo, which remains the major unmet need and a key challenge. The 2008 publication of perfusion-based decellularization and partial recellularization of a rat heart revolutionized the tissue engineering field by showing that it was feasible to rebuild an organ using a decellularized extracellular matrix scaffold. Toward the goal of clinical translation of bioengineered tissues and organs, there is increasing recognition of the underlying need to better integrate basic science domains and industry. From the perspective of a research group focusing on whole heart engineering, we discuss the current approaches and advances in whole organ engineering research as they relate to this multidisciplinary field's 3 major pillars: organ scaffolds, large numbers of cells, and biomimetic bioreactor systems. The success of whole organ engineering will require optimization of protocols to produce biologically-active scaffolds for multiple organ systems, and further technological innovation both to produce the massive quantities of high-quality cells needed for recellularization and to engineer a bioreactor with physiologic stimuli to recapitulate organ function. Also discussed are the challenges to building an implantable vascularized solid organ. [ABSTRACT FROM AUTHOR]

Organ-on-a-chip (OOAC) is an emerging technology based on microfluid platforms and in vitro cell culture that has a promising future in the healthcare industry. The numerous advantages of OOAC over conventional systems make it highly popular. The chip is an innovative combination of novel technologies, including lab-on-a-chip, microfluidics, biomaterials, and tissue engineering. This paper begins by analyzing the need for the development of OOAC followed by a brief introduction to the technology. Later sections discuss and review the various types of OOACs and the fabrication materials used. The implementation of artificial intelligence in the system makes it more advanced, thereby helping to provide a more accurate diagnosis as well as convenient data management. We introduce selected OOAC projects, including applications to organ/disease modelling, pharmacology, personalized medicine, and dentistry. Finally, we point out certain challenges that need to be surmounted in order to further develop and upgrade the current systems. [ABSTRACT FROM AUTHOR]

An organ-on-a-chip is a device that combines micro-manufacturing and tissue engineering to replicate the critical physiological environment and functions of the human organs. Therefore, it can be used to predict drug responses and environmental effects on organs. Microfluidic technology can control micro-scale reagents with high precision. Hence, microfluidics have been widely applied in organ-on-chip systems to mimic specific organ or multiple organs in vivo. These models integrated with various sensors show great potential in simulating the human environment. In this review, we mainly introduce the typical structures and recent research achievements of several organ-on-a-chip platforms. We also discuss innovations in models applied to the fields of pharmacokinetics/pharmacodynamics, nano-medicine, continuous dynamic monitoring in disease modeling, and their further applications in other fields. [ABSTRACT FROM AUTHOR]