Search

We report on a simple and efficient method for the selective positioning of Au/DNA hybrid nanocircuits using a sequential combination of electron-beam lithography (EBL), plasma ashing, and a molecular patterning process. The nanostructures produced by the EBL and ashing process could be uniformly formed over a 12.6 in2 substrate with sub-10 nm patterning with good pattern fidelity. In addition, DNA molecules were immobilized on the selectively nanopatterned regions by alternating surface coating procedures of 3-(aminopropyl)triethoxysilane (APS) and diamond like carbon (DLC), followed by deposition of DNA molecules into a well-defined single DNA nanowire. These single DNA nanowires were used not only for fabricating Au/DNA hybrid nanowires by the conjugation of Au nanoparticles with DNA, but also for the formation of Au/DNA hybrid nanocircuits. These nanocircuits prepared from Au/DNA hybrid nanowires demonstrate conductivities of up to 4.3 × 105 S/m in stable electrical performance. This selective and precise positioning method capable of controlling the size of nanostructures may find application in making sub-10 nm DNA wires and metal/DNA hybrid nanocircuits. A fabrication method for manufacturing nanoscale circuits with high spatial resolution could provide a boost for high-sensitivity molecular detection devices. Electron-beam lithography (EBL) is a powerful tool for patterning nanowire circuits, but its performance declines at spatial scales of less than 10 nanometers. Researchers led by Dr. Deok-Ho Kim at the Johns Hopkins University and Dr. Hyung Jin Kim at the Gumi Electronics and information Technology Research Institute have overcome this limitation by coupling EBL with a plasma ashing procedure. Using this approach, they were able to precisely pattern nanostructures in a silicon substrate. They subsequently performed a series of chemical modifications that enabled them to couple DNA strands to these patterns, which in turn served as a template for gold nanowires. This strategy could accelerate the production of nanocircuits for biomolecular detection in diagnostics and other applications.

Many cellular processes, including adhesion, alignment, migration, and differentiation, can be manipulated by nanotopography, which is generally fabricated on a support substrate and may restricts the potential applications of the scaffold. In this study, we developed free-standing nanopatterned poly(ϵ-caprolactone) (PCL) thin films for regulation of cellular alignment. We characterized the surface topography, wettability, and thermal properties of free-standing nanopatterned PCL thin films. Surface nanotopography on PCL thin film efficiently aligns cells with well-ordered orientation of actin and the nucleus. The developed free-standing thin films were nicely attached to human skin via water droplet. We further functionalized free-standing thin film by introducing magnetic nanoparticles to be manipulated by magnetic field. The techniques described herein would be useful for preparation of highly functionalized scaffolds because of the simple, versatile, and fully scalable features of the system.

The cell reprogramming of somatic cells into pluripotent states and subsequent differentiation into certain phenotypes has helped progress regenerative medicine research and other medical applications. Recent research has used viral vectors to induce this reprogramming; however, limitations include low efficiency and safety concerns. In this review, we discuss how biomaterial methods offer potential avenues for either increasing viability and downstream applicability of viral methods, or providing a safer alternative. The use of non-viral delivery systems, such as electroporation, micro/nanoparticles, nucleic acids and the modulation of culture substrate topography and stiffness, has generated valuable insights regarding cell reprogramming.

Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies.

Living cells and the extracellular matrix (ECM) can display complex interactions that define key developmental, physiological and pathological processes. Here, we report a new type of directed migration — which we term ‘topotaxis’ — by which cell movement is guided by the gradient of the nanoscale topographic features in the cells’ ECM environment. We show that the direction of topotaxis is reflective of the effective cell stiffness, and that it depends on the balance of the ECM-triggered signalling pathways PI3K-Akt and ROCK-MLCK. In melanoma cancer cells, this balance can be altered by different ECM inputs, pharmacological perturbations or genetic alterations, particularly a loss of PTEN in aggressive melanoma cells. We conclude that topotaxis is a product of the material properties of cells and the surrounding ECM, and propose that the invasive capacity of many cancers may depend broadly on topotactic responses, providing a potentially attractive mechanism for controlling invasive and metastatic behaviour.

In this study, we report nanopatterned Nafion microelectrode arrays for in vitro cardiac electrophysiology. With the aim of defining sophisticated Nafion nanostructures with highly ionic conductivity, fabrication parameters such as Nafion concentration and curing temperature were optimized. By increasing curing temperature and Nafion concentration, we were able to control the replication fidelity of Nafion nanopatterns when copied from a PDMS master mold. We also found that cross-sectional morphology and ion current density of nanopatterned Nafion strongly depends on the fabrication parameters. To investigate this dependency, current-voltage analysis was conducted using organic electrochemical transistors (OECT) overlaid with patterned Nafion substrates. Nanopatterned Nafion was found to allow higher ion current densities than unpatterned surfaces. Furthermore, higher curing temperatures were found to render Nafion layers with higher ion/electrical transfer properties. To optimize nanopattern dimensions, electrical current flows, and film uniformity, a final configuration consisting of 5% nanopatterned Nafion cured at 65°C was chosen. Multielectrode arrays (MEAs) were then covered with optimized Nafion nanopatterns and used for electrophysiological analysis of two types of induced pluripotent stem cell-derived cardiomyocytes (iPSCs-CMs). These data highlight the suitability of nanopatterned Nafion, combined with MEAs, for enhancing the cellular environment of iPSC-CMs for use in electrophysiological analysis in vitro.

Advancing as a biosensing nanotechnology, nanohybrids present a new class of functional materials with high selectivity and sensitivity, enabling integration of nanoscale chemical/biological interactions with biomedical devices. The unique properties of ZnO combined with metal oxide nanostructures were recently demonstrated to be an efficient approach for sensor device fabrication with accurate, real-time and high-throughput biosensing, creating new avenues for diagnosis, disease management and therapeutics. This review article collates recent advances in the modified ZnO nanostructured metal oxide nanohybrids for efficient enzymatic and non-enzymatic biosensor applications. Furthermore, we also discussed future prospects for nanohybrid materials to yield high-performance biosensor devices.

Despite possessing substantial regenerative capacity, skeletal muscle can suffer from loss of function due to catastrophic traumatic injury or degenerative disease. In such cases, engineered tissue grafts hold the potential to restore function and improve patient quality of life. Requirements for successful integration of engineered tissue grafts with the host musculature include cell alignment that mimics host tissue architecture and directional functionality, as well as vascularization to ensure tissue survival. Here we have developed biomimetic nanopatterned poly(lactic-co-glycolic acid) (PLGA) substrates conjugated with sphingosine-1-phosphate (S1P), a potent angiogenic and myogenic factor, to enhance myoblast and endothelial maturation. Primary muscle cells cultured on these functionalized S1P nanopatterned substrates developed a highly aligned and elongated morphology, and exhibited higher expression levels of myosin heavy chain, in addition to genes characteristic of mature skeletal muscle. We also found that S1P enhanced angiogenic potential in these cultures, as evidenced by elevated expression of endothelial-related genes. Computational analyses of live-cell videos showed a significantly improved functionality of tissues cultured on S1P-functionalized nanopatterns as indicated by greater myotube contraction displacements and velocities. In summary, our study demonstrates that biomimetic nanotopography and S1P can be combined to synergistically regulate the maturation and vascularization of engineered skeletal muscles.

A lack of electrical conductivity and structural organization in currently available biomaterial scaffolds limits their utility for generating physiologically representative models of functional cardiac tissue. Here we report on the development of scalable, graphene-functionalized topographies with anisotropic electrical conductivity for engineering the structural and functional phenotypes of macroscopic cardiac tissue constructs. Guided by anisotropic electroconductive and topographic cues, the tissue constructs displayed structural property enhancement in myofibrils and sarcomeres, and exhibited significant increases in the expression of cell–cell coupling and calcium handling proteins, as well as in action potential duration and peak calcium release.

Human tissues are sophisticated ensembles of many distinct cell types embedded in the complex, but well-defined, structures of the extracellular matrix (ECM). Dynamic biochemical, physicochemical, and mechano-structural changes in the ECM define and regulate tissue-specific cell behaviors. To recapitulate this complex environment in vitro, dynamic polymer-based biomaterials have emerged as powerful tools to probe and direct active changes in cell function. The rapid evolution of polymerization chemistries, structural modulation, and processing technologies, as well as the incorporation of stimuli-responsiveness, now permit synthetic microenvironments to capture much of the dynamic complexity of native tissue. These platforms are comprised not only of natural polymers chemically and molecularly similar to ECM, but those fully synthetic in origin. Here, we review recent in vitro efforts to mimic the dynamic microenvironment comprising native tissue ECM from the viewpoint of material design. We also discuss how these dynamic polymer-based biomaterials are being used in fundamental cell mechanobiology studies, as well as towards efforts in tissue engineering and regenerative medicine.

Vascular smooth muscle cells (vSMCs) retain the ability to undergo modulation in their phenotypic continuum, ranging from a mature contractile state to a proliferative, secretory state. vSMC differentiation is modulated by a complex array of microenvironmental cues, which include the biochemical milieu of the cells and the architecture and stiffness of the extracellular matrix. In this study, we demonstrate that by using UV-assisted capillary force lithography (CFL) to engineer a polyurethane substratum of defined nanotopography and stiffness, we can facilitate the differentiation of cultured vSMCs, reduce their inflammatory signature, and potentially promote the optimal functioning of the vSMC contractile and cytoskeletal machinery. Specifically, we found that the combination of medial tissue-like stiffness (11 MPa) and anisotropic nanotopography (ridge width_groove width_ridge height of 800_800_600 nm) resulted in significant upregulation of calponin, desmin, and smoothelin, in addition to the downregulation of intercellular adhesion molecule-1, tissue factor, interleukin-6, and monocyte chemoattractant protein-1. Further, our results allude to the mechanistic role of the RhoA/ROCK pathway and caveolin-1 in altered cellular mechanotransduction pathways via differential matrix nanotopography and stiffness. Notably, the nanopatterning of the stiffer substrata (1.1 GPa) resulted in the significant upregulation of RhoA, ROCK1, and ROCK2. This indicates that nanopatterning an 800_800_600 nm pattern on a stiff substratum may trigger the mechanical plasticity of vSMCs resulting in a hypercontractile vSMC phenotype, as observed in diabetes or hypertension. Given that matrix stiffness is an independent risk factor for cardiovascular disease and that CFL can create different matrix nanotopographic patterns with high pattern fidelity, we are poised to create a combinatorial library of arterial test beds, whether they are healthy, diseased, injured, or aged. Such high-throughput testing environments will pave the way for the evolution of the next generation of vascular scaffolds that can effectively crosstalk with the scaffold microenvironment and result in improved clinical outcomes.

Adult stem cells hold great promise as a source of diverse terminally differentiated cell types for tissue engineering applications. However, due to the complexity of chemical and mechanical cues specifying differentiation outcomes, development of arbitrarily complex geometric and structural arrangements of cells, adopting multiple fates from the same initial stem cell population, has been difficult. Here, we show that the topography of the cell adhesion substratum can be an instructive cue to adult stem cells and topographical variations can strongly bias the differentiation outcome of the cells towards adipocyte or osteocyte fates. Switches in cell fate decision from adipogenic to osteogenic lineages were accompanied by changes in cytoskeletal stiffness, spanning a considerable range in the cell softness/rigidity spectrum. Our findings suggest that human mesenchymal stem cells (hMSC) can respond to the varying density of nanotopographical cues by regulating their internal cytoskeletal network and use these mechanical changes to guide them toward making cell fate decisions. We used this finding to design a complex two-dimensional pattern of co-localized cells preferentially adopting two alternative fates, thus paving the road for designing and building more complex tissue constructs with diverse biomedical applications.

We report a simple and versatile method for in vitro fabrication of scaffold-free tissue-engineered constructs with predetermined cellular alignment, by combining magnetic cell levitation with thermoresponsive nanofabricated substratum (TNFS) based cell sheet engineering technique. The TNFS based nanotopography provides contact guidance cues for regulation of cellular alignment and enables cell sheet transfer, while magnetic nanoparticles facilitate the magnetic levitation of the cell sheet. The temperature-mediated change in surface wettability of the thermoresponsive poly(N-isopropylacrylamide), PNIPAM, substratum enables the spontaneous detachment of cell monolayers, which can then be easily manipulated through use of a ring or disk shaped magnet. Our developed platform could be readily applicable to production of tissue-engineered constructs containing complex physiological structures for the study of tissue structure-function relationships, drug screening, and regenerative medicine.

Part I. Micro/Nano Techniques in Cell Mechanobiology: 1. Nanotechnologies and FRET imaging in live cells Eddie Y. Chung, Qin Qin, Agamoni Bhattacharyya, Shaoying Lu and Yingxiao Wang 2. Electron microscopy and three-dimensional single-particle analysis as tools for understanding the structural basis of mechanobiology Niels Volkmann and Dorit Hanein 3. Stretchable micropost array cytometry: a powerful tool for cell mechanics and mechanobiology research Yue Shao, Shinuo Weng and Jianping Fu 4. Microscale generation of dynamic forces in cell culture systems Christopher Moraes, Luke A. MacQueen, Yu Sun and Craig A. Simmons 5. Multi-scale topographical approaches for cell mechanobiology studies Koichiro Uto, Elliot Fisher, Hong-Nam Kim, Chang Ho Seo and Deok-Ho Kim 6. Hydrogels with dynamically tunable properties Murat Guvendiren and Jason A. Burdick 7. Micro-engineered tools for studying cell migration in electric fields Jiandong Wu and Francis Lin 8. Laser ablation to investigate cell and tissue mechanics in vivo Teresa Zulueta-Coarasa and Rodrigo Fernandez-Gonzalez 9. Computational image analysis techniques for cell mechanobiology Ge Yang and Hao-Chih Lee 10. Micro- and nanotools to probe cancer cell mechanics and mechanobiology Yasaman Nematbakhsh and Chwee Teck Lim 11. Stimuli-responsive polymeric substrates for cell-matrix mechanobiology Mitsuhiro Ebara and Koichiro Uto Part II. Recent Progress in Cell Mechanobiology: 12. Forces of nature: understanding the role of mechanotransduction in stem cell differentiation Andrew W. Holle, Jennifer L. Young and Yu Suk Choi 13. Heart valve mechanobiology Craig Simmons 14. Bone cell mechanobiology using micro- and nano-techniques Chao Liu, Kevin Middleton and Lidan You 15. Molecular mechanisms of cellular mechanotransduction in wound healing Vincent F. Fiore, Dwight M. Chambers and Thomas H. Barker 16. Micropost arrays as a means to assess cardiac muscle cells Andrea Leonard, Marita L. Rodriguez and Nathan J. Sniadecki 17. Micro/nanofabrication for the study of biochemical and biomechanical regulation of T cell activation Hye Mi Kim and Junsang Doh 18. Study of tumor angiogenesis using microfluidic approaches Yoojin Shin, Sewoon Han, Hyo-Eun Jeong, Jeong Ah Kim, Jessie S. Jeon and Sid Chung 19. Neuromechanobiology of the brain: mechanics of neuronal structure, function, and pathophysiology Jerel Mueller and William Tyler.

Nanotechnology-based tools are beginning to emerge as promising platforms for quantitative high-throughput analysis of live cells and tissues. Despite unprecedented progress made over the last decade, a challenge still lies in integrating emerging nanotechnology-based tools into macroscopic biomedical apparatuses for practical purposes in biomedical sciences. In this review, we discuss the recent advances and limitations in the analysis and control of mechanical, biochemical, fluidic, and optical interactions in the interface areas of nanotechnology-based materials and living cells in both in vitro and in vivo settings.

Nanoscale topography of artificial substrates can greatly influence the fate of stem cells including adhesion, proliferation, and differentiation. Thus the design and manipulation of nanoscale stem cell culture platforms or scaffolds are of great importance as a strategy in stem cell and tissue engineering applications. In this report, we propose that a graphene oxide (GO) film is an efficient platform for modulating structure and function of human adipose-derived stem cells (hASCs). Using a self-assembly method, we successfully coated GO on glass for fabricating GO films. The hASCs grown on the GO films showed increased adhesion, indicated by a large number of focal adhesions, and higher correlation between the orientations of actin filaments and vinculin bands compared to hASCs grown on the glass (uncoated GO substrate). It was also found that the GO films showed the stronger affinity for hASCs than the glass. In addition, the GO film proved to be a suitable environment for the time-dependent viability of hASCs. The enhanced differentiation of hASCs included osteogenesis, adipogenesis, and epithelial genesis, while chondrogenic differentiation of hASCs was decreased, compared to tissue culture polystyrene as a control substrate. The data obtained here collectively demonstrates that the GO film is an efficient substratum for the adhesion, proliferation, and differentiation of hASCs.

Shape-memory systems represent an exciting class of “smart” materials that possess the unique capability to change from a temporary distorted structure back to a memorized permanent shape upon application of an external stimulus. Though metallic shape-memory alloys have gained traction as solid-state alternatives to conventional actuators in the automotive and robotics industry, shape-memory polymers (SMPs) are a cheap and efficient alternative with a diverse number of applications in the biomedical sector. Building on a variety of polymeric SMPs that offer one-way control over material geometry, exciting new research in reversible shape-memory systems has dramatically expanded the complexity over which researchers can dictate dynamic material properties. Such spatiotemporal control over material geometry will prove invaluable in the promising fields of drug delivery, tissue engineering, and regenerative medicine. In this chapter, we examine a variety of soft SMP and supramolecular biomaterial systems from the viewpoint of materials nanoarchitectonics.

The phenotypic expression and function of stem cells are regulated by their integrated response to variable microenvironmental cues, including growth factors and cytokines, matrix-mediated signals, and cell–cell interactions. Recently, growing evidence suggests that matrix-mediated signals include mechanical stimuli such as strain, shear stress, substrate rigidity and topography, and these stimuli have a more profound impact on stem cell phenotypes than had previously been recognized, e.g. self-renewal and differentiation through the control of gene transcription and signaling pathways. Using a variety of cell culture models enabled by micro and nanoscale technologies, we are beginning to systematically and quantitatively investigate the integrated response of cells to combinations of relevant mechanobiological stimuli. This paper reviews recent advances in engineering physical stimuli for stem cell mechanobiology and discusses how micro- and nanoscale engineered platforms can be used to control stem cell niche environments and regulate stem cell fate and function.

Live cells are exquisitely sensitive to both the sub- stratum rigidity and texture. To explore cell responses to both these types of inputs in a precisely controlled fashion, we analyzed the responses of Chinese hamster ovary (CHO) cells to nanotopographically defined substrata of different rigidities, ranging from 1.8 MPa to 1.1 GPa. Parallel arrays of nanogrooves (800-nm width, 800-nm space, and 800-nm depth) on polyurethane (PU)-based material surfaces were fabricated by UV-assisted capillary force lithography (CFL) over an area of 5 mm × 3 mm. We observed dramatic morphological responses of CHO cells, evident in their elongation and polarization along the nanogrooves direction. The cells were progressively more spread and elongated as the sub- stratum rigidity increased, in an integrin β1 dependent manner. However, the degree of orientation was independent of substratum rigidity, suggesting that the cell shape is primarily determined by the topographical cues.

Polymers provide a versatile platform for mimicking various aspects of physiological extracellular matrix properties such as chemical composition, rigidity, and topography for use in cell and tissue engineering applications. In this review, we provide a brief overview of patterning methods of various polymers with a particular focus on biocompatibility and processability. The materials highlighted here are widely used polymers including thermally curable polydimethyl siloxane, ultraviolet-curable polyurethane acrylate and polyethylene glycol, thermo-sensitive poly(N-isopropylacrylamide) and thermoplastic and conductive polymers. We also discuss how micro- and nanofabricated polymeric substrates of tunable elastic modulus can be used to engineer cell and tissue structure and function. Such synergistic effect of topography and rigidity of polymers may be able to contribute to constructing more physiologically relevant microenvironment.

It is becoming increasingly evident that cell biology research can be considerably advanced through the use of bioengineered tools enabled by nanoscale technologies. Recent advances in nanopatterning techniques pave the way for engineering biomaterial surfaces that control cellular interactions from the nano- to the microscale, allowing more precise quantitative experimentation capturing multi-scale aspects of complex tissue physiology in vitro. The spatially and temporally controlled display of extracellular signaling cues on nanopatterned surfaces (e. g., cues in the form of chemical ligands, controlled stiffness, texture, etc.) that can now be achieved on biologically relevant length scales is particularly attractive enabling experimental platform for investigating fundamental mechanisms of adhesion-mediated cell signaling. Here, we present an overview of bio-nanopatterning methods, with the particular focus on the recent advances on the use of nanofabrication techniques as enabling tools for studying the effects of cell adhesion and signaling on cell function. We also highlight the impact of nanoscale engineering in controlling cell-material interfaces, which can have profound implications for future development of tissue engineering and regenerative medicine.

Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell–cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell–material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

This chapter introduces innovative organ-on-chip platforms for chemical assay and toxicity testing that measures the physiological properties of live, engineered muscular tissue samples. The advantages of using such engineered tissues for drug screening compared to more conventional cell-based assays are discussed. Specifically, this chapter will outline recent developments and applications of cardiac and skeletal muscle organ-on-chip systems. Recent advances in micro- and nanofabrication techniques, along with their biological applications with regard to organ-on-chips, are also reviewed in this chapter.

Electrically conductive materials provide a suitable platform for the in vitro study of excitable cells, such as skeletal muscle cells, due to their inherent conductivity and electro-activity. Here we demonstrate that bioinspired electroconductive nanopatterned substrates enhanced myogenic differentiation and maturation. The topographical cues from the highly-aligned collagen bundles that form the extracellular matrix (ECM) of skeletal muscle tissue were mimicked using nanopatterns created with capillary force lithography. Electron beam deposition was then utilized to conformally coat nanopatterned substrates with a thin layer of either gold or titanium to create electroconductive substrates with well-defined, large-area nanotopographical features. C2C12 cells, a myoblast cell line, were cultured for 7 days on substrates, and the effects of topography and electrical conductivity on cellular morphology and myogenic differentiation were assessed. We found that biomimetic nanotopography enhanced the formation of aligned myotubes, and the addition of an electroconductive coating promoted myogenic differentiation and maturation, as indicated by the upregulation of myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). These results suggest the suitability of electroconductive nanopatterned substrates as a biomimetic platform for the in vitro engineering of skeletal muscle tissue.

Endothelial physiology is regulated not only by humoral factors, but also by mechanical factors such as fluid shear stress and the underlying cellular matrix microenvironment. The purpose of the present study was to examine the effects of matrix topographical cues on the endothelial secretion of cytokines/chemokines in vitro. Human endothelial cells were cultured on nanopatterned polymeric substrates with different ratios of ridge to groove widths (1:1, 1:2, and 1:5) and with different stiffnesses (6.7 MPa and 2.5 GPa) in the presence and absence of 1.0 ng/mL TNF-α. The levels of cytokines/chemokines secreted into the conditioned media were analyzed with a multiplexed bead-based sandwich immunoassay. Of the nanopatterns tested, the 1:1 and 1:2 type patterns were found to induce the greatest degree of endothelial cell elongation and directional alignment. The 1:2 type nanopatterns lowered the secretion of inflammatory cytokines such as IL-1β, IL-3, and MCP-1, compared to unpatterned substrates. Additionally, of the two polymers tested, it was found that the stiffer substrate resulted in significant decreases in the secretion of IL-3 and MCP-1. These results suggest that substrates with specific extracellular nanotopographical cues or stiffnesses may provide anti-atherogenic effects like those seen with laminar shear stresses by suppressing the endothelial secretion of cytokines and chemokines involved in vascular inflammation and remodeling.

In this paper, we report a microfabrication approach to patterned three-dimensional growth of muscle cells for powering biohybrid microdevices. The microwells confined by adhesion-resistant polyethylene glycol (PEG) microstructures were patterned on SiO2-based substrate with controllable surface topography for muscle cell culturing. The morphology and motility of neonatal rat cardiac myocytes patterned within microwells were analyzed with different topographical heights of the PEG barrier. It was found that isolated aggregates of cardiac muscles showed various beating activities depending on the morphology and the number of cells. Furthermore, three-dimensionally grown cardiac muscle cells mediated by a higher physical barrier of PEG microstructures generated higher contraction force with faster beating frequency than those of cells attached to the collagen-coated surface on the culture dish (control), suggesting that control over surface topography of microwells for cell growth would be potentially useful in engineering cell motors. Thus the technique presented here would provide a valuable platform for optimizing the activity and functions of patterned muscle cells in building up integrated bioactuated microdevices.

Most cells in the body secrete, or are in intimate contact with extracellular matrix (ECM), which provides structure to tissues and regulates various cellular phenotypes. Cells are well known to respond to biochemical signals from the ECM, but recent evidence has highlighted the mechanical properties of the matrix, including matrix elasticity and nanotopography, as fundamental instructive cues regulating signal transduction pathways and gene transcription. Recent observations also highlight the importance of matrix nanotopography as a regulator of cellular functions, but lack of facile experimental platforms has resulted in a continued negligence of this important microenvironmental cue in tissue culture experimentation. In this review, we present our opinion on the importance of nanotopography as a biological cue, contexts in which it plays a primary role influencing cell behavior, and detail advanced techniques to incorporate nanotopography into the design of experiments, or in cell culture environments. In addition, we highlight signal transduction pathways that are involved in conveying the extracellular matrix nanotopography information within the cells to influence cell behavior.

A simple method was developed to fabricate polyethylene glycol (PEG) nanostructures using capillary lithography mediated by ultraviolet (UV) exposure. Acrylate-containing PEG monomers, such as PEG dimethacrylate (PEG-DMA, MW = 330), were photo-cross-linked under UV exposure to generate patterned structures. In comparison to unpatterned PEG films, hydrophobicity of PEG nanostructure modified surfaces was significantly enhanced. This could be attributed to trapped air in the nanostructures as supported by water contact angle measurements. Proteins (fibronectin, immunoglobulin, and albumin) and cells (fibroblasts and P19 EC cells) were examined on the modified surfaces to test for the level of protein adsorption and cell adhesion. It was found that proteins and cells preferred to adhere on nanostructured PEG surfaces in comparison to unpatterned PEG films; however, this level of adhesion was significantly lower than that of glass controls. These results suggest that capillary lithography can be used to fabricate PEG nanostructures capable of modifying protein and cell adhesive properties of surfaces.

The nanoscale sensing and manipulation have become a challenging issue in micro/nanorobotic applications. In particular, a feedback sensor-based manipulation is necessary for realizing an efficient and reliable handling of particles under uncertain environment in a micro/ nano scale. This paper presents a piezoresistive MEMS cantilever for nanoscale force measurement in microrobotics. A piezoresistive MEMS cantilever enables sensing of gripping and contact forces in nanonewton resolution by measuring changes in the stress-induced electrical resistances. The calibration of a piezoresistive MEMS cantilever is experimentally carried out. In addition, as part of the work on nanomanipulation with a piezoresistive MEMS cantilever, the analysis on the interaction forces between a tip and a material, and the associated manipulation strategies are investigated. Experiments and simulations show that a piezoresistive MEMS cantilever integrated into a microrobotic system can be effectively used in nanoscale force measurements and a sensor-based manipulation.

The papers in this special section were presented at the 8th International Conference on Nano/Molecular Medicine and Engineering (IEEE-NANOMED 2014).

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models.

The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method., The application of 3D printing to biomaterials presents interesting possibilities for tissue engineering. Here, the authors show that a printing medium derived from an extracellular matrix can be applied to printing tissue analogues with enhanced cell compatibility.

The ability of the living body to heal and regenerate itself following trauma is astonishing. Numerous events of repair and regeneration occur during our lifetime, most of which we are never aware of. Unfortunately, in some cases, the injury or defect cannot be adequately repaired solely by nature and medical intervention is required.

Eung-Sam Kim,1,2 Eun Hyun Ahn,3,4 Tal Dvir,5,6 Deok-Ho Kim1,4,71Department of Bioengineering, University of Washington, Seattle, WA, USA; 2Department of Biological Sciences, Chonnam National University, Gwangju, Korea; 3Department of Pathology, 4Institute of Stem Cell and Regenerative Medicine, School of Medicine, University of Washington, Seattle, WA, USA; 5Department of Molecular Microbiology and Biotechnology, 6Center for Nanoscience and Nanotechnology, Tel Aviv University, Tel Aviv, Israel; 7Center for Cardiovascular Biology, University of Washington, Seattle, WA, USAThe history of human kind suggests that there has been a correlation between global population growth and major events in science and technology over the last three centuries. Sharp increases in the world’s population have been triggered by the industrial revolution and scientific and technological breakthroughs including: the advent of the railways, discovery of penicillin and deoxyribonucleic acid (DNA), and the invention of the computer.1 Since the 20th century, interdisciplinary areas in the physical and biological sciences have accelerated the progress of biomedical applications. The recent integration of emerging nanotechnology into biology and biomedicine has resulted in a range of innovative nanoengineering efforts for the repair and regeneration of tissues and organs.2 Thus, it is expected that nanoengineering approaches to biomedical applications can contribute to addressing the present issue of personal and global health care and its economic burden for more than 7 billion people.Why are we paying attention to nanoengineering for biomedical applications? The size of most biomolecules ranges from 0.2 nm to 200 nm (Figure 1). Research has focused on control of the interaction and localization of biomolecules even at the single-molecule level using ever-evolving nanotechnology.3 The evidence indicates that cells can respond to nanoscale changes in the dynamic extracellular matrix and vice versa. Biomimetic nanopatterns alone can direct the differentiation of stem cells without involvement of exogenous soluble biochemical factors.4,5 This regulation of cellular behavior by nanotechnology is one of many examples demonstrating the significant applications of nanoengineering in biomedicine. This special issue includes four review papers and seven research articles that provide an insight into current nanoengineering approaches to the repair or regeneration of tissues and organs.

Current tissue engineering methods lack the ability to properly recreate scaffold-free, cell-dense tissues with physiological structures. Recent studies have shown that the use of nanoscale cues allows for precise control over large-area 2D tissue structures without restricting cell growth or cell density. In this study, we developed a simple and versatile platform combining a thermoresponsive nanofabricated substratum (TNFS) incorporating nanotopographical cues and the gel casting method for the fabrication of scaffold-free 3D tissues. Our TNFS allows for the structural control of aligned cell monolayers which can be spontaneously detached via a change in culture temperature. Utilizing our gel casting method, viable, aligned cell sheets can be transferred without loss of anisotropy or stacked with control over individual layer orientations. Transferred cell sheets and individual cell layers within multilayered tissues robustly retain structural anisotropy, allowing for the fabrication of scaffold-free, 3D tissues with hierarchical control of overall tissue structure.

Electrospun nanofibers have been developed into a variety of forms for tissue engineering scaffolds to regulate the cellular functions guided by nanotopographical cues. Here, we have successfully fabricated nanofiber-based scaffold complexes of rod and sheet type by combining the three microfabrication techniques of electrospinning, spin coating, and polymer melt deposition. It was demonstrated that this hybrid fabrication could produce uniaxially aligned nanofiber scaffolds supported by a thin film, allowing for a mechanically enforced substrate for cell culture as well as facile scaffold manipulation. The results of cell analysis indicated that nanofibers on spin-coated films could provide contact guidance effects on cells and retain them even after manipulation. As an application of the cell-laden nanofiber film, we built a rod-type structure by rolling up the film around a mechanically supporting core microfiber, which was incorporated by polymer melt deposition. A biocompatible and biodegradable polymer, polycaprolactone, was used throughout the processes and thus could be used as a directly implantable substitute in tissue regeneration.

Skeletal muscle is a highly organized tissue in which the extracellular matrix (ECM) is composed of highly-aligned cables of collagen with nanoscale feature sizes, and provides structural and functional support to muscle fibers. As such, the transplantation of disorganized tissues or the direct injection of cells into muscles for regenerative therapy often results in suboptimal functional improvement due to a failure to integrate with native tissue properly. Here, we present a simple method in which biodegradable, biomimetic substrates with precisely controlled nanotopography were fabricated using solvent-assisted capillary force lithography (CFL) and were able to induce the proper development and differentiation of primary mononucleated cells to form mature muscle patches. Cells cultured on these nanopatterned substrates were highly-aligned and elongated, and formed more mature myotubes as evidenced by up-regulated expression of the myogenic regulatory factors Myf5, MyoD and myogenin (MyoG). When transplanted into mdx mice models for Duchenne muscular dystrophy (DMD), the proposed muscle patches led to the formation of a significantly greater number of dystrophin-positive muscle fibers, indicating that dystrophin replacement and myogenesis is achievable in vivo with this approach. These results demonstrate the feasibility of utilizing biomimetic substrates not only as platforms for studying the influences of the ECM on skeletal muscle function and maturation, but also to create transplantable muscle cell patches for the treatment of chronic and acute muscle diseases or injuries.

Microfluidic platforms provide several unique advantages for drug development. In the production of drug carriers, physical properties such as size and shape, and chemical properties such as drug composition and pharmacokinetic parameters, can be modified simply and effectively by tuning the flow rate and geometries. Large numbers of carriers can then be fabricated with minimal effort and with little to no batch-to-batch variation. Additionally, cell or tissue culture models in microfluidic systems can be used as in vitro drug screening tools. Compared to in vivo animal models, microfluidic drug screening platforms allow for high-throughput and reproducible screening at a significantly lower cost, and when combined with current advances in tissue engineering, are also capable of mimicking native tissues. In this review, various microfluidic platforms for drug and gene carrier fabrication are reviewed to provide guidelines for designing appropriate carriers. In vitro microfluidic drug screening platforms designed for high-throughput analysis and replication of in vivo conditions are also reviewed to highlight future directions for drug research and development.

Biological examples both motivate and stimulate new scientific research. Biomimetics is the study and simulation of biological systems for desired functional properties. It involves the transformation of the underlying principles discovered in nature into man-made technology. In recent times, bio-inspiration has given rise to a new promising direction for solving the tribological issues in micro/nano-devices such as Micro/Nano-Electro-Mechanical Systems (MEMS/NEMS). This chapter highlights on the various bio-inspired approaches undertaken based on the ‘Lotus Effect’ to create biomimetic surfaces as novel tribological solutions for micro/nano-devices. It also presents the recent advancements in this field. Examples presented in the chapter clearly indicate that by learning from natural surfaces, the development of biomimetic surfaces would remarkably enhance the smooth operation, long-term wear durability and reliability of micro/nano-devices.

Human tissues are intricate ensembles of multiple cell types embedded in complex and well-defined structures of the extracellular matrix (ECM). The organization of ECM is frequently hierarchical from nano to macro, with many proteins forming large scale structures with feature sizes up to several hundred microns. Inspired from these natural designs of ECM, nanotopography-guided approaches have been increasingly investigated for the last several decades. Results demonstrate that the nanotopography itself can activate tissue-specific function in vitro as well as promote tissue regeneration in vivo upon transplantation. In this review, we provide an extensive analysis of recent efforts to mimic functional nanostructures in vitro for improved tissue engineering and regeneration of injured and damaged tissues. We first characterize the role of various nanostructures in human tissues with respect to each tissue-specific function. Then, we describe various fabrication methods in terms of patterning principles and material characteristics. Finally, we summarize the applications of nanotopography to various tissues, which are classified into four types depending on their functions: protective, mechano-sensitive, electro-active, and shear stress-sensitive tissues. Some limitations and future challenges are briefly discussed at the end.

Tunneling nanotubes (TNTs) have previosly been observed as long and thin transient structures forming between cells and intercellular protein transfer through them has been experimentally verified. It is hypothesized that this may be a physiologically important means of cell–cell communication. This paper attempts to give a simple model for the rates of transfer of molecules across these TNTs at different distances. We describe the transfer of both cytosolic and membrane bound molecules between neighboring populations of cells and argue how the lifetime of the TNT, the diffusion rate, distance between cells, and the size of the molecules may affect their transfer. The model described makes certain predictions and opens a number of questions to be explored experimentally.

Current techniques used in stem cell research only crudely mimic the physiological complexity of the stem cell niches. Recent advances in the field of micro- and nanoengineering have brought an array of in vitro cell culture models that have enabled development of novel, highly precise and standardized tools that capture physiological details in a single platform, with greater control, consistency, and throughput. In this review, we describe the micro- and nanotechnology-driven modern toolkit for stem cell biologists to design novel experiments in more physiological microenvironments with increased precision and standardization, and caution them against potential challenges that the modern technologies might present.

The introduction of microtechnology and microfluidic platforms for cell culture can dramatically enhance the pace of stem cell research. With the use of microfluidic-based techniques, extracellular microenvironments can be controlled in a precise manner, and their influence on various cellular behaviors can be studied. Microfluidic devices made of transparent materials allow real-time and high-throughput monitoring of cell functions and cell fate by using fluorescence microscopy and other optical techniques. This chapter gives a perspective on the considerable capability of microfluidic devices, which remain an underutilized technology for stem cell research. It provides stem cell researchers with a brief review of basic microtechnology and the application of microfluidics to stem cell research, as well as highlights to engineers the peculiarities of stem cell culture and experimental capabilities of microfluidics. In addition, it provides insights into creating integrated, modular, and easy-to-use microfluidic devices to perturb stem cells with biochemico/mechanical stimuli in a precise, controlled, combinatorial, and high-throughput fashion.

We have examined the effects of surface nanotopography on in vitro osteogenesis of human mesenchymal stem cells (hMSCs). UV-assisted capillary force lithography was employed to fabricate a scalable (4 cm × 5 cm), well-defined nanostructured substrate of a UV curable polyurethane polymer with dots (150-, 400-, 600-nm diameter) and lines (150-, 400-, 600-nm width). The influence of osteogenic differentiation of hMSCs was characterized at day 8 by alkaline phosphatase (ALP) assay, RT-PCR, and real time PCR analysis. We found that hMSCs cultured on the nanostructured surfaces in osteogenic induction media showed significantly higher ALP activity compared to unpatterned PUA surface (control group). In particular, the hMSCs on the 400-nm dot pattern showed the highest level of ALP activity. Further investigation with real-time quantitative RT-PCR analysis demonstrated significantly higher expression of core binding factor 1 (Cbfa1), osteopontin (OP), and osteocalcin (OC) levels in hMSCs cultured on the 400-nm dot pattern in osteogenic induction media. These findings suggest that surface nanotopography can enhance osteogenic differentiation synergistically with biochemical induction substance.