Molecularly Imprinted Polymer-Based Sensors for the Detection of Skeletal- and Cardiac-Muscle-Related Analytes.

Molecularly imprinted polymers (MIPs) are synthetic polymers with specific binding sites that present high affinity and spatial and chemical complementarities to a targeted analyte. They mimic the molecular recognition seen naturally in the antibody/antigen complementarity. Because of their specificity, MIPs can be included in sensors as a recognition element coupled to a transducer part that converts the interaction of MIP/analyte into a quantifiable signal. Such sensors have important applications in the biomedical field in diagnosis and drug discovery, and are a necessary complement of tissue engineering for analyzing the functionalities of the engineered tissues. Therefore, in this review, we provide an overview of MIP sensors that have been used for the detection of skeletal- and cardiac-muscle-related analytes. We organized this review by targeted analytes in alphabetical order. Thus, after an introduction to the fabrication of MIPs, we highlight different types of MIP sensors with an emphasis on recent works and show their great diversity, their fabrication, their linear range for a given analyte, their limit of detection (LOD), specificity, and reproducibility. We conclude the review with future developments and perspectives.

A Patch of Detachable Hybrid Microneedle Depot for Localized Delivery of Mesenchymal Stem Cells in Regeneration Therapy.

Mesenchymal stem cells (MSCs) have been widely used for regenerative therapy. In most current clinical applications, MSCs are delivered by injection but face significant issues with cell viability and penetration into the target tissue due to a limited migration capacity. Some therapies have attempted to improve MSC stability by their encapsulation within biomaterials; however, these treatments still require an enormous number of cells to achieve therapeutic efficacy due to low efficiency. Additionally, while local injection allows for targeted delivery, injections with conventional syringes are highly invasive. Due to the challenges associated with stem cell delivery, a local and minimally invasive approach with high efficiency and improved cell viability is highly desired. In this study, we present a detachable hybrid microneedle depot (d-HMND) for cell delivery. Our system consists of an array of microneedles with an outer poly(lactic-co-glycolic) acid (PLGA) shell and an internal gelatin methacryloyl (GelMA)-MSC mixture (GMM). The GMM was characterized and optimized for cell viability and mechanical strength of the d-HMND required to penetrate mouse skin tissue was also determined. MSC viability and function within the d-HMND was characterized in vitro and the regenerative efficacy of the d-HMND was demonstrated in vivo using a mouse skin wound model.

3D Bioprinting in Skeletal Muscle Tissue Engineering.

Skeletal muscle tissue engineering (SMTE) aims at repairing defective skeletal muscles. Until now, numerous developments are made in SMTE; however, it is still challenging to recapitulate the complexity of muscles with current methods of fabrication. Here, after a brief description of the anatomy of skeletal muscle and a short state-of-the-art on developments made in SMTE with "conventional methods," the use of 3D bioprinting as a new tool for SMTE is in focus. The current bioprinting methods are discussed, and an overview of the bioink formulations and properties used in 3D bioprinting is provided. Finally, different advances made in SMTE by 3D bioprinting are highlighted, and future needs and a short perspective are provided.

In situ three-dimensional printing for reparative and regenerative therapy.

Three-dimensional (3D) bioprinting is an emerging biofabrication technology, driving many innovations and opening new avenues in regenerative therapeutics. The aim of 3D bioprinting is to fabricate grafts in vitro, which can then be implanted in vivo. However, the tissue culture ex vivo carries safety risks and thereby complicated manufacturing equipment and practice are required for tissues to be implanted in the humans. The implantation of printed tissues also adds complexities due to the difficulty in maintaining the structural integrity of fabricated constructs. To tackle this challenge, the concept of in situ 3D bioprinting has been suggested in which tissues are directly printed at the site of injury or defect. Such approach could be combined with cells freshly isolated from patients to produce custom-made grafts that resemble target tissue and fit precisely to target defects. Moreover, the natural cellular microenvironment in the body can be harnessed for tissue maturation resulting in the tissue regeneration and repair. Here, we discuss literature reports on in situ 3D printing and we describe future directions and challenges for in situ 3D bioprinting. We expect that this novel technology would find great attention in different biomedical fields in near future.

Development of Silver-Based Bactericidal Composite Nanofibers by Airbrushing.

In this article, we report a simple, cost-effective and eco-friendly method of airbrushing for the fabrication of antibacterial composite nanofibers using Nylon-6 and silver chloride (AgCl). The Nylon-6 is a widely used polymer for various biomedical applications because of its excellent biocompatibility and mechanical properties. Similarly, silver has also been known for their antibacterial, antifungal, antiviral, and anti-inflammatory properties. In order to enhance the antibacterial functionality of the Nylon-6, composite nanofibers in combination with AgCl have been fabricated using airbrush method. The chemical functional groups and morphological studies of the airbrushed Nylon-6/AgCl composite nanofibers were carried out by FTIR and SEM, respectively. The antibacterial activity of airbrushed Nylon-6/AgCl composite nanofibers was evaluated using Gram +ve (Staphylococcus aureus) and Gram -ve (Escherichia coli) bacterial strains. The results showed that the airbrushed Nylon-6/AgCl composite nanofibers have better antibacterial activity against the tested bacterial strains than the airbrushed Nylon-6 nanofibers. Therefore, the airbrushed Nylon-6/AgCl composite nanofibers could be used as a potential antibacterial scaffolding system for tissue engineering and regenerative medicine.

Bioconjugated Hydrogels for Tissue Engineering and Regenerative Medicine.

Hydrogels are hydrophilic polymer networks with high water content, which have played an important role as scaffolds for cells, as carriers for various biomolecules (e.g., drugs, genes, and soluble factors), and as injectable biomaterials in tissue engineering (TE) and regenerative medicine. Bioconjugation is an approach for improving the performance of hydrogels using cell-responsive components, such as proteins and peptides, which have high affinity to regulate cellular behaviors and tissue morphogenesis. However, the current knowledge on the role of those bioconjugated moieties in controlling cellular functions and tissue morphogenesis and bioconjugation methods are limited in the context of TE and organogenesis. Moreover, micro- and nanofabrication techniques have been used to manipulate bioconjugated hydrogels for regulating cell behaviors and function. This Review therefore describes synthesis, characteristics, and manipulation of various bioconjugated hydrogels and their potential in TE applications with special emphasis on preclinical/clinical translation.

Fiber-assisted molding (FAM) of surfaces with tunable curvature to guide cell alignment and complex tissue architecture.

A simple and robust method termed "fiber-assisted molding (FAM)" is presented to create biomimetic three-dimensional surfaces with controllable curvature and helical twist. The alignment of muscle fibrils and the assembly of helically patterned extracellular matrix by cells demonstrate the potential of this method for tissue engineering and other materials science applications.

Biomimetic microfluidic device for in vitro antihypertensive drug evaluation.

Microfluidic devices have emerged as revolutionary, novel platforms for in vitro drug evaluation. In this work, we developed a facile method for evaluating antihypertensive drugs using a microfluidic chip. This microfluidic chip was generated using the elastic material poly(dimethylsiloxane) (PDMS) and a microchannel structure that simulated a blood vessel as fabricated on the chip. We then cultured human umbilical vein endothelial cells (HUVECs) inside the channel. Different pressures and shear stresses could be applied on the cells. The generated vessel mimics can be used for evaluating the safety and effects of antihypertensive drugs. Here, we used hydralazine hydrochloride as a model drug. The results indicated that hydralazine hydrochloride effectively decreased the pressure-induced dysfunction of endothelial cells. This work demonstrates that our microfluidic system provides a convenient and cost-effective platform for studying cellular responses to drugs under mechanical pressure.

Microfluidic generation of polydopamine gradients on hydrophobic surfaces.

Engineered surface-bound molecular gradients are of great importance for a range of biological applications. In this paper, we fabricated a polydopamine gradient on a hydrophobic surface. A microfluidic device was used to generate a covalently conjugated gradient of polydopamine (PDA), which changed the wettabilty and the surface energy of the substrate. The gradient was subsequently used to enable the spatial deposition of adhesive proteins on the surface. When seeded with human adipose mesenchymal stem cells, the PDA-graded surface induced a gradient of cell adhesion and spreading. The PDA gradient developed in this study is a promising tool for controlling cellular behavior and may be useful in various biological applications.

Engineered nanomembranes for directing cellular organization toward flexible biodevices.

Controlling the cellular microenvironment can be used to direct the cellular organization, thereby improving the function of synthetic tissues in biosensing, biorobotics, and regenerative medicine. In this study, we were inspired by the microstructure and biological properties of the extracellular matrix to develop freestanding ultrathin polymeric films (referred as "nanomembranes") that were flexible, cell adhesive, and had a morphologically tailorable surface. The resulting nanomembranes were exploited as flexible substrates on which cell-adhesive micropatterns were generated to align C2C12 skeletal myoblasts and embedded fibril carbon nanotubes enhanced the cellular elongation and differentiation. Functional nanomembranes with tunable morphology and mechanical properties hold great promise in studying cell-substrate interactions and in fabricating biomimetic constructs toward flexible biodevices.

A contactless electrical stimulator: application to fabricate functional skeletal muscle tissue.

Engineered skeletal muscle tissues are ideal candidates for applications in drug screening systems, bio-actuators, and as implantable constructs in tissue engineering. Electrical field stimulation considerably improves the differentiation of muscle cells to muscle myofibers. Currently used electrical stimulators often use direct contact of electrodes with tissue constructs or their culture medium, which may cause hydrolysis of the culture medium, joule heating of the medium, contamination of the culture medium due to products of electrodes corrosion, and surface fouling of electrodes. Here, we used an interdigitated array of electrodes combined with an isolator coverslip as a contactless platform to electrically stimulate engineered muscle tissue, which eliminates the aforementioned problems. The effective stimulation of muscle myofibers using this device was demonstrated in terms of contractile activity and higher maturation as compared to muscle tissues without applying the electrical field. Due to the wide array of potential applications of electrical stimulation to two- and three-dimensional (2D and 3D) cell and tissue constructs, this device could be of great interest for a variety of biological applications as a tool to create noninvasive, safe, and highly reproducible electric fields.

Latest developments in engineered skeletal muscle tissues for drug discovery and development.

INTRODUCTION: With the advances in skeletal muscle tissue engineering, new platforms have arisen with important applications in biology studies, disease modeling, and drug testing. Current developments highlight the quest for engineering skeletal muscle tissues with higher complexity . These new human skeletal muscle tissue models will be powerful tools for drug discovery and development and disease modeling. AREAS COVERED: The authors review the latest advances in in vitro models of engineered skeletal muscle tissues used for testing drugs with a focus on the use of four main cell culture techniques: Cell cultures in well plates, in microfluidics, in organoids, and in bioprinted constructs. Additional information is provided on the satellite cell niche. EXPERT OPINION: In recent years, more sophisticated in vitro models of skeletal muscle tissues have been fabricated. Important developments have been made in stem cell research and in the engineering of human skeletal muscle tissue. Some platforms have already started to be used for drug testing, notably those based on the parameters of hypertrophy/atrophy and the contractibility of myotubes. More developments are expected through the use of multicellular types and multi-materials as matrices . The validation and use of these models in drug testing should now increase.

Bioprinting and biomaterials for dental alveolar tissue regeneration.

Three dimensional (3D) bioprinting is a powerful tool, that was recently applied to tissue engineering. This technique allows the precise deposition of cells encapsulated in supportive bioinks to fabricate complex scaffolds, which are used to repair targeted tissues. Here, we review the recent developments in the application of 3D bioprinting to dental tissue engineering. These tissues, including teeth, periodontal ligament, alveolar bones, and dental pulp, present cell types and mechanical properties with great heterogeneity, which is challenging to reproduce in vitro. After highlighting the different bioprinting methods used in regenerative dentistry, we reviewed the great variety of bioink formulations and their effects on cells, which have been established to support the development of these tissues. We discussed the different advances achieved in the fabrication of each dental tissue to provide an overview of the current state of the methods. We conclude with the remaining challenges and future needs.

Minimally Invasive Sub-Retinal Transplantation of RPE-J Cells on a Biodegradable Composite PCL/Collagen Nanosheet.

Retinal cells are irreparably damaged by diseases such as age-related macular degeneration (AMD). A promising method to restore partial or whole vision is through cell-based transplantation to the damaged location. However, cell transplantation using conventional vitreous surgery is an invasive procedure that may induce infections and has a high failure rate of cell engraftment. In this study, we describe the fabrication of a biodegradable composite nanosheet used as a substrate to support retinal pigment epithelial (RPE-J) cells, which can be grafted to the sub-retinal space using a minimally invasive approach. The nanosheet was fabricated using polycaprolactone (PCL) and collagen in 80:20 weight ratio, and had size of 200 µm in diameter and 300 nm in thickness. These PCL/collagen nanosheets showed excellent biocompatibility and mechanical strength in vitro. Using a custom designed 27-gauge glass needle, we successfully transplanted an RPE-J cell loaded nanosheet into the sub-retinal space of a rat model with damaged photoreceptors. The cell loaded nanosheet did not trigger immunological reaction within 2 weeks of implantation and restored the retinal environment. Thus, this composite PCL/collagen nanosheet holds great promise for organized cell transplantation, and the treatment of retinal diseases.

3D Bioprinting tissue analogs: Current development and translational implications.

Three-dimensional (3D) bioprinting is a promising and rapidly evolving technology in the field of additive manufacturing. It enables the fabrication of living cellular constructs with complex architectures that are suitable for various biomedical applications, such as tissue engineering, disease modeling, drug screening, and precision regenerative medicine. The ultimate goal of bioprinting is to produce stable, anatomically-shaped, human-scale functional organs or tissue substitutes that can be implanted. Although various bioprinting techniques have emerged to develop customized tissue-engineering substitutes over the past decade, several challenges remain in fabricating volumetric tissue constructs with complex shapes and sizes and translating the printed products into clinical practice. Thus, it is crucial to develop a successful strategy for translating research outputs into clinical practice to address the current organ and tissue crises and improve patients' quality of life. This review article discusses the challenges of the existing bioprinting processes in preparing clinically relevant tissue substitutes. It further reviews various strategies and technical feasibility to overcome the challenges that limit the fabrication of volumetric biological constructs and their translational implications. Additionally, the article highlights exciting technological advances in the 3D bioprinting of anatomically shaped tissue substitutes and suggests future research and development directions. This review aims to provide readers with insight into the state-of-the-art 3D bioprinting techniques as powerful tools in engineering functional tissues and organs.

Enhanced Maturation of 3D Bioprinted Skeletal Muscle Tissue Constructs Encapsulating Soluble Factor-Releasing Microparticles.

Several microfabrication technologies have been used to engineer native-like skeletal muscle tissues. However, the successful development of muscle remains a significant challenge in the tissue engineering field. Muscle tissue engineering aims to combine muscle precursor cells aligned within a highly organized 3D structure and biological factors crucial to support cell differentiation and maturation into functional myotubes and myofibers. In this study, the use of 3D bioprinting is proposed for the fabrication of muscle tissues using gelatin methacryloyl (GelMA) incorporating sustained insulin-like growth factor-1 (IGF-1)-releasing microparticles and myoblast cells. This study hypothesizes that functional and mature myotubes will be obtained more efficiently using a bioink that can release IGF-1 sustainably for in vitro muscle engineering. Synthesized microfluidic-assisted polymeric microparticles demonstrate successful adsorption of IGF-1 and sustained release of IGF-1 at physiological pH for at least 21 days. Incorporating the IGF-1-releasing microparticles in the GelMA bioink assisted in promoting the alignment of myoblasts and differentiation into myotubes. Furthermore, the myotubes show spontaneous contraction in the muscle constructs bioprinted with IGF-1-releasing bioink. The proposed bioprinting strategy aims to improve the development of new therapies applied to the regeneration and maturation of muscle tissues.

Controlled release of drugs from gradient hydrogels for high-throughput analysis of cell-drug interactions.

In this paper, we report a method to fabricate microengineered hydrogels that contain a concentration gradient of a drug for high-throughput analysis of cell-drug interactions. A microfluidic gradient generator was used to create a concentration gradient of okadaic acid (OA) as a model drug within poly(ethylene glycol) diacrylate hydrogels. These hydrogels were then incubated with MC3T3-E1 cell seeded glass slides to investigate the cell viability through the spatially controlled release of OA. The drug was released from the hydrogel in a gradient manner and induced a gradient of the cell viability. The drug concentration gradient containing hydrogels developed in this study have the potential to be used for drug discovery and diagnostics applications due to their ability to simultaneously test the effects of different concentrations of various chemicals.

Integration of a pump and an electrical sensor into a membrane-based PDMS microbioreactor for cell culture and drug testing.

To the extent possible, artificial organs should have characteristics that match those of the in vivo system. To this end, microfabrication techniques allow us to create microenvironments that can help maintain cell organization and functionality in in vitro cultures. We present three new microbioreactors, each of which allows cells to be cultured in a perfused microenvironment similar to that found in vivo. Our microbioreactors use new technology that permits integration onto the chip (35 mm × 20 mm) of an electrical sensor, in addition to one or more pumping systems and associated perfusion circuitry. The monitoring of Caco-2 cell cultures using electrical impedance spectroscopy (EIS) has allowed us to measure the effects of cell growth, cellular barrier formation and the presence of chemical compounds and/or toxins. Specifically, we have investigated the ability of the electrical sensor to maintain appropriate sensitivity and precision. Our results show that the sensor was very sensitive not only to the presence or the absence of the cells, but also to changes in cell state. Our perfused microbioreactors are highly efficient miniaturized tools that are easy to operate. We anticipate that they will offer promising new opportunities in many types of cell culture research, including drug screening and tissue engineering.

Cardiac Differentiation of Mesenchymal Stem Cells: Impact of Biological and Chemical Inducers.

Cardiovascular disorders (CVDs) are the leading cause of global death, widely occurs due to irreparable loss of the functional cardiomyocytes. Stem cell-based therapeutic approaches, particularly the use of Mesenchymal Stem Cells (MSCs) is an emerging strategy to regenerate myocardium and thereby improving the cardiac function after myocardial infarction (MI). Most of the current approaches often employ the use of various biological and chemical factors as cues to trigger and modulate the differentiation of MSCs into the cardiac lineage. However, the recent advanced methods of using specific epigenetic modifiers and exosomes to manipulate the epigenome and molecular pathways of MSCs to modify the cardiac gene expression yield better profiled cardiomyocyte like cells in vitro. Hitherto, the role of cardiac specific inducers triggering cardiac differentiation at the cellular and molecular level is not well understood. Therefore, the current review highlights the impact and recent trends in employing biological and chemical inducers on cardiac differentiation of MSCs. Thereby, deciphering the interactions between the cellular microenvironment and the cardiac inducers will help us to understand cardiomyogenesis of MSCs. Additionally, the review also provides an insight on skeptical roles of the cell free biological factors and extracellular scaffold assisted mode for manipulation of native and transplanted stem cells towards translational cardiac research.

Healthy and diseased in vitro models of vascular systems.

Irregular hemodynamics affects the progression of various vascular diseases, such atherosclerosis or aneurysms. Despite the extensive hemodynamics studies on animal models, the inter-species differences between humans and animals hamper the translation of such findings. Recent advances in vascular tissue engineering and the suitability of in vitro models for interim analysis have increased the use of in vitro human vascular tissue models. Although the effect of flow on endothelial cell (EC) pathophysiology and EC-flow interactions have been vastly studied in two-dimensional systems, they cannot be used to understand the effect of other micro- and macro-environmental parameters associated with vessel wall diseases. To generate an ideal in vitro model of the vascular system, essential criteria should be included: 1) the presence of smooth muscle cells or perivascular cells underneath an EC monolayer, 2) an elastic mechanical response of tissue to pulsatile flow pressure, 3) flow conditions that accurately mimic the hemodynamics of diseases, and 4) geometrical features required for pathophysiological flow. In this paper, we review currently available in vitro models that include flow dynamics and discuss studies that have tried to address the criteria mentioned above. Finally, we critically review in vitro fluidic models of atherosclerosis, aneurysm, and thrombosis.