Fabrication and Applications of Micro/Nanostructured Devices for Tissue Engineering
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Nanotechnology allows the realization of new materials and devices with basic structural unit in the range of 1–100 nm and characterized by gaining control at the atomic, molecular, and supramolecular level. Reducing the dimensions of a material into the nanoscale range usually results in the change of its physiochemical properties such as reactivity, crystallinity, and solubility. This review treats the convergence of last research news at the interface of nanostructured biomaterials and tissue engineering for emerging biomedical technologies such as scaffolding and tissue regeneration. The present review is organized into three main sections. The introduction concerns an overview of the increasing utility of nanostructured materials in the field of tissue engineering. It elucidates how nanotechnology, by working in the submicron length scale, assures the realization of a biocompatible interface that is able to reproduce the physiological cell–matrix interaction. The second, more technical section, concerns the design and fabrication of biocompatible surface characterized by micro- and submicroscale features, using microfabrication, nanolithography, and miscellaneous nanolithographic techniques. In the last part, we review the ongoing tissue engineering application of nanostructured materials and scaffolds in different fields such as neurology, cardiology, orthopedics, and skin tissue regeneration.
KeywordsNanomaterials Nanostructures Microfabrication Nanofabrication Device Tissue engineering
Electron beam lithography
Focused ion beam lithography
Central nervous system
Peripheral nervous system
The field of regenerative medicine includes tissue engineering (TE) and research on self-healing events, during which the organism uses its repairing systems and/or foreign biocompatible material to maintain or regrow tissues and organs. The main target of TE is to combine biocompatible materials, cells, drugs, and active molecules to restore or improve biological functions. This field is expanding on a daily basis, through integrating knowledge such as cell biology, chemistry, materials science, nanotechnology, and micro- and nanofabrication [1, 2, 3].
In the last few years, because of the combination of their bulk and surface properties to the overall behavior, nanomaterials have become increasingly important for the realization of novel TE tools. The development of functional nanostructured materials accomplishes the needs of sick or damaged tissues in bone, cartilage, muscle, and neuronal system [4, 5] during the recovery. Tissue function and physiopathology must be outlined in in vitro experiments and integrated in vivo into host tissue. Materials for TE must be chosen so that the extracellular space results as close as possible to the original environment. The ideal candidate should accommodate cells by ensuring them the possibility to reform an extracellular matrix (ECM) and, therefore, to physiologically attach, proliferate, and differentiate . Cells in contact with an appropriate biomaterial [7, 8], can proliferate, differentiate, and reproduce by secreting ECM molecules and factors and remodeling their environments. The principal components of the ECM are the proteoglycans-rich interfibrillar matrix, structural fibrillar proteins as elastin and collagen and other proteins as laminin, fibrillin, and fibronectin . Since proteins and others biological molecules are nanoscaled structures, cells are programmed to interact with nanophase materials; the mutual interaction between the ECM components and the receptors present on the cell surface regulates cell behavior and its development .
Surfaces characterized by submicron scale features have been used to study cells' response to nanometer-scale topographical cues that can influence a wide range of cellular functions such as morphology, adhesion, and migration [11, 12, 13]. Nowadays, nanoscale technology, together with the most advanced microfabrication and post-processing modification techniques, supports the realization of a wide range of two -and three-dimensional (2D and 3D) bioengineered substitutes for in vitro [14, 15, 16, 17, 18] and implantation tests [19, 20, 21]. During the last 15 years, the scientific publication rate of paper treating arguments regarding the application of nanostructure materials in the TE field increases year by year. In detail, by making a search in the Web of Science bibliographic database using the words “nanostructured, materials, tissue, engineering,” it resulted that the number of the papers published in 2014 was more than double what it was published in 2012. Nanostructured materials allowed the creation of efficient biocompatible scaffolds, thanks to their large surface area and small dimension. As well as native tissues that are composed of nanosized biomolecules and of cells that interact with the ECM, nanomaterials such as nanoparticles [22, 23], nanotubes [24, 25], nanofibers [26, 27, 28, 29] and other nanostructured fabricated devices with features smaller than 500 nm, can closely mimic native biological system by assuring cell growth and tissue regeneration [14, 30].
With the development of nanotechnology coupled with advance microfabrication techniques, biocompatible-nanostructured materials closely fulfill the requirement in the recovery of native tissues for TE applications. The goal of this review is to discuss the fabrication and application of novel nanostructured natural and synthetic polymers and scaffolds for TE research. Microfabrication, nanolithography, and miscellaneous nanolithographic techniques are discussed in detail along with different applications in many branches of the modern medicine such as neuroscience, cardiology, orthopedics, skin, and dermatology.
2 Microfabrication and Nanolithography Techniques in TE
2.1 Replica Molding
Standard microfabrication processing methods such as optical lithography, deposition, and etching, optimized for materials such as silicon and glass, are usually not suitable for natural and synthetic biomaterials without being coupled with other techniques. Unconventional nanofabrication routes, such as replica molding (RM) and embossing, have been developed for patterning nanoscale structures. The formation of this kind of structures requires a high-resolution master, typically generated by conventional nanofabrication techniques that can be replicated by molding or embossing . RM and soft lithography techniques allowed the obtainment of replicas, realized using biocompatible polymers (polydimethylsiloxane, polystyrene, and so on), characterized by nanometric features (down to 50 nm) . Hot embossing, thermal forming, solvent casting, and injection molding require dedicated tools such as a hot press or injection molding machines and can be used to create small patterns into different thermoplastics biocompatible materials.
2.2 Etching and Direct-Write Techniques
Nanostructured materials for TE application are realized by means of different nanotechnology techniques that can be briefly summarized in two approaches, bottom-up and top-down. Supramolecular chemistry and surface science are considered bottom-up approaches; they enable the combination of various layers that could be made from different materials and/or incorporate different biochemical cues.
Nanostructured materials can be synthesized with precisely controlled morphologies, sizes, and seemingly limitless chemical functional groups . In contrast to bottom-up technologies, biocompatible-nanostructured surface has been fabricated with a top-down approach, via etching away bulk material to achieve the required smaller structural architectures  or through electrospinning by directly shaping the materials into the desired structure . Electrospinning and novel direct-write 3D electrospinning technique, by enabling the production of micro/nanoscale-shaped structures such as continuous, self-aligned, fiber-by-fiber, and template-free manner fibers are showing growing potential in tissue engineering applications .
Plasma etching represents a nanolithographic process that, in conjunction with soft lithography, nanoimprint lithography, and dip-pen lithography, as well as conventional techniques such as EBL and FIBL has been utilized for patterning surfaces with nanometer resolution.
More specifically, EBL and FIBL represent two of the most used direct-write fabrication techniques that are able to realize nanometric detailed patterning by means of focused accelerated particle beams to scan across a surface. In particular, EBL has been used for the creation of nanopillared molds, which were consequently used to hot emboss PCL surfaces with nanopits , and for the fabrication of grooves ranging from 20 to 1000 nm width . Cells grown on the patterns with 80–350 nm grooves showed the most alignment to the pattern and alignment decreased as the depth and pitch of the grooves decreased. The depth of the grooves appeared to play a greater role than the pitch in guiding cell alignment, as cells seeded on 600-nm deep grooves showed greater orientation than on the 150-nm deep patterns . EBL is often also used in conjunction with other lithographies such as nanoimprint and capillary lithography to drastically improve their natural resolutions .
FIBL uses electrostatic and magnetic fields to focus a low-energy ion beam commonly down to diameters of 50 nm; it can be passed through a stencil onto a surface and also used in free-form milling processes due to the complexities of ion beam optics . FIBL is more frequently used for direct-write on hard surfaces, such as silicon glass and metals without using polymer resists that are needed for patterning. Yet, this kind of nanolithography is successfully used also for biomedical milling tasks, such as tune hydrogel surface morphology and modulus. During irradiation, unique nanoscale porous features in regular formation were observed and the pore parameters were found to be dependent on ion incident angles . Modern FIB instruments, equipped with electrostatic optics and a liquid metal ion source, can achieve a resolution comparable to that of EBL . Applications on a polydimethylsiloxane resulted on topographical modification for potential TE applications .
3 Applications of Nanostructured Materials in TE
A micro- and nanofabrication technological approach can successfully be applied to TE biomedical demands through producing nanoparticles, nanofibers, and scaffolds with nanometric features, which are able to mimicking native tissues by optimizing biomaterials and structuring their geometry. Tissue damage and organ failure is a serious medical condition; the request for tissue regeneration and organ replacement is expanding year by year because of the significant shortage of organs available for transplantation. In order to treat patients with failing or failed organs, organ-assist medical devices that replicate the functions of organs have been realized; micro- and nanofabricated systems can mimic components of native tissue resulting in effectively in vitro models as well as successful medical devices for in vivo test.
3.1 Neural Tissue
The physiology of the nervous system presents demanding challenges to TE research addressing central and peripheral nervous system injuries (CNS and PNS). The main difference between the PNS and CNS is the capacity for the first one to regenerate. The inability of CNS neurons to regenerate and the formation of a gliotic scar are the major contributing factors to the irreparable nature of spinal cord injury .
Through the combined efforts of physicians, biologists, biotechnologists, and engineers, experimental work in neurological TE application has made great progress. Micro- and nanotechnology are providing microstructured scaffolds to promote regeneration and direct repair by reconnecting axons. By combining traditional microfabrication techniques with nanotechnological tools, it is possible to realize implantable scaffolds with precisely formed architectures and to control their surface chemistries, creating a physiological microenvironment able to promote neurites’ outgrowth. A completely different repair strategy tries to repair nerves through the reconnection of damaged axons immediately after an injury. This strategy uses microfabrication to realize microelectromechanical systems that serve as ultramicrosurgical solutions to manipulate individual axons without incurring damage . When cultured on different substrates, neurons are shown to rearrange their axon growth orientation and network shape according to the imposed topography , while the cell body does not necessarily follow the same adjustment . It seems that a discontinuous topographical pattern could promote Schwann cell and axonal alignment, provided that it hosts anisotropic geometrical features, even though their sizes range at the subcellular length scale .
By means of photