Alex Jiao

Nanotopography-guided tissue engineering and regenerative medicine ☆

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.

Patterning Methods for Polymers in Cell and Tissue Engineering

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.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells

Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700-1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies.

Thermoresponsive nanofabricated substratum for the engineering of three-dimensional tissues with layer-by-layer architectural control.

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.

Fabrication of poly(ethylene glycol): gelatin methacrylate composite nanostructures with tunable stiffness and degradation for vascular tissue engineering

Although synthetic polymers are desirable in tissue engineering applications for the reproducibility and tunability of their properties, synthetic small diameter vascular grafts lack the capability to endothelialize in vivo. Thus, synthetically fabricated biodegradable tissue scaffolds that reproduce important aspects of the extracellular environment are required to meet the urgent need for improved vascular grafting materials. In this study, we have successfully fabricated well-defined nanopatterned cell culture substrates made of a biodegradable composite hydrogel consisting of poly(ethylene glycol) dimethacrylate (PEGDMA) and gelatin methacrylate (GelMA) by using UV-assisted capillary force lithography. The elasticity and degradation rate of the composite PEG-GelMA nanostructures were tuned by varying the ratios of PEGDMA and GelMA. Human umbilical vein endothelial cells (HUVECs) cultured on nanopatterned PEG-GelMA substrates exhibited enhanced cell attachment compared with those cultured on unpatterned PEG-GelMA substrates. Additionally, HUVECs cultured on nanopatterned PEG-GelM substrates displayed well-aligned, elongated morphology similar to that of native vascular endothelial cells and demonstrated rapid and directionally persistent migration. The ability to alter both substrate stiffness and degradation rate and culture endothelial cells with increased elongation and alignment is a promising next step in recapitulating the properties of native human vascular tissue for tissue engineering applications.

Initiated chemical vapor deposition of thermoresponsive poly (N-vinylcaprolactam) thin films for cell sheet engineering

Poly(N-vinylcaprolactam) (PNVCL) is a thermoresponsive polymer known to be nontoxic, water soluble and biocompatible. Here, PNVCL homopolymer was successfully synthesized for the first time by use of a one-step vapor-phase process, termed initiated chemical vapor deposition (iCVD). Fourier transform infrared spectroscopy results showed that radical polymerization took place from N-vinylcaprolactam monomers without damaging the functional caprolactam ring. A sharp lower critical solution temperature transition was observed at 31°C from the iCVD poly(N-vinylcaprolactam) (PNVCL) film. The thermoresponsive PNVCL surface exhibited a hydrophilic/hydrophobic alteration with external temperature change, which enabled the thermally modulated attachment and detachment of cells. The conformal coverage of PNVCL film on various substrates with complex topography, including fabrics and nanopatterns, was successfully demonstrated, which can further be utilized to fabricate cell sheets with aligned cell morphology. The advantage of this system is that cells cultured on such thermoresponsive surfaces could be recovered as an intact cell sheet by simply lowering the temperature, eliminating the need for conventional enzymatic treatments.

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide(1). Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The hearts extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale(2). Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering(3-5). A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling(2). Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart(6-9). Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)(8) and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function(10-14). Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively(15,16). Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (λ = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 °C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS(2).

Regulation of skeletal myotube formation and alignment by nanotopographically controlled cell-secreted extracellular matrix: REGULATION OF MYOTUBE FORMATION BY MATRIX NANOTOPOGRAPHY

Skeletal muscle has a well-organized tissue structure comprised of aligned myofibers and an encasing extracellular matrix (ECM) sheath or lamina, within which reside satellite cells. We hypothesize that the organization of skeletal muscle tissues in culture can affect both the structure of the deposited ECM and the differentiation potential of developing myotubes. Furthermore, we posit that cellular and ECM cues can be a strong determinant of myoblast fusion and morphology in 3D tissue culture environments. To test these, we utilized a thermoresponsive nanofabricated substratum to engineer anisotropic sheets of myoblasts which could then be transferred and stacked into multilayered tissues. Within such engineered tissues, we found that myoblasts rapidly sense topography and deposit structurally organized ECM proteins. Furthermore, the initial tissue structure was found to exert significant control over myoblast fusion and eventual myotube organization. These results highlight the importance of ECM structure on myoblast fusion and organization, and provide insights into substrate-mediated control of myotube formation in the development of novel, more effective, engineered skeletal muscle tissues.

Biomimetic approaches for engineered organ chips and skin electronics for in vitro diagnostics

Two kinds of biomimetic systems including engineered organ chip and flexible electronic sensor are presented. First, in vivo, renal tubular epithelial cells are exposed to luminal fluid shear stress (FSS) and a transepithelial osmotic gradient. In this study, we used a simple collecting-duct-on-a-chip to investigate the role of an altered luminal microenvironment in the translocation of aquaporin-2 (AQP2) and the reorganization of actin cytoskeleton (F-actin) in primary cultured inner medullary collecting duct (IMCD) cells of rat kidney. We demonstrate that several factors (i.e., luminal FSS, hormonal stimulation, transepithelial osmotic gradient) collectively exert a profound effect on the AQP2 trafficking in the collecting ducts, which is associated with actin cytoskeletal reorganization. Furthermore, with this kidney-mimicking chip, renal toxicity of cisplatin was tested under static and fluidic conditions, suggesting the physiological relevancy of fluidic environment compared to static culture. Second, we present a simple architecture for a flexible and highly sensitive strain sensor that enables the detection of pressure, shear and torsion. The device is based on two interlocked arrays of high-aspect-ratio Pt-coated polymeric nanofibres that are supported on thin polydimethylsiloxane layers. When different sensing stimuli are applied, the degree of interconnection and the electrical resistance of the sensor changes in a reversible, directional manner with specific, discernible strain-gauge factors. We show that the sensor can be used to monitor signals ranging from human heartbeats to the impact of a bouncing water droplet on a superhydrophobic surface.