Cellular alignment and fusion: Quantifying the effect of macrophages and fibroblasts on myoblast terminal differentiation.

Successful skeletal muscle wound repair requires the alignment and fusion of myoblasts to generate multinucleated myofibers. In vitro, the accurate quantification of cellular alignment remains a challenge. Here we present the application of ImageJ and ct-FIRE to quantify muscle cell orientation by means of an alignment index (AI). Our optimised method, which does not require programming skills, allows the alignment of myoblasts in vitro to be determined independently of a predefined reference point. Using this method, we demonstrate that co-culture of myoblasts with macrophages, but not fibroblasts, promotes myoblast alignment in a cell density-dependent manner. Interestingly, myoblast fusion was significantly decreased in response to co-culture with macrophages, while the effect of fibroblasts on fusion was density-dependent. At lower numbers, fibroblasts significantly increased myoblast fusion, whereas at higher numbers a significant decrease was observed. Finally, triple co-culture revealed that the effect of macrophages on myoblast alignment and fusion is unaltered by the additional presence of fibroblasts. Application of our optimised method has therefore revealed quantitative differences in the roles of macrophages versus fibroblasts during alignment and fusion: while successful myoblast alignment is promoted by increasing macrophage numbers, regenerative fusion coincides with a decreasing macrophage population and initial rise in fibroblast numbers.

Developing Porous Fibrin Scaffolds with Tunable Anisotropic Features to Direct Myoblast Orientation.

Functional regeneration of anisotropically aligned tissues such as ligaments, microvascular networks, myocardium, or skeletal muscle requires a temporal and spatial series of biochemical and biophysical cues to direct cell functions that promote native tissue regeneration. When these cues are lost during traumatic injuries such as volumetric muscle loss (VML), scar formation occurs, limiting the regenerative capacity of the tissue. Currently, autologous tissue transfer is the gold standard for treating injuries such as VML but can result in adverse outcomes including graft failure, donor site morbidity, and excessive scarring. Tissue-engineered scaffolds composed of biomaterials, cells, or both have been investigated to promote functional tissue regeneration but are still limited by inadequate tissue ingrowth. These scaffolds should provide precisely tuned topographies and stiffnesses using proregenerative materials to encourage tissue-specific functions such as myoblast orientation, followed by aligned myotube formation and recovery of functional contraction. In this study, we describe the design and characterization of novel porous fibrin scaffolds with anisotropic microarchitectural features that recapitulate the native tissue microenvironment and offer a promising approach for regeneration of aligned tissues. We used directional freeze-casting with varied fibrin concentrations and freezing temperatures to produce scaffolds with tunable degrees of anisotropy and strut widths. Nanoindentation analyses showed that the moduli of our fibrin scaffolds varied as a function of fibrin concentration and were consistent with native skeletal muscle tissue. Quantitative morphometric analyses of myoblast cytoskeletons on scaffold microarchitectures demonstrated enhanced cell alignment as a function of microarchitectural morphology. The ability to precisely control the anisotropic features of fibrin scaffolds promises to provide a powerful tool for directing aligned tissue ingrowth and enhance functional regeneration of tissues such as skeletal muscle.

Engineering highly-aligned three-dimensional (3D) cardiac constructs for enhanced myocardial infarction repair.

Native myocardium exhibits well-organized cellular orientations and highly vascularized architectures, which is important for tissue survival and synchronic contraction activities. Mimicking such structural organizations to engineer functional cardiac constructs is a promising approach to treat myocardial infarctionin vivo. Here we propose a novel strategy to engineer highly-aligned three-dimensional (3D) cardiac constructs by co-culturing cardiomyocytes and rat aortic endothelial cells (RAECs) along with native extracellular matrix-derived fibrin within electrohydrodynamic-printed microfibrous architectures. Cell-laden fibrin with a relatively rapid gelation rate enables uniform cellular distribution in 3D and can re-organize to form multidirectionally aligned 3D cardiac bands with similar orientations to the printed microfibers. The resultant 3D cardiac constructs show enhanced cardiomyocyte-specific protein expression, synchronous contraction and low excitation threshold. The addition of RAECs significantly increases the width of cardiac bands and enhances their beating frequency. The engineered 3D cardiac constructs with layer-specific orientations were found to effectively reduce infracted area, enhance neovascularization and eventually realize functional repair of infarcted myocardiumin vivo. This exploration provides a promising strategy to engineer 3D cardiac constructs with tissue-specific cellular orientations for the functional repair of infarcted myocardium.

