Surface Micro- and Nanoengineering: Applications of Layer-by-Layer Technology as a Versatile Tool to Control Cellular Behavior.

Extracellular matrix (ECM) cues have been widely investigated for their impact on cellular behavior. Among mechanics, physics, chemistry, and topography, different ECM properties have been discovered as important parameters to modulate cell functions, activating mechanotransduction pathways that can influence gene expression, proliferation or even differentiation. Particularly, ECM topography has been gaining more and more interest based on the evidence that these physical cues can tailor cell behavior. Here, an overview of bottom-up and top-down approaches reported to produce materials capable of mimicking the ECM topography and being applied for biomedical purposes is provided. Moreover, the increasing motivation of using the layer-by-layer (LbL) technique to reproduce these topographical cues is highlighted. LbL assembly is a versatile methodology used to coat materials with a nanoscale fidelity to the geometry of the template or to produce multilayer thin films composed of polymers, proteins, colloids, or even cells. Different geometries, sizes, or shapes on surface topography can imply different behaviors: effects on the cell adhesion, proliferation, morphology, alignment, migration, gene expression, and even differentiation are considered. Finally, the importance of LbL assembly to produce defined topographical cues on materials is discussed, highlighting the potential of micro- and nanoengineered materials to modulate cell function and fate.

Bioinspired multilayer membranes as potential adhesive patches for skin wound healing.

Bioinspired and adhesive multilayer membranes are produced using the layer-by-layer (LbL) assembly of chitosan (CHT), alginate (ALG) and hyaluronic acid modified with dopamine (HA-DN). Freestanding multilayer membranes without DN are also produced as a control. The success of the synthesis of HA-DN was confirmed using UV-visible spectroscopy. Scanning electron microscopy images indicate that the surface of the DN-containing membranes is more porous than the control ones; they also present a higher average thickness value for the same number of CHT/ALG/CHT/HA(-DN) tetralayers (n = 100). Also, water uptake, mechanical strength and adhesion are enhanced with the introduction of DN moieties along the nano-layers. Besides, human dermal fibroblast viability, enhanced adhesion and proliferation were confirmed by immunofluorescence assays and by measuring both the metabolic activity and DNA content. Moreover, in vivo assays with such kinds of DN-containing multilayer membranes were performed; the application of these membranes in the treatment of dermal wounds induced in Wistar rats results in the highest decrease of inflammation of rat skin, compared with the control conditions. Overall, this investigation suggests that these mussel-inspired freestanding multilayer membranes may enhance either their mechanical performance or cellular adhesion and proliferation, leading to an improved wound healing process, being a promising material to restore the structural and functional properties of wounded skin.

Adhesive free-standing multilayer films containing sulfated levan for biomedical applications.

This work is the first reporting the use of layer-by-layer to produce adhesive free-standing (FS) films fully produced using natural-based macromolecules: chitosan (CHI), alginate (ALG) and sulfated levan (L-S). The deposition conditions of the natural polymers were studied through zeta potential measurements and quartz crystal microbalance with dissipation monitoring analysis. The properties of the FS films were evaluated and compared with the control ones composed of only CHI and ALG in order to assess the influence of levan polysaccharide introduced in the multilayers. Tensile tests, dynamic mechanical analysis and single lap shear strength tests were performed to evaluate the mechanical properties of the prepared FS films. The presence of L-S conferred both higher tensile strength and shear strength to the developed FS membranes. The results showed an adhesion strength 4 times higher than the control (CHI/ALG) FS films demonstrating the adhesive character of the FS films containing L-S. Morphological and topography studies were carried out revealing that the crosslinking reaction granted the L-S based FS film with a higher roughness and surface homogeneity. Preliminary biological assays were performed by cultivating myoblasts cells on the surface of the produced FS films. Both crosslinked and uncrosslinked FS films containing L-S were cytocompatible and myoconductive. STATEMENT OF SIGNIFICANCE: Sutures remain as the "gold standard" for wound closure and bleeding control; however they still have limitations such as, high infection rate, inconvenience in handling, and concern over possible transmission of blood-borne disease through the use of needles. One of the challenges of tissue engineering consist on the design and development of biocompatible tissue adhesives and sealants with high adhesion properties to repair or attach devices to tissues. In this work, the introduction of sulfated levan (L-S) on multilayered free-standing membranes was proposed to confer adhesive properties. Moreover, the films were myoconductive even in the absence of crosslinking just by the presence of L-S. This study provides a promising strategy to develop biological adhesives and for cardiac tissue engineering applications.

Multilayered membranes with tuned well arrays to be used as regenerative patches.

Membranes have been explored as patches in tissue repair and regeneration, most of them presenting a flat geometry or a patterned texture at the nano/micrometer scale. Herein, a new concept of a flexible membrane featuring well arrays forming pore-like environments to accommodate cell culture is proposed. The processing of such membranes using polysaccharides is based on the production of multilayers using the layer-by-layer methodology over a patterned PDMS substrate. The detached multilayered membrane exhibits a layer of open pores at one side and a total thickness of 38±2.2µm. The photolithography technology used to produce the molds allows obtaining wells on the final membranes with a tuned shape and micro-scale precision. The influence of post-processing procedures over chitosan/alginate films with 100 double layers, including crosslinking with genipin or fibronectin immobilization, on the adhesion and proliferation of human osteoblast-like cells is also investigated. The results suggest that the presence of patterned wells affects positively cell adhesion, morphology and proliferation. In particular, it is seen that cells colonized preferentially the well regions. The geometrical features with micro to sub-millimeter patterned wells, together with the nano-scale organization of the polymeric components along the thickness of the film will allow to engineer highly versatile multilayered membranes exhibiting a pore-like microstructure in just one of the sides, that could be adaptable in the regeneration of multiple tissues. STATEMENT OF SIGNIFICANCE: Flexible multilayered membranes containing multiple micro-reservoirs are found as potential regenerative patches. Layer-by-layer (LbL) methodology over a featured PDMS substrate is used to produce patterned membranes, composed only by natural-based polymers, that can be easily detached from the PDMS substrate. The combination of nano-scale control of the polymeric organization along the thickness of the chitosan/alginate (CHT/ALG) membranes, provided by LbL, together with the geometrical micro-scale features of the patterned membranes offers a uniqueness system that allows cells to colonize 3-dimensionally. This study provides a promising strategy to control cellular spatial organization that can face the region of the tissue to regenerate.

Control of Cell Alignment and Morphology by Redesigning ECM-Mimetic Nanotopography on Multilayer Membranes.

Inspired by native extracellular matrix (ECM) together with the multilevel architecture observed in nature, a material which topography recapitulates topographic features of the ECM and the internal architecture mimics the biological materials organization is engineered. The nanopatterned design along the XY plane is combined with a nanostructured organization along the Z axis on freestanding membranes prepared by layer-by-layer deposition of chitosan and chondroitin sulfate. Cellular behavior is monitored using two different mammalian cell lines, fibroblasts (L929) and myoblasts (C2C12), in order to perceive the response to topography. Viability, proliferation, and morphology of L929 are sensitively controlled by topography; also differentiation of C2C12 into myotubes is influenced by the presence of nanogrooves. This kind of nanopatterned structure has also been associated with strong cellular alignment. To the best of the knowledge, it is the first time that such a straightforward and inexpensive strategy is proposed to produce nanopatterned freestanding multilayer membranes. Controlling cellular alignment plays a critical role in many human tissues, such as muscles, nerves, or blood vessels, so these membranes can be potentially useful in specific tissue regeneration strategies.

Cell-Adhesive Bioinspired and Catechol-Based Multilayer Freestanding Membranes for Bone Tissue Engineering.

Mussels are marine organisms that have been mimicked due to their exceptional adhesive properties to all kind of surfaces, including rocks, under wet conditions. The proteins present on the mussel's foot contain 3,4-dihydroxy-l-alanine (DOPA), an amino acid from the catechol family that has been reported by their adhesive character. Therefore, we synthesized a mussel-inspired conjugated polymer, modifying the backbone of hyaluronic acid with dopamine by carbodiimide chemistry. Ultraviolet-visible (UV-vis) spectroscopy and nuclear magnetic resonance (NMR) techniques confirmed the success of this modification. Different techniques have been reported to produce two-dimensional (2D) or three-dimensional (3D) systems capable to support cells and tissue regeneration; among others, multilayer systems allow the construction of hierarchical structures from nano- to macroscales. In this study, the layer-by-layer (LbL) technique was used to produce freestanding multilayer membranes made uniquely of chitosan and dopamine-modified hyaluronic acid (HA-DN). The electrostatic interactions were found to be the main forces involved in the film construction. The surface morphology, chemistry, and mechanical properties of the freestanding membranes were characterized, confirming the enhancement of the adhesive properties in the presence of HA-DN. The MC3T3-E1 cell line was cultured on the surface of the membranes, demonstrating the potential of these freestanding multilayer systems to be used for bone tissue engineering.