Tissue-engineered Constructs Research Articles

The Innovative Biomaterials and Technologies for Developing Corneal Endothelium Tissue Engineering Scaffolds: A Review and Prospect.

Corneal transplantation is the only treatment for corneal endothelial blindness. However, there is an urgent need to find substitutes for corneal endothelium grafts due to the global shortage of donor corneas. An emerging research field focuses on the construction of scaffold-based corneal endothelium tissue engineering (CETE). Long-term success in CETE transplantation may be achieved by selecting the appropriate biomaterials as scaffolds of corneal endothelial cells and adding bioactive materials to promote cell activity. This article reviews the research progress of CETE biomaterials in the past 20 years, describes the key characteristics required for corneal endothelial scaffolds, and summarizes the types of materials that have been reported. Based on these, we list feasible improvement strategies for biomaterials innovation. In addition, we describe the improved techniques for the scaffolds' surface topography and drug delivery system. Some promising technologies for constructing CETE are proposed. However, some questions have not been answered yet, and clinical trials and industrialization should be carried out with caution.

MicroAge Mission: Examining the Effects of Microgravity and Electrical Stimulation on the Proteome of Human Tissue-Engineered Muscle Constructs

MicroAge Mission: Examining the Effects of Microgravity and Electrical Stimulation on the Proteome of Human Tissue-Engineered Muscle Constructs

Integrating shear flow and trypsin treatment to assess cell adhesion strength.

Cell adhesion is of fundamental importance in cell and tissue organization and for designing cell-laden constructs for tissue engineering. Prior methods to assess cell adhesion strength for strongly adherent cells using hydrodynamic shear flow either involved the use of specialized flow devices to generate high shear stress or used simpler implementations like larger height parallel plate chambers that enable multihour cell culture but generate low wall shear stress and are, hence, more applicable for weakly adherent cells. Here, we propose a shear flow assay for adhesion strength assessment of strongly adherent cells that employs off-the-shelf parallel plate chambers for shear flow as well as simultaneous trypsin treatment to tune down the adhesion strength of cells. We implement the assay with a strongly adherent cell type and show that wall shear stress in the 0.07-7 Pa range is sufficient to dislodge the cells with simultaneous trypsin treatment. Imaging of cells over a square centimeter area allows cell morphological analysis of hundreds of cells. We show that the cell area of cells that are dislodged, on average, does not monotonically increase with wall shear stress at the higher end of wall shear stresses used and suggest that this can be explained by the likely higher resistance of high circularity cells to trypsin digestion. The adhesion strength assay proposed can be used to assess the adhesion strength of both weakly and strongly adherent cell types and has the potential to be adapted for substrate stiffness-dependent adhesion strength assessment in mechanobiology studies.

From Static to Dynamic: Smart Materials Pioneering Additive Manufacturing in Regenerative Medicine.

The emerging field of regenerative medicine holds immense promise for addressing complex tissue and organ regeneration challenges. Central to its advancement is the evolution of additive manufacturing techniques, which have transcended static constructs to embrace dynamic, biomimetic solutions. This manuscript explores the pivotal role of smart materials in this transformative journey, where materials are endowed with dynamic responsiveness to biological cues and environmental changes. By delving into the innovative integration of smart materials, such as shape memory polymers and stimulus-responsive hydrogels, into additive manufacturing processes, this research illuminates the potential to engineer tissue constructs with unparalleled biomimicry. From dynamically adapting scaffolds that mimic the mechanical behavior of native tissues to drug delivery systems that respond to physiological cues, the convergence of smart materials and additive manufacturing heralds a new era in regenerative medicine. This manuscript presents an insightful overview of recent advancements, challenges, and future prospects, underscoring the pivotal role of smart materials as pioneers in shaping the dynamic landscape of regenerative medicine and heralding a future where tissue engineering is propelled beyond static constructs towards biomimetic, responsive, and regenerative solutions.

Fundamentals and Applications of Raman-Based Techniques for the Design and Development of Active Biomedical Materials.

Raman spectroscopy is an analytical method based on light-matter interactions that can interrogate the vibrational modes of matter and provide representative molecular fingerprints. Mediated by its label-free, non-invasive nature, and high molecular specificity, Raman-based techniques have become ubiquitous tools for in situ characterization of materials. This review comprehensively describes the theoretical and practical background of Raman spectroscopy and its advanced variants. The numerous facets of material characterization that Raman scattering can reveal, including biomolecular identification, solid-to-solid phase transitions, and spatial mapping of biomolecular species in bioactive materials, are highlighted. The review illustrates the potential of these techniques in the context of active biomedical material design and development by highlighting representative studies from the literature. These studies cover the use of Raman spectroscopy for the characterization of both natural and synthetic biomaterials, including engineered tissue constructs, biopolymer systems, ceramics, and nanoparticle formulations, among others. To increase the accessibility and adoption of these techniques, the present review also provides the reader with practical recommendations on the integration of Raman techniques into the experimental laboratory toolbox. Finally, perspectives on how recent developments in plasmon- and coherently-enhanced Raman spectroscopy can propel Raman from underutilized to critical for biomaterial development areprovided.

Immunobiology of foreign body response to composite PLACL/gelatin electrospun nanofiber meshes with mesenchymal stem/stromal cells in a mouse model: Implications in pelvic floor tissue engineering and regeneration

Pelvic Organ Prolapse (POP) is a common gynaecological disorder where pelvic organs protrude into the vagina. While transvaginal mesh surgery using non-degradable polymers was a commonly accepted treatment for POP, it has been associated with high rates of adverse events such as mesh erosion, exposure and inflammation due to serious foreign body response and therefore banned from clinical use after regulatory mandates. This study proposes a tissue engineering strategy using uterine endometrium-derived mesenchymal stem/stromal cells (eMSC) delivered with degradable poly L-lactic acid-co-poly ε-caprolactone (PLACL) and gelatin (G) in form of a composite electrospun nanofibrous mesh (P + G nanomesh) and evaluates the immunomodulatory mechanism at the material interfaces. The study highlights the critical acute and chronic inflammatory markers along with remodelling factors that determine the mesh surgery outcome. We hypothesise that such a bioengineered construct enhances mesh integration and mitigates the Foreign Body Response (FBR) at the host interface associated with mesh complications. Our results show that eMSC-based nanomesh significantly increased 7 genes associated with ECM synthesis and cell adhesion including, Itgb1, Itgb2, Vcam1, Cd44, Cdh2, Tgfb1, Tgfbr1, 6 genes related to angiogenesis including Ang1, Ang2, Vegfa, Pdgfa, Serpin1, Cxcl12, and 5 genes associated with collagen remodelling Col1a1, Col3a1, Col6a1, Col6a2, Col4a5 at six weeks post-implantation. Our findings suggest that cell-based tissue-engineered constructs potentially mitigate the FBR response elicited by biomaterial implants. From a clinical perspective, this construct provides an alternative to current inadequacies in surgical outcomes by modulating the immune response, inducing angiogenesis and ECM synthesis during the acute and chronic phases of the FBR.

Repair of osteochondral defects: efficacy of a tissue-engineered hybrid implant containing both human MSC and human iPSC-cartilaginous particles

Both mesenchymal stromal cells (MSC) and induced pluripotent stem cells (iPSC) offer the potential for repair of damaged connective tissues. The use of hybrid implants containing both human MSC and iPSC was investigated to assess their combined potential to yield enhanced repair of osteochondral defects. Human iPSC-CP wrapped with tissue engineered constructs (TEC) containing human MSC attained secure defect filling with good integration to adjacent tissue in a rat osteochondral injury model. The presence of living MSC in the hybrid implants was required for effective biphasic osteochondral repair. Thus, the TEC component of such hybrid implants serves several critical functions including, adhesion to the defect site via the matrix and facilitation of the repair via live MSC, as well as enhanced angiogenesis and neovascularization. Based on these encouraging studies, such hybrid implants may offer an effective future intervention for repair of complex osteochondral defects.

Review: Tailored microstructures in the system tricalcium phosphate-wollastonite-diopside for bone regeneration scaffolds

Tissue engineering constructs (TECs) combining resorbable scaffolds, cells and/or reactive agents are required for bone regeneration. New green ceramic processing methods allow fabricating layered and gradient porous structures. Dense bioceramics with sufficient strength that keep their microstructure when incorporating porosity are required. Bioceramics in the system tricalcium phosphate-wollastonite-diopside are reviewed. Tailoring of microstructures to improve their mechanical and biological behaviors and their potential for TECs are analyzed. First, a brief discussion on bone properties and requirements for biomaterials and the capabilities and limits of glasses and glass-ceramics formulated in the system is done. Then, the relationships between the microstructure and the mechanical behavior of single-phase and composite bioceramics are discussed. TCP-D-W microstructures with adequate mechanical performance support viability and osteogenic differentiation of human mesenchymal stem cells (hASCs) and their colonization by hASCs presents specific morphological features that make them adequate for TECs.

Biological properties of macroporous cryostructurate based on extracellular matrix components

Objective: to study the biological properties of macroporous cryostructurate from multicomponent concentrated collagen-containing solution (MCCS) as a promising matrix for the formation of cell- and tissue-engineered constructs.Materials and methods. A macroporous spongy carrier was obtained by cryostructuring of collagencontaining extract, prepared by acetic acid hydrolysis of chicken connective tissue (BIOMIR Service, Russian Federation). N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (Sigma-Aldrich, USA) was used to make the cryostructurate water insoluble. The micromorphology of the sponge surface was studied using scanning electron microscopy. The cytotoxicity of the carrier was evaluated by reaction of the mouse NIH 3T3 fibroblast cell culture using automated microscope IncuCyte ZOOM (EssenBioscience, USA). Biocompatibility of the macroporous carrier was studied on cultures of human adipose tissue-derived mesenchymal stromal cells (AD-MSC), human hepatocellular carcinoma cell line HepG2 and human umbilical vein endothelial cell line EA.hy926. The metabolic activity of cells was determined using PrestoBlue™ reagents (Invitrogen™, USA). Cell population development during long-term cultivation of the cell-engineered construct (CEC) was assessed by fluorescencelifetime imaging microscopy over the entire surface of the sample using a Leica Dmi8 inverted microscope with Leica Thunder software (Leica Microsystems, Germany).Results. Optical microscopy and scanning electron microscopy (SEM) showed the presence of pores of different sizes in the resulting biopolymer material: large pores with 237 ± 32 μm diameter, medium-sized pores with 169 ± 23 μm diameter, and small-sized pores with 70 ± 20 μm diameter; large and medium-sized pores were predominant. The studied media did not exhibit cytotoxicity. Cell adhesion and proliferation on the surface of the material and their penetration into the underlying layers during long-term cultivation were observed. The highest metabolic activity of the cells was observed for human AD-MSC on day 14, which corresponds to the normal dynamics of development of a population of cells of this type. The functional activity of HepG2 cells – albumin and urea production – was shown in the liver CEC model.Conclusion. The good adhesion and active proliferation that were shown for the three cell types indicate that the resulting biopolymer carrier is biocompatible, and that the spread of the cells into the inner volume of the sponge and active population of the sponge under prolonged culturing indicates that this material can be used to create cell- and tissue-engineered constructs.

Biofabrication of Composite Tendon Constructs with the Fibrous Arrangement, High Cell Density, and Enhanced Cell Alignment.

Current tissue-engineered tendons are mostly limited to the replication of fibrous organizations of native tendons, which lack the biomimicry of a densely packed cell arrangement. In this study, composite tendon constructs (CTCs) with fibrous arrangement, high cell density, and enhanced cell alignment were developed by integrating the electrohydrodynamic jet 3D printing (e-jetting) technique and the fabrication of tissue strands (TSs). A tubular polycaprolactone (PCL) scaffold was created using e-jetting, followed by coating a thin layer of alginate. Human mesenchymal stem cells were then microinjected into the PCL scaffolds, aggregated into TSs, and formed CTCs with a core-shell structure. Owing to the presence of TSs, CTCs demonstrated the anatomically relevant cell density and morphology, and cells migrated from the TSs onto e-jetted scaffolds. Also, the mechanical strength of CTCs approached that of native tendons due to the existence of e-jetted scaffolds (Young's modulus: ∼21 MPa, ultimate strength: ∼5 MPa). During the entire culture period, CTCs maintained high survival rates and good structural integrity without the observation of necrotic cores and disintegration of two portions. In addition, CTCs that were cultured with uniaxial cyclic stretching revealed not only the increased expression of tendon-related proteins but also the enhanced cellular orientation. The promising results demonstrated the potential of this novel biofabrication strategy for building tissue-engineered tendon constructs with the proper biological, mechanical, and histological relevance..

Characterization of 3D organotypic epithelial tissues reveals tonsil-specific differences in tonic interferon signaling.

Three-dimensional (3D) culturing techniques can recapitulate the stratified nature of multicellular epithelial tissues. Organotypic 3D epithelial tissue culture methods have several applications, including the study of tissue development and function, drug discovery and toxicity testing, host-pathogen interactions, and the development of tissue-engineered constructs for use in regenerative medicine. We grew 3D organotypic epithelial tissues from foreskin, cervix, and tonsil-derived primary cells and characterized the transcriptome of these in vitro tissue equivalents. Using the same 3D culturing method, all three tissues yielded stratified squamous epithelium, validated histologically using basal and superficial epithelial cell markers. The goal of this study was to use RNA-seq to compare gene expression patterns in these three types of epithelial tissues to gain a better understanding of the molecular mechanisms underlying their function and identify potential therapeutic targets for various diseases. Functional profiling by over-representation and gene set enrichment analysis revealed tissue-specific differences: i.e., cutaneous homeostasis and lipid metabolism in foreskin, extracellular matrix remodeling in cervix, and baseline innate immune differences in tonsil. Specifically, tonsillar epithelia may play an active role in shaping the immune microenvironment of the tonsil balancing inflammation and immune responses in the face of constant exposure to microbial insults. Overall, these data serve as a resource, with gene sets made available for the research community to explore, and as a foundation for understanding the epithelial heterogeneity and how it may impact their in vitro use. An online resource is available to investigate these data (https://viz.datascience.arizona.edu/3DEpiEx/).

Hydroxyapatite cross-linked in situ polyvinyl alcohol hydrogel for bionic calcified cartilage layer.

Many tissue engineering constructs have been investigated for repairing the calcified cartilage layer in the recent years, but engineering a consistent and stable interface to facilitate a graft-to-bone fixation remains a concern. In the work, hydroxyapatite (HA) is modified by diisocyanate (HDI) and integrated with polyvinyl alcohol (PVA) to prepare the hydrogel. The IR shows that HDI is grafted onto HA and helps HA to cross-link in situ in the PVA gel network. When the mass ratio of HA/PVA is 3.5wt%, the swelling rate in the PBS of different pH reduced with time prolong, and the hydrogel takes on good swelling resistance. The tensile strength and toughness are 1890 KPa and 264 KJ/m-3, respectively, while the compression strength reaches 1125 KPa at compressive strain of 60%. The hydrogel not only is well durable via continuous 100 rounds of compression-recovery, but also has excellent bioactivity. The work will provide a platform for developing multifunctional soft tissue scaffold.

Emerging development in polymeric electrospun nanoscale mats for tissue regeneration: narrative review of the literature

Tissue engineering is a cutting-edge discipline that brings together scientific and health-related, biological, and engineering principles in order to build tissue-engineered constructions able to restore or sustain the physiological properties of native tissue, or to marginally enhance those properties. This field is called "regenerative medicine". By constructing structures that are analogous to the extracellular matrix, it will be possible to improve the transmission of oxygen and nutrients, as well as the release of toxins during the process of tissue healing, all while simultaneously maturing tissues. Over the past few years, various studies have concentrated on looking at nanostructures in three dimensions with the goal of using them in tissue engineering. In this group of methods, electrospinning stands out as one of the most successful options. Over the course of the past few decades, a great number of nanofibrous scaffolds have been produced for the purpose of restoring and repairing damaged tissue. In this article, the engineering of new tissues using nanofibrous textures as scaffolds are reviewed. In addition, recent developments in tissue regeneration and the difficulties related to electrospinning are discussed in this article, along with their respective solutions.

Bio-Inspired Nanomaterials for Micro/Nanodevices: A New Era in Biomedical Applications.

Exploring bio-inspired nanomaterials (BINMs) and incorporating them into micro/nanodevices represent a significant development in biomedical applications. Nanomaterials, engineered to imitate biological structures and processes, exhibit distinctive attributes such as exceptional biocompatibility, multifunctionality, and unparalleled versatility. The utilization of BINMs demonstrates significant potential in diverse domains of biomedical micro/nanodevices, encompassing biosensors, targeted drug delivery systems, and advanced tissue engineering constructs. This article thoroughly examines the development and distinctive attributes of various BINMs, including those originating from proteins, DNA, and biomimetic polymers. Significant attention is directed toward incorporating these entities into micro/nanodevices and the subsequent biomedical ramifications that arise. This review explores biomimicry's structure-function correlations. Synthesis mosaics include bioprocesses, biomolecules, and natural structures. These nanomaterials' interfaces use biomimetic functionalization and geometric adaptations, transforming drug delivery, nanobiosensing, bio-inspired organ-on-chip systems, cancer-on-chip models, wound healing dressing mats, and antimicrobial surfaces. It provides an in-depth analysis of the existing challenges and proposes prospective strategies to improve the efficiency, performance, and reliability of these devices. Furthermore, this study offers a forward-thinking viewpoint highlighting potential avenues for future exploration and advancement. The objective is to effectively utilize and maximize the application of BINMs in the progression of biomedical micro/nanodevices, thereby propelling this rapidly developing field toward its promising future.

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Generation of Patterned Cocultures in 2D and 3D: State of the Art.

Cells inside the body are embedded into a highly structured microenvironment that consists of cells that lie in direct or close contact with other cell types that regulate the overall tissue function. Therefore, coculture models are versatile tools that can generate tissue engineering constructs with improved mimicking of in vivo conditions. While there are many reviews that have focused on pattering a single cell type, very few reviews have been focused on techniques for coculturing multiple cell types on a single substrate with precise control. In this regard, this Review covers various technologies that have been utilized for the development of these patterned coculture models while mentioning the limitations associated with each of them. Further, the application of these models to various tissue engineering applications has been discussed.

Successful endothelial monolayer formation on melt electrowritten scaffolds under dynamic conditions to mimic tunica intima

The lack of transplantable tissues and organs as well as the limitations of synthetic implants highlight the need for tissue-engineered constructs to obtain safe, long-lasting, and limitless tissue replacements. Scaffolds for cardiovascular applications, such as for a tissue-engineered vascular graft (TEVG), are thus highly required. For TEVGs, tubular scaffolds should support the formation of confluent endothelial layers in particular under dynamic conditions to prevent thrombosis and maintain hemostasis. For that purpose, a porous and highly diffusible scaffold structure is necessary to allow optimal cell adhesion as well as oxygen and nutrient exchange with the surrounding tissue. Here, we present a three-dimensional-printed scaffold made by a combination of fused deposition modeling (FDM) and melt electrowriting (MEW) out of polycaprolactone that enables monolayer formation and alignment of endothelial cells in the direction of medium flow under a shear stress of up to 10 dyn cm-2. Pore size and coating with human fibrin were optimized to enable confluent endothelial layers on the printed scaffold structures. Cell orientation and shape analysis showed a characteristic alignment and elongation of the tested endothelial cells with the direction of flow after dynamic cultivation. In contrast, melt electrospun scaffolds based on the same CAD design under comparable printing and cultivation conditions were not sufficient to form gapless cell layers. Thus, the new scaffold fabricated by MEW/FDM approach appears most suitable for TEVGs as a template for the innermost vascular wall layer, the tunica intima.

The Influence of Technological Factors and Polar Molecules on the Structure of Fibrillar Matrices Based on Ultrafine Poly-3-hydroxybutyrate Fibers Obtained via Electrospinning

The article examines the regularities of structure formation of ultrafine fibers based on poly-3-hydroxybutyrat under the influence of technological (electrical conductivity, viscosity), molecular (molecular weight), and external factors (low-molecular and nanodispersed substances of different chemical natures). Systems with polar substances are characterized by the presence of intermolecular interactions and the formation of a more perfect crystalline fiber structure. Changes in technological and molecular characteristics affect the fiber formation process, resulting in alterations in the morphology of the nonwoven fabric, fiber geometry, and supramolecular fiber structure. Polymer molecular weight, electrical conductivity, and solution viscosity influence fiber formation and fiber diameter. The fiber structure is heterogeneous, consisting of both crystalline and non-equilibrium amorphous phases. This article shows that with an increase in the molecular weight and concentration of the polymer, the diameter of the fiber increases. At the same time, the increase in the productivity of the electrospinning process does not affect the fiber geometry. The chemical structure of the solvent and the concentration of polar substances play a decisive role in the formation of fibers of even geometry. As the polarity of the solvent increases, the intermolecular interaction with the polar groups of poly-3-hydroxybutyrate increases. As a result of this interaction, the crystallites are improved, and the amorphous phase of the polymer is compacted. The action of polar molecules on the polymer is similar to the action of polar nanoparticles. They increase crystallinity via a nucleation mechanism. This is significant in the development of matrix-fibrillar systems for drug delivery, bioactive substances, antiseptics, tissue engineering constructs, tissue engineering scaffolds, artificial biodegradable implants, sorbents, and other applications.

Morphology and gene expression of sex‐determining region Y‐box 9 and runt‐related transcription factor 2 in centrifugally compressed cell collagen‐combined constructs (C6)

AbstractThe authors previously developed a scaffold‐free tissue‐engineered construct (TEC) from mesenchymal stem cells (MSCs). Although the TEC exhibited even cell distribution and was successfully applied for cartilage repair in animal models, it is unsuitable for relatively large‐scale cartilage defects due to its small size. To solve the problem, the authors recently developed a novel biomaterial, a centrifugally compressed cell‐collagen combined construct (C6) from a mixture of MSCs and atelocollagen, both of which are subjected to centrifugation. The results of the previous study indicated that C6 exhibited high cell viability (70 %) and sufficient cell distribution similar to that of the TEC. In the present study, the morphology and gene expression of C6 were investigated. Histological examination indicated that C6 is six times thicker (approximately 1 mm) than the TEC after a 7‐day culture. The C6 remained unchanged in scale with increased cell density after a 21‐day culture. Scanning electron microscopic observation indicated that C6 exhibited interconnected and porous microstructures, while the TEC had close‐knit microstructures. Reverse transcriptase‐polymerase chain reaction analysis indicated that the expression of sex‐determining region Y‐box 9 and runt‐related transcription factor 2 was significantly higher in C6 than that in TEC.

Surface tension-induced biomimetic assembly of cell-laden fibrous bundle construct for muscle tissue engineering.

The field of tissue engineering has been long seeking to develop functional muscle tissue that closely resembles natural muscle. This study used a bio-inspired assembly based on the surface tension mechanism to develop a novel method for engineering muscle tissue. This approach enabled uniaxially ordered electrospun fibers to naturally collide into an aligned bundle without the need for manual handling, thereby reducing cell damage during the cell culture procedure. During the assembly procedure, C2C12 myoblasts were cultured in a viscous collagen hydrogel that caused wetting while providing adequate structural stability for the cell-fiber construct. In addition, gene expression analysis of the resulting muscle-like fibril bundle revealed improved myogenic differentiation. These findings highlight the potential of using a collagen hydrogel and the surface tension mechanism to construct biologically relevant muscle tissue, offering a promising strategy that may outperform existing approaches. Overall, this study contributes to the development of advanced tissue engineering methods and brings us a step closer to creating functional muscle tissue for therapeutic and regenerative medicine applications.