Fabricating Tissues In Situ with the Controlled Cellular Alignments.

Tissue engineering techniques have enabled to replicate the geometrical architecture of native tissues but usually fail to reproduce their exact cellular arrangements during the fabricating process, while it is critical for manufacturing physiologically relevant tissues. To address this problem, a "sewing-like" method of controlling cellular alignment during the fabricating process is reported here. By integrating the stretching step into the fabricating process, a static mechanical environment is created which, in turn, regulates the subsequent cellular alignment, elongation, and differentiation in the generated tissues. With this method, patterned cellular constructs can be fabricated with controlled cellular alignment. Moreover, this method shows a potent capability to fabricate physiologically relevant skeletal muscle constructs in vitro by mechanically inducing myoblast fusion and maturation. As a potential clinical application, aligned myofibers are directly fabricated onto injured muscles in vivo, which repair the damaged tissues effectively. This study shows that the "sewing-like" method can produce engineered tissues with precise control of cellular arrangements and more clinically viable functionalities.

Fabrication of a mimetic vascular graft using melt spinning with tailorable fiber parameters.

Smooth muscle cells play a pivotal role in maintaining blood pressure and remodeling of the extracellular matrix. These cells have a characteristic spindle shape and are aligned in the radial direction to aid in the constriction of any artery. Tissue engineered grafts have the potential to recreate this alignment and offer a viable alternative to non-resorbable or autologous grafts. Specifically, with melt spinning small diameter fibers can be created that can align circumferentially on the scaffolds. In this study, a set of simplified equations were formulated to predict the final fiber parameters. Smooth muscle cell alignment was monitored on the fabricated scaffolds. Finally, a co-culture of smooth muscle cells in direct contact with endothelial cells was performed to assess the influence of the smooth muscle cell alignment on the morphology of the endothelial cells. The results show that the equations were able to accurately predict the fiber diameter, distance and angle. Primary vascular smooth muscle cells aligned according to the fiber direction mimicking the native orientation. The co-culture with endothelial cells showed that the aligned smooth muscle cells did not have an influence on the morphology of the endothelial cells. In conclusion, we formulated a series of equations that can predict the fiber parameters during melt spinning. Furthermore, the method described here can create a vascular graft with smooth muscle cells aligned circumferentially that morphologically mimics the native orientation.

Biomimicked large-area anisotropic grooves fromDracaena sanderianaleaf enhances cellular alignment and subsequent differentiation.

Cellular alignment is important for the proper functioning of different tissues such as muscles or blood vessel walls. Hence, in tissue engineering, sufficient effort has been made to control cellular orientation and alignment. It has been shown that micro-and nanoscale anisotropic topological features on cell culture substrates can control cellular orientation. Such substrates are fabricated using various lithography techniques such as photolithography and soft lithography. Although such techniques are suitable for creating patterns in small areas to establish a proof-of-concept, patterning large areas with intricate features is an unsolved problem. In this work, we report that a replica of the groove-like anisotropic patterns of the abaxial side of aDracaena sanderiana(bamboo) leaf can be used for large-area patterning of cells. We imprinted the leaf on polydimethylsiloxane (PDMS) and characterised its surface topography using scanning electron microscopy. We further cultured bone marrow human mesenchymal cells (BM-hMSCs), skeletal muscle cells (C2C12), and neuroblastoma cells (SHSY5Y) on the patterned PDMS on which the cells orient along the direction of the grooved pattern. Further, we observed enhanced neuronal differentiation of SHSY5Y cells on biomimicked pattern compared to flat PDMS as measured by percentage of cells with neurites, neurite length and the expression of neuronal differentiation marker beta-III tubulin (TUJ1). This process is simple, frugal, and can be adopted by laboratories with resource constraints. This one-step technique to fabricate large-area anisotropic surface patterns from bamboo leaves can be used as a platform to study cellular alignment and its effect on various cellular functions, including differentiation.

Formulation of Dermal Tissue Matrix Bioink by a Facile Decellularization Method and Process Optimization for 3D Bioprinting toward Translation Research.

Decellularized extracellular matrices (ECMs) are being extensively used for tissue engineering purposes and detergents are predominantly used for this. A facile detergent-free decellularization method is developed for dermal matrix and compared it with the most used detergent-based decellularization methods. An optimized, single-step, cost-effective Hypotonic/Hypertonic (H/H) Sodium Chloride (NaCl) solutions-based method is employed to decellularize goat skin that resulted in much higher yield than other methods. The ECM composition, mechanical property, and cytocompatibility are evaluated and compared with other decellularization methods. Furthermore, this H/H-treated decellularized dermal ECM (ddECM) exhibits a residual DNA content of <50 ng mg-1  of dry tissue. Moreover, 85.64 ± 3.01% of glycosaminoglycans and 65.53 ± 2.9% collagen are retained compared to the native tissue, which is higher than the ddECMs prepared by other methods. The cellular response is superior in ddECM (H/H) than other ddECMs prepared by detergent-based methods. Additionally, a bioink is formulated with the ddECM (H/H), showing good shear thinning and shear recovery properties. Process optimization in terms of print speed, flow rate, and viscosity is done to obtain a bioprinting window for ddECM bioink. The printed constructs with optimized parameters have adequate mechanical and cell adhesive properties and excellent isotropic cellular alignment.

Electrohydrodynamic jet 3D printing in biomedical applications.

Electrohydrodynamic Jet 3D Printing (e-jetting) is a promising technique developed from electrospinning, which enables precise fiber deposition in a layer-by-layer fashion with customized designs. Several studies have verified that e-jetted scaffolds were able to support cell attachment, proliferation, and extracellular matrix formation, as well as cell infiltration into the scaffold due to the well-defined pores. Besides, e-jetting has also been combined with other techniques to incorporate biomaterials (e.g., hydrogels and cell spheroids) that could not be e-jetted, to promote the biological performance of the scaffold. In the recent decade, applying e-jetting in the fabrication of tissue-engineered scaffolds has drawn a lot of interest. Moreover, efforts have been put to develop varied scaffolds for some specific biomedical applications such as cartilage, tendon, and blood vessel, which exhibited superior mechanical properties and promoted cell behaviors including cellular alignment and differentiation. This review article also provides the reader with some crucial considerations and major limitations of e-jetting, such as scaffold design, printability of large-scale constructs, applicable biomaterials, and cell behaviors. Overall, this review article expounds on perspectives in the context of development and biomedical applications of this technique. STATEMENT OF SIGNIFICANCE: E-jetting technique is able to produce fibers with diameter in micrometer scale, which has been considered as a promising 3D printing technique. This technique has shown promise for regeneration of tissue engineered scaffolds with well-defined structures, which has been reported to apply in regeneration of different tissue types. The superior controllability of the process endows the feasibility of constructing multi-scale scaffolds with great biological mimicry and cellular infiltration. The incorporation of other biomaterials into the e-jetted networks further reinforces the scope of applications as compared to e-jetted scaffolds only. There is no doubt that e-jetting will be a great tool for tissue engineered scaffolding, and this review article will give overall perspectives in this topic.

Multi-directional cellular alignment in 3D guided by electrohydrodynamically-printed microlattices.

Recapitulating aligned cellular architectures of native tissues in vitro is important to engineer artificial tissue analogs with desired biological functions. Here a novel strategy is presented to direct three-dimensional (3D) cellular alignment by embedding cell/collagen hydrogel into the predefined electrohydrodynamically-printed microlattices. The cell/collagen hydrogel, originally filled within the printed microlattices uniformly, was found to gradually develop into densely-populated and highly-aligned bands along the longitudinal direction of the printed microlattices. The cellular alignment was highly dependent on the height, spacing and orientation of the microlattices. The presented method was applicable to multiple cell types including primary cardiomyocytes and the gaps formed between the aligned bands and the lateral walls of the microlattice facilitated the subsequent seeding and rapid alignment of other cell types which enables to engineer anisotropic multicellular tissue constructs. The engineered cardiac patches expressed mature cardiomyocyte-specific phenotypes and exhibited synchronous contractive activities. Multilayer cellular alignment with varied orientation in 3D collagen hydrogel was successfully achieved by using electrohydrodynamically-printed microlattices with layer-specific orientations. This exploration offers a promising way to engineer complex 3D tissue constructs with predefined cellular alignments. STATEMENT OF SIGNIFICANCE: Fabrication of biomimetic highly-aligned complex cellular architectures has a great significance to recapitulate the unique mechanical and physiological functions of the engineered tissues (e.g., heart tissue, neuron, muscle). Here, we introduced a novel strategy to direct 3D cellular alignment by embedding cell/collagen hydrogel into the predefined electrohydrodynamically-printed microlattices without any external stimuli. The microscopical study of the dynamic alignment process of cells and collagen fibers contributed to exploring the mechanism of autonomous formation of highly-aligned cellular bands. Multilayer cellular alignment with varied orientation in 3D collagen hydrogel was successfully achieved by using the microlattices with layer-specific orientations, which showed a promising way to engineer complex 3D tissue constructs with predefined cellular alignments.

Uniaxial Stretching of Cell-Laden Microfibers for Promoting C2C12 Myoblasts Alignment and Myofibers Formation.

Fiber-shaped cellular constructs have attracted increasing attention in the regeneration of blood vessels, nerve networks, and skeletal myofibers. Nevertheless, the generation of functional fiber-shaped cellular constructs suffers from limited appropriate microfiber-based fabrication approaches and the maintenance of regenerated tissue functions. Herein, we demonstrate a silicone-tube-based coagulant bath free method to fabricate tens of centimeters long cell-laden microfibers using single UV exposure without pretreatment of nozzles or microchannels. By modulating the exposure time, the gelatin methacrylate microfibers with tissue-like microstructures and mechanical properties are obtained. Then, a culture system integrated with a pillar well-array based stretching device is used to apply uniaxial stretching with various strain ratios in situ to cell-laden microfibers in a 60 mm petri dish. Cells with improved spreading, elongation, and alignment are obtained under uniaxial stretching. Moreover, the promotional effects of uniaxial stretching on the differentiation of C2C12 myoblasts, the formation, and contractility of myofibers become more pronounced with increasing strain ratio and achieve saturation level as strain ratio up to ∼35%.

Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Drug-Induced Inotropic Effects in Early Drug Development. Part 2: Designing and Fabricating Microsystems for Assaying Cardiac Contractility With Physiological Relevance Using Human iPSC-Cardiomyocytes.

Contractility of the myocardium engines the pumping function of the heart and is enabled by the collective contractile activity of its muscle cells: cardiomyocytes. The effects of drugs on the contractility of human cardiomyocytes in vitro can provide mechanistic insight that can support the prediction of clinical cardiac drug effects early in drug development. Cardiomyocytes differentiated from human-induced pluripotent stem cells have high potential for overcoming the current limitations of contractility assays because they attach easily to extracellular materials and last long in culture, while having human- and patient-specific properties. Under these conditions, contractility measurements can be non-destructive and minimally invasive, which allow assaying sub-chronic effects of drugs. For this purpose, the function of cardiomyocytes in vitro must reflect physiological settings, which is not observed in cultured cardiomyocytes derived from induced pluripotent stem cells because of the fetal-like properties of their contractile machinery. Primary cardiomyocytes or tissues of human origin fully represent physiological cellular properties, but are not easily available, do not last long in culture, and do not attach easily to force sensors or mechanical actuators. Microengineered cellular systems with a more mature contractile function have been developed in the last 5 years to overcome this limitation of stem cell-derived cardiomyocytes, while simultaneously measuring contractile endpoints with integrated force sensors/actuators and image-based techniques. Known effects of engineered microenvironments on the maturity of cardiomyocyte contractility have also been discovered in the development of these systems. Based on these discoveries, we review here design criteria of microengineered platforms of cardiomyocytes derived from pluripotent stem cells for measuring contractility with higher physiological relevance. These criteria involve the use of electromechanical, chemical and morphological cues, co-culture of different cell types, and three-dimensional cellular microenvironments. We further discuss the use and the current challenges for developing and improving these novel technologies for predicting clinical effects of drugs based on contractility measurements with cardiomyocytes differentiated from induced pluripotent stem cells. Future research should establish contexts of use in drug development for novel contractility assays with stem cell-derived cardiomyocytes.

Surface Topography of PDMS Replica Transferred from Various Decellularized Aortic Lumens Affects Cellular Orientation.

Cells sense and respond to various surface topographies of substrates. Many types of topographical architectures have been developed for understanding cell-extracellular matrix (ECM) interactions and for their application in biomaterials. In the present study, as a topographical surface similar to native tissue, we developed a PDMS replica prepared using the transferring method of the decellularized aorta, which is an ECM assembly, and its cellular behaviors, such as orientation and elongation on it. Decellularized aortas were prepared by high hydrostatic pressure (HHP) and sodium dodecyl sulfate (SDS) methods for use as templates. Scanning electron microscopic observation of the SDS replica showed a randomly rough surface. Further, microscaled linear structures along the direction of the aortic longitudinal axis were observed on the HHP replica. These results indicated that the topographical surface of the HHP and SDS decellularized aorta could be replicated to their replicas at a microscale. Fibroblasts (NIH3T3) and endothelial cells (HUVECs) were cultured on their surfaces. Although they were randomly aligned on the SDS replica and flat surface, the high cellular alignment along with the direction of the aortic longitudinal axis was shown in the HHP replica and HHP decellularized aorta. These results suggest that the topographical structure similar to a native aorta could effectively induce the cell alignment, which is important to regulate cellular functions, and could provide important methodologies and knowledge for vascular biomaterials or culture substrates.

Fibre-based scaffolding techniques for tendon tissue engineering.

Tendon refers to a band of tough, regularly arranged, and connective tissue connecting muscle and bone, transferring strength from muscle to bone, and enabling articular stability and movement. The limitations of natural tendon grafts motivate the scaffold-based tissue engineering (TE) approaches, which aim to build patient-specific biological substitutes that can repair the damaged or diseased tissues. Advances in engineering and knowledge of chemistry and biology have brought forth numerous fibre-based technologies, including electrospinning, electrohydrodynamic jet printing, electrochemical alignment technique, and other fibre-assembly technologies, which enable the fabrication of tendon tissue structure in 3-dimension. Textile techniques such as knitting and braiding have also been performed based on the fibrous materials to produce more complex structure. These scaffolds showed great similarity with native tendons in architectural features, mechanical properties, and facilitate biological functionality such as cellular adhesion, ingrowth, proliferation, and differentiation towards tendon tissue. Herein, we review the techniques that have been used to assemble fibres into scaffolds for tendon TE application. The morphological structures, mechanical properties, materials, degradation characteristics, and biological activities of the induced scaffolds were compared. The existing challenges and future prospects of fibre-based tendon TE have also been discussed.

Interwoven Aligned Conductive Nanofiber Yarn/Hydrogel Composite Scaffolds for Engineered 3D Cardiac Anisotropy.

Mimicking the anisotropic cardiac structure and guiding 3D cellular orientation play a critical role in designing scaffolds for cardiac tissue regeneration. Significant advances have been achieved to control cellular alignment and elongation, but it remains an ongoing challenge for engineering 3D cardiac anisotropy using these approaches. Here, we present a 3D hybrid scaffold based on aligned conductive nanofiber yarns network (NFYs-NET, composition: polycaprolactone, silk fibroin, and carbon nanotubes) within a hydrogel shell for mimicking the native cardiac tissue structure, and further demonstrate their great potential for engineering 3D cardiac anisotropy for cardiac tissue engineering. The NFYs-NET structures are shown to control cellular orientation and enhance cardiomyocytes (CMs) maturation. 3D hybrid scaffolds were then fabricated by encapsulating NFYs-NET layers within hydrogel shell, and these 3D scaffolds performed the ability to promote aligned and elongated CMs maturation on each layer and individually control cellular orientation on different layers in a 3D environment. Furthermore, endothelialized myocardium was constructed by using this hybrid strategy via the coculture of CMs on NFYs-NET layer and endothelial cells within hydrogel shell. Therefore, these 3D hybrid scaffolds, containing NFYs-NET layer inducing cellular orientation, maturation, and anisotropy and hydrogel shell providing a suitable 3D environment for endothelialization, has great potential in engineering 3D cardiac anisotropy.

Matrix Topographical Cue-Mediated Myogenic Differentiation of Human Embryonic Stem Cell Derivatives.

Biomaterials varying in physical properties, chemical composition and biofunctionalities can be used as powerful tools to regulate skeletal muscle-specific cellular behaviors, including myogenic differentiation of progenitor cells. Biomaterials with defined topographical cues (e.g., patterned substrates) can mediate cellular alignment of progenitor cells and improve myogenic differentiation. In this study, we employed soft lithography techniques to create substrates with microtopographical cues and used these substrates to study the effect of matrix topographical cues on myogenic differentiation of human embryonic stem cell (hESC)-derived mesodermal progenitor cells expressing platelet-derived growth factor receptor alpha (PDGFRA). Our results show that the majority (>80%) of PDGFRA+ cells on micropatterned polydimethylsiloxane (PDMS) substrates were aligned along the direction of the microgrooves and underwent robust myogenic differentiation compared to those on non-patterned surfaces. Matrix topography-mediated alignment of the mononucleated cells promoted their fusion resulting in mainly (~86%⁻93%) multinucleated myotube formation. Furthermore, when implanted, the cells on the micropatterned substrates showed enhanced in vivo survival (>5⁻7 times) and engraftment (>4⁻6 times) in cardiotoxin-injured tibialis anterior (TA) muscles of NOD/SCID mice compared to cells cultured on corresponding non-patterned substrates.

Collagen Substrate Stiffness Anisotropy Affects Cellular Elongation, Nuclear Shape, and Stem Cell Fate toward Anisotropic Tissue Lineage.

Rigidity of substrates plays an important role in stem cell fate. Studies are commonly carried out on isotropically stiff substrate or substrates with unidirectional stiffness gradients. However, many native tissues are anisotropically stiff and it is unknown whether controlled presentation of stiff and compliant material axes on the same substrate governs cytoskeletal and nuclear morphology, as well as stem cell differentiation. In this study, electrocompacted collagen sheets are stretched to varying degrees to tune the stiffness anisotropy (SA) in the range of 1 to 8, resulting in stiff and compliant material axes orthogonal to each other. The cytoskeletal aspect ratio increased with increasing SA by about fourfold. Such elongation was absent on cellulose acetate replicas of aligned collagen surfaces indicating that the elongation was not driven by surface topography. Mesenchymal stem cells (MSCs) seeded on varying anisotropy sheets displayed a dose-dependent upregulation of tendon-related markers such as Mohawk and Scleraxis. After 21 d of culture, highly anisotropic sheets induced greater levels of production of type-I, type-III collagen, and thrombospondin-4. Therefore, SA has direct effects on MSC differentiation. These findings may also have ramifications of stem cell fate on other anisotropically stiff tissues, such as skeletal/cardiac muscles, ligaments, and bone.

Conducting polymer-based multilayer films for instructive biomaterial coatings.

AIM: To demonstrate the design, fabrication and testing of conformable conducting biomaterials that encourage cell alignment. MATERIALS & METHODS: Thin conducting composite biomaterials based on multilayer films of poly(3.4-ethylenedioxythiophene) derivatives, chitosan and gelatin were prepared in a layer-by-layer fashion. Fibroblasts were observed with fluorescence microscopy and their alignment (relative to the dipping direction and direction of electrical current passed through the films) was determined using ImageJ. RESULTS: Fibroblasts adhered to and proliferated on the films. Fibroblasts aligned with the dipping direction used during film preparation and this was enhanced by a DC current. CONCLUSION: We report the preparation of conducting polymer-based films that enhance the alignment of fibroblasts on their surface which is an important feature of a variety of tissues.

Patterning Cellular Alignment through Stretching Hydrogels with Programmable Strain Gradients.

The graded mechanical properties (e.g., stiffness and stress/strain) of excellular matrix play an important role in guiding cellular alignment, as vital in tissue reconstruction with proper functions. Though various methods have been developed to engineer a graded mechanical environment to study its effect on cellular behaviors, most of them failed to distinguish stiffness effect from stress/strain effect during mechanical loading. Here, we construct a mechanical environment with programmable strain gradients by using a hydrogel of a linear elastic property. When seeding cells on such hydrogels, we demonstrate that the pattern of cellular alignment can be rather precisely tailored by substrate strains. The experiment is in consistency with a theoritical prediction when assuming that focal adhesions (FAs) would drive a cell to reorient to the directions where they are most stable. A fundamental theory has also been developed and is excellent in agreement with the complete temporal alignment of cells. This work not only provides important insights into the cellular response to the local mechanical microenvironment but can also be utilized to engineer patterned cellular alignment that can be critical in tissue remodeling and regenerative medicine applications.

Orthogonally oriented scaffolds with aligned fibers for engineering intestinal smooth muscle.

Controlling cellular alignment is critical in engineering intestines with desired structure and function. Although previous studies have examined the directional alignment of cells on the surface (x-y plane) of parallel fibers, quantitative analysis of the cellular alignment inside implanted scaffolds with oriented fibers has not been reported. This study examined the cellular alignment in the x-z and y-z planes of scaffolds made with two layers of orthogonally oriented fibers. The cellular orientation inside implanted scaffolds was evaluated with immunofluorescence. Quantitative analysis of coherency between cell orientation and fiber direction confirmed that cells aligned along the fibers not only on the surface (x-y plane) but also inside the scaffolds (x-z & y-z planes). Our study demonstrated that two layers of orthogonally aligned scaffolds can generate the histological organization of cells similar to that of intestinal circular and longitudinal smooth muscle.

Dual-Microstructured Porous, Anisotropic Film for Biomimicking of Endothelial Basement Membrane.

Human endothelial basement membrane (BM) plays a pivotal role in vascular development and homeostasis. Here, a bioresponsive film with dual-microstructured geometries was engineered to mimic the structural roles of the endothelial BM in developing vessels, for vascular tissue engineering (TE) application. Flexible poly(ε-caprolactone) (PCL) thin film was fabricated with microscale anisotropic ridges/grooves and through-holes using a combination of uniaxial thermal stretching and direct laser perforation, respectively. Through optimizing the interhole distance, human mesenchymal stem cells (MSCs) cultured on the PCL film's ridges/grooves obtained an intact cell alignment efficiency. With prolonged culturing for 8 days, these cells formed aligned cell multilayers as found in native tunica media. By coculturing human umbilical vein endothelial cells (HUVECs) on the opposite side of the film, HUVECs were observed to build up transmural interdigitation cell-cell contact with MSCs via the through-holes, leading to a rapid endothelialization on the PCL film surface. Furthermore, vascular tissue construction based on the PCL film showed enhanced bioactivity with an elevated total nitric oxide level as compared to single MSCs or HUVECs culturing and indirect MSCs/HUVECs coculturing systems. These results suggested that the dual-microstructured porous and anisotropic film could simulate the structural roles of endothelial BM for vascular reconstruction, with aligned stromal cell multilayers, rapid endothelialization, and direct cell-cell interaction between the engineered stromal and endothelial components. This study has implications of recapitulating endothelial BM architecture for the de novo design of vascular TE scaffolds.