Bi‐Layered Tubular Microfiber Scaffolds as Functional Templates for Engineering Human Intestinal Smooth Muscle Tissue

Designing biomimetic scaffolds with in vivo–like microenvironments using biomaterials is an essential component of successful tissue engineering approaches. The intestinal smooth muscle layers exhibit a complex tubular structure consisting of two concentric muscle layers in which the inner circular layer is orthogonally oriented to the outer longitudinal layer. Here, a 3D bi‐layered tubular scaffold is presented based on flexible, mechanically robust, and well aligned silk protein microfibers to mimic the native human intestinal smooth muscle structure. The scaffolds are seeded with primary human intestinal smooth muscle cells to replicate intestinal muscle tissues in vitro. Characterization of the tissue constructs reveals good biocompatibility and support for cell alignment and elongation in the different scaffold layers to enhance cell differentiation and functions. Furthermore, the engineered smooth muscle constructs support oriented neurite outgrowth, a requisite step to achieve functional innervation. These results suggest these microfiber scaffolds as functional templates for in vitro regeneration of human intestinal smooth muscle systems. The scaffolding provides a crucial step toward engineering functional human intestinal tissue in vitro, as well as engineering other types of smooth muscles in terms of their similar phenotypes. Such utility may lead to a better understanding of smooth muscle associated diseases and treatments.     

Mechanical Instabilities as a Central Issue for InSilico Analysis of Cell Dynamics

Even if the leading role of mechanical forces in tissue growth and remodeling is well admitted, it is only recently that the effects of physical forces on gene expression and cell dynamics have been more extensively investigated. In this multiscale context, developing in silico models of cells and tissues is necessary to understand how biochemical and biomechanical cell signaling pathways interplay to specify a wide range of cellular dynamical processes and tissue architectures. In this paper, we review modeling approaches exemplifying how mechanical instabilities may drive emergent biological processes at different scales. At the cell level,we discuss the self-organized character of cell protrusions dynamics and the associated oscillatory cell shape changes modulated by the cell cortex contractility. At the cell population level, we analyze how the in vitro morphogenesis of endothelial cell networks can be enhanced or inhibited by modifications of the mechanical homeostasis of the cell/extracellular matrix medium. We finally outline how our understanding of such mechanical instabilities may support tissue engineering approaches in which transduction of mechanical forces has to be optimized in order to obtain specific cellular responses and tissue functional properties.     

Evaluation of scaffolds based on α-tricalcium phosphate cements for tissue engineering applications

Growth of cells in 3-D porous scaffolds has gained importance in the field of tissue engineering. The scaffolds guide cellular growth, synthesize extracellular matrix and other biological molecules, and make the formation of tissues and functional organs easier. The aim of this study is to use α-tricalcium phosphate cement in order to obtain new types of scaffolds with the aid of paraffin spheres as pore generators. The porosity of the scaffolds produced with paraffin spheres was analyzed and compared to the literature, and the study of scaffold permeability using the Forchheimer equation allowed the analysis of pore interconnectivity. In vitro tests showed the behavior of scaffolds in solutions of simulated body fluid, and viability and cell proliferation were also evaluated. The results show the potential use of the materials developed for scaffolds for use in tissue engineering applications.     

Layer‐by‐Layer Assembly of 3D Tissue Constructs with Functionalized Graphene

Carbon‐based nanomaterials have been considered promising candidates to mimic certain structure and function of native extracellular matrix materials for tissue engineering. Significant progress has been made in fabricating carbon nanoparticle‐incorporated cell culture substrates, but only a limited number of studies have been reported on the development of 3D tissue constructs using these nanomaterials. Here, a novel approach to engineer 3D multilayer constructs using layer‐by‐layer (LbL) assembly of cells separated with self‐assembled graphene oxide (GO)‐based thin films is presented. The GO‐based structures are shown to serve as cell adhesive sheets that effectively facilitate the formation of multilayer cell constructs with interlayer connectivity. By controlling the amount of GO deposited in forming the thin films, the thickness of the multilayer tissue constructs could be tuned with high cell viability. Specifically, this approach could be useful for creating dense and tightly connected cardiac tissues through the co‐culture of cardiomyocytes and other cell types. In this work, the fabrication of stand‐alone multilayer cardiac tissues with strong spontaneous beating behavior and programmable pumping properties is demonstrated. Therefore, this LbL‐based cell construct fabrication approach, utilizing GO thin films formed directly on cell surfaces, has great potential in engineering 3D tissue structures with improved organization, electrophysiological function, and mechanical integrity.     

Additive Manufacturing Approaches for Hydroxyapatite‐Reinforced Composites

Additive manufacturing (AM) techniques have gained interest in the tissue engineering field, thanks to their versatility and unique possibilities of producing constructs with complex macroscopic geometries and defined patterns. Recently, composite materials—namely, heterogeneous biomaterials identified as continuous phase (matrix) and reinforcement (filler)—have been proposed as inks that can be processed by AM to obtain scaffolds with improved biomimetic and bioactive properties. Significant efforts have been dedicated to hydroxyapatite (HA)‐reinforced composites, especially targeting bone tissue engineering, thanks to the chemical similarities of HA with respect to mineral components of native mineralized tissues. Herein, applications of AM techniques to process HA‐reinforced composites and biocomposites for the production of scaffolds with biological matrices, including cellular tissues, are reviewed. The primary outcomes of recent investigations in terms of morphological, structural, and in vitro and in vivo biological properties of the materials are discussed. The approaches based on the nature of the matrices employed to embed the HA reinforcements and produce the tissue substitutes are classified, and a critical discussion is provided on the presented state of the art as well as the future perspectives, to offer a comprehensive picture of the strategies investigated as well as challenges in this emerging field of materiomics.     

Advances and Applications of Biodegradable Elastomers in Regenerative Medicine

When elastomers were first proposed as useful materials for regenerative medicine a few decades ago, their high versatility and suitability for a diverse and wide range of in vivo applications could not have been predicted. Due to their ability to recover after deformation, these materials were first introduced in tissue engineering in an attempt to mimic the mechanical properties of the extracellular matrix. Furthermore, elastomeric characteristics have been described as important criteria for cell interaction by modulating cellular behavior. From soft to hard tissues, elastomers have demonstrated degradation, mechanical, and biocompatibility requirements in accordance with the target tissue. In this feature article, biodegradable synthetic polyester elastomers that have been reported in the literature are discussed, with special focus on those that show promise for in vivo tissue replacement. Their satisfactory performance in vivo shows the promise of elastomers for use in regenerative medicine. However, further investigation is required to demonstrate the prospect of elastomer‐based therapies in clinical trials.     

Ultrarapid Engineering of Biomimetic Materials and Tissues: Fabrication of Nano‐ and Microstructures by Plastic Compression

Currently, the concept of engineered tissues depends on the ability of cultured cells to fabricate new tissue around a scaffold. This is inherently slow and expensive and has had limited success so far. We report here a new process for the cell‐independent, controlled engineering of biomimetic scaffolds by rapid removal of fluid from hyperhydrated collagen gel (or other) constructs, using plastic compression (PC). PC fabrication produces dense, cellular, mechanically strong native collagen structures with controllable nano‐ and microscale biomimetic structures. The huge‐scale shrinkage (> 100‐fold) provides the ability to introduce controllable mechanical properties, microlayering, and embossed interface topography without cell participation, but with high cell viability. Critically, this takes minutes rather than the conventional days and weeks. The rapidity and biomimetic potential of the PC fabrication process at the mesoscale opens a new route for the production of biomaterials and patient‐customized tissues. It also represents a new concept in ‘engineering’ tissues.     

Integration of Self‐Assembled Microvascular Networks with Microfabricated PEG‐Based Hydrogels

Despite tremendous efforts, tissue engineered constructs are restricted to thin, simple tissues sustained only by diffusion. The most significant barrier in tissue engineering is insufficient vascularization to deliver nutrients and metabolites during development in vitro and to facilitate rapid vascular integration in vivo. Tissue engineered constructs can be greatly improved by developing perfusable microvascular networks in vitro in order to provide transport that mimics native vascular organization and function. Here a microfluidic hydrogel is integrated with a self‐assembling pro‐vasculogenic co‐culture in a strategy to perfuse microvascular networks in vitro. This approach allows for control over microvascular network self‐assembly and employs an anastomotic interface for integration of self‐assembled microvascular networks with fabricated microchannels. As a result, transport within the system shifts from simple diffusion to vessel supported convective transport and extra‐vessel diffusion, thus improving overall mass transport properties. This work impacts the development of perfusable prevascularized tissues in vitro and ultimately tissue engineering applications in vivo.     

Microfluidic Spinning of Cell‐Responsive Grooved Microfibers

Engineering living tissues that simulate their natural counterparts is a dynamic area of research. Among the various models of biological tissues being developed, fiber‐shaped cellular architectures, which can be used as artificial blood vessels or muscle fibers, have drawn particular attention. However, the fabrication of continuous microfiber substrates for culturing cells is still limited to a restricted number of polymers (e.g., alginate) having easy processability but poor cell–material interaction properties. Moreover, the typical smooth surface of a synthetic fiber does not replicate the micro‐ and nanofeatures observed in vivo, which guide and regulate cell behavior. In this study, a method to fabricate photocrosslinkable cell‐responsive methacrylamide‐modified gelatin (GelMA) fibers with exquisite microstructured surfaces by using a microfluidic device is developed. These hydrogel fibers with microgrooved surfaces efficiently promote cell encapsulation and adhesion. GelMA fibers significantly promote the viability of cells encapsulated in/or grown on the fibers compared with similar grooved alginate fibers used as controls. Importantly, the grooves engraved on the GelMA fibers induce cell alignment. Furthermore, the GelMA fibers exhibit excellent processability and could be wound into various shapes. These microstructured GelMA fibers have great potential as templates for the creation of fiber‐shaped tissues or tissue microstructures.     

Evaluation of the Biocompatibility of PLACL/Collagen Nanostructured Matrices with Cardiomyocytes as a Model for the Regeneration of Infarcted Myocardium

Pioneering research suggests various modes of cellular therapeutics and biomaterial strategies for myocardial tissue engineering. Despite several advantages, such as safety and improved function, the dynamic myocardial microenvironment prevents peripherally or locally administered therapeutic cells from homing and integrating of biomaterial constructs with the infarcted heart. The myocardial microenvironment is highly sensitive due to the nanoscale cues that it exerts to control bioactivities, such as cell migration, proliferation, differentiation, and angiogenesis. Nanoscale control of cardiac function has not been extensively analyzed in the field of myocardial tissue engineering. Inspired by microscopic analysis of the ventricular organization in native tissue, a scalable in‐vitro model of nanoscale poly(L ‐lactic acid)‐co ‐poly(? ‐caprolactone)/collagen biocomposite scaffold is fabricated, with nanofibers in the order of 594 ± 56 nm to mimic the native myocardial environment for freshly isolated cardiomyocytes from rabbit heart, and the specifically underlying extracellular matrix architecture: this is done to address the specificity of the underlying matrix in overcoming challenges faced by cellular therapeutics. Guided by nanoscale mechanical cues provided by the underlying random nanofibrous scaffold, the tissue constructs display anisotropic rearrangement of cells, characteristic of the native cardiac tissue. Surprisingly, cell morphology, growth, and expression of an interactive healthy cardiac cell population are exquisitely sensitive to differences in the composition of nanoscale scaffolds. It is shown that suitable cell–material interactions on the nanoscale can stipulate organization on the tissue level and yield novel insights into cell therapeutic science, while providing materials for tissue regeneration.     

Elastomeric Fibrous Hybrid Scaffold Supports In Vitro and In Vivo Tissue Formation

Biomimetic materials with biomechanical properties resembling those of native tissues while providing an environment for cell growth and tissue formation, are vital for tissue engineering (TE). Mechanical anisotropy is an important property of native cardiovascular tissues and directly influences tissue function. This study reports fabrication of anisotropic cell‐seeded constructs while retaining control over the construct's architecture and distribution of cells. Newly synthesized poly‐4‐hydroxybutyrate (P4HB) is fabricated with a dry spinning technique to create anelastomeric fibrous scaffold that allows control of fiber diameter, porosity, and rate ofdegradation. To allow cell and tissue ingrowth, hybrid scaffolds with mesenchymalstem cells (MSCs) encapsulated in a photocrosslinkable hydrogel were developed. Culturing the cellularized scaffolds in a cyclic stretch/flexure bioreactor resulted in tissue formation and confirmed the scaffold's performance under mechanical stimulation. In vivo experiments showed that the hybrid scaffold is capable of withstanding physiological pressures when implanted as a patch in the pulmonary artery. Aligned tissue formation occurred on the scaffold luminal surface without macroscopic thrombus formation. This combination of a novel, anisotropic fibrous scaffold and a tunable native‐like hydrogel for cellular encapsulation promoted formation of 3D tissue and provides a biologically functional composite scaffold for soft‐tissue engineering applications.     

Evolution of Bioengineered Lung Models: Recent Advances and Challenges in Tissue Mimicry for Studying the Role of Mechanical Forces in Cell Biology

Mechanical stretch under both physiological (breathing) and pathophysiological (ventilator‐induced) conditions is known to significantly impact all cellular compartments in the lung, thereby playing a pivotal role in lung growth, regeneration and disease development. In order to evaluate the impact of mechanical forces on the cellular level, in vitro models using lung cells on stretchable membranes have been developed. Only recently have some of these cell‐stretching devices become suitable for air–liquid interface cell cultures, which is required to adequately model physiological conditions for the alveolar epithelium. To reach this goal, a multi‐functional membrane for cell growth balancing biophysical and mechanical properties is critical to mimic (patho)physiological conditions. In this review, i) the relevance of cyclic mechanical forces in lung biology is elucidated, ii) the physiological range for the key parameters of tissue stretch in the lung is described, and iii) the currently available in vitro cell‐stretching devices are discussed. After assessing various polymers, it is concluded that natural‐synthetic copolymers are promising candidates for suitable stretchable membranes used in cell‐stretching models. This work provides guidance on future developments in biomimetic in vitro models of the lung with the potential to function as a template for other organ models (e.g., skin, vessels).     

Composite Living Fibers for Creating Tissue Constructs Using Textile Techniques

The fabrication of cell‐laden structures with anisotropic mechanical properties while having a precise control over the distribution of different cell types within the constructs is important for many tissue engineering applications. Automated textile technologies for making fabrics allow simultaneous control over the color pattern and directional mechanical properties. The use of textile techniques in tissue engineering, however, demands the presence of cell‐laden fibers that can withstand the mechanical stresses during the assembly process. Here, the concept of composite living fibers (CLFs) in which a core of load bearing synthetic polymer is coated by a hydrogel layer containing cells or microparticles is introduced. The core thread is drawn sequentially through reservoirs containing a cell‐laden prepolymer and a crosslinking reagent. The thickness of the hydrogel layer increases linearly with to the drawing speed and the prepolymer viscosity. CLFs are fabricated and assembled using regular textile processes including weaving, knitting, braiding, winding, and embroidering, to form cell‐laden structures. Cellular viability and metabolic activity are preserved during CLF fabrication and assembly, demonstrating the feasibility of using these processes for engineering functional 3D tissue constructs.     

Rapid Biofabrication of Printable Dense Collagen Bioinks of Tunable Properties

Recent convergence of the 3D printing of tissue‐like bioinks and regenerative medicine offers promise in the high‐throughput engineering of in vitro tissue models and organoids for drug screening and discovery research, and of potentially implantable neo‐tissues with tailored structural, biological, and mechanical properties. However, the current printing approaches are not compatible with collagen, the native scaffolding material. Herein, a unique biofabrication approach that uses automated gel aspiration‐ejection (GAE) is reported to potentially overcome these challenges. Automated‐GAE generates highly defined, aligned, dense collagen gel bioinks of various geometries (i.e., cylindrical, quadrangular, and tubular), dimensions, as well as tunable microstructural and mechanical properties that modulate seeded cellular responses. By densifying initial naturally derived reconstituted collagen hydrogels incorporating cells, automated‐GAE generates mini‐tissue building blocks with tailored protein fibril density and alignment, as well as cell loading, density and orientation according to the intended use. Surprisingly, a simple mathematical relationship defining the bioink compaction factor is found to be highly effective in predicting the initial and temporal properties of the bioinks in culture. Therefore, automated‐GAE will potentially also enable a fourth dimension to biofabrication, where cell–cell communications and cell‐extracellular matrix interactions as a function of time in culture can be predicted and modeled.     

Rapid Self‐Assembly of Macroscale Tissue Constructs at Biphasic Aqueous Interfaces

An entirely new approach to tissue engineering is presented that uses the interfacial forces between aqueous solutions of phase‐separating polymers to confine cells and promote their assembly into interconnected, macroscopic tissue constructs. This simple and inexpensive general procedure creates free‐standing, centimeter‐scale constructs from cell suspensions at the interface between poly(ethylene glycol) and dextran aqueous two‐phase systems in as little as 2 h. Using this method, skin constructs are produced that integrate with decellularized dermal matrices, on which they differentiate and stratify into skin equivalents. It is demonstrated that the constructs produced by this method have appropriate integrity and mechanical properties for use as in vitro tissue models.     

Three-dimensional in silico breast phantoms for multimodal image simulations

Anatomic simulators have provided researchers with the realistic objects needed to develop and evaluate medical imaging approaches. Today we have new insights into the cellular biology of breast tissues that is driving many new targeted diagnostic and therapeutic approaches, including molecular imaging. We report on our initial efforts to build a scalable framework for the construction of realistic 3-D in silico breast phantoms (ISBP) capable of leveraging existing knowledge and yet adaptable to fully integrate future discoveries. The ISBP frames are developed with scalable anatomical shapes and morphologic features as adapted from a rich literature on this topic. Frames are populated with tissue subtypes essential for imaging and object contrast functions are assigned. These data can be resampled to match the intrinsics scales of various imaging modalities; we explore mammography, sonography and computed tomography. Initial comparisons between simulated and clinical images demonstrate reasonable agreement and provides guidance for future development of a more realistic ISBP. An end-to-end simulation of breast images is described to demonstrate techniques for including stochastic variability and deterministic physical principles on which image formation is based.     

Functional Human Vascular Network Generated in Photocrosslinkable Gelatin Methacrylate Hydrogels

The generation of functional, 3D vascular networks is a fundamental prerequisite for the development of many future tissue engineering-based therapies. Current approaches in vascular network bioengineering are largely carried out using natural hydrogels as embedding scaffolds. However, most natural hydrogels present a poor mechanical stability and a suboptimal durability, which are critical limitations that hamper their widespread applicability. The search for improved hydrogels has become a priority in tissue engineering research. Here, the suitability of a photopolymerizable gelatin methacrylate (GelMA) hydrogel to support human progenitor cell-based formation of vascular networks is demonstrated. Using GelMA as the embedding scaffold, it is shown that 3D constructs containing human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs) generate extensive capillary-like networks in vitro. These vascular structures contain distinct lumens that are formed by the fusion of ECFC intracellular vacuoles in a process of vascular morphogenesis. The process of vascular network formation is dependent on the presence of MSCs, which differentiate into perivascular cells occupying abluminal positions within the network. Importantly, it is shown that implantation of cell-laden GelMA hydrogels into immunodeficient mice results in a rapid formation of functional anastomoses between the bioengineered human vascular network and the mouse vasculature. Furthermore, it is shown that the degree of methacrylation of the GelMA can be used to modulate the cellular behavior and the extent of vascular network formation both in vitro and in vivo. These data suggest that GelMA hydrogels can be used for biomedical applications that require the formation of microvascular networks, including the development of complex engineered tissues.     

Tissue Regeneration through Cyber-Physical Systems and Microbots

Tissue engineering is a systematic approach of assembling cells onto a 3D scaffold to form a functional tissue in the presence of critical growth factors. The scaffolding system guides stem cells through topological, physiochemical, and mechanical cues to differentiate and integrate to form a functional tissue. However, cellular communication during tissue formation taking place in a reactor needs to be understood properly to enable appropriate positioning of the cells in a 3D environment. Hence, sensors and actuators integrated with cyber-physical system (CPS) may be able to sense the tissue microenvironment and direct cells/cellular aggregates to an appropriate position, respectively. This can facilitate better cell-to-cell communication and cell–extracellular matrix communication for proper tissue morphogenesis. Advancements are made in the field of smart scaffolds that can morph cells/cellular aggregates after sensing the cellular microenvironment in a desired 3D architecture by providing appropriate cues. Recent scientific developments in the additive manufacturing technology have enabled the fabrication of smart scaffolds to create structural and functional tissue constructs. Sensors/actuators, cyber-systems, smart materials, and additive manufacturing put together is expected to lead to improved tissue-engineered medical products. The present review aims to highlight the possibilities of advancement of BioCPS for tissue engineering and regenerative medicine.     

Photosynthetic Cyanobacteria can Clearly Induce Efficient Muscle Tissue Regeneration of Bioprinted Cell-Constructs

Tissue engineering strategies using cell-laden constructs have shown promising results in the treatment of various types of damaged tissues. However, inadequate oxygen delivery to the macroscale 3D cell-constructs for regenerating skeletal muscle tissue has remained a multiplex issue owing to the pivotal factors including cell metabolism and several regulatory intercellular pathways that eventually influence various cellular activities and determines cell phenotype. To overcome this issue, a photosynthetic cyanobacterium (Synechococcus elongatus) is employed in a methacrylated gelatin bioink. Furthermore, to effectively induce cell alignment in the bioink, in situ electric field stimulation is used in a bioprinting system to fabricate cell-laden scaffolds for regenerating skeletal muscle tissue. Owing to the synergistic effects of the bioactive microenvironment that rescues cells from hypoxic conditions and activations of voltage-gated ion channels, highly aligned, multi-nucleated myofibers are obtained as well as significant upregulation (7–10-fold) of myogenic-related genes compared with conventionally prepared cell-constructs. In addition, in vivo studies using a mouse volumetric muscle loss model demonstrate considerable restoration of muscle functionality and regeneration.     

Manipulation of Stem Cells Fates: The Master and Multifaceted Roles of Biophysical Cues of Biomaterials

Owing to their self-renewal and differentiation ability, stem cells are conducive for repairing injured tissues, making them a promising source of seed cells for tissue engineering. The extracellular microenvironment (ECM) is under dynamic mechanical control, which is closely related to stem cell behaviors. During the design and fabrication of biomaterials for regenerative medicine, the physiochemical properties of the natural ECM should be closely mimicked, which can reinforce stem cell lineage choice and tissue engineering. By reproducing the biophysical stimulations that stem cells may experience in vivo, many studies have highlighted the key role of biophysical cues in regulation of cell fate. Optimization of biophysical factors leads to desirable stem cell functions, which can maximize the effectiveness of regenerative treatment. In this review, the main biophysical cues of biomaterials, including stiffness, topography, mechanical force, and external physical fields are summarized, and their individual and synergistic influence on stem cell behavior is discussed. Subsequently, the current progress in tissue regeneration using biomaterials is presented, which directs the design and fabrication of functional biomaterial. The mechanisms via which biophysical cues activate cellular responses are also analyzed. Finally, the challenges in basic research as well as for clinical translation in this field are discussed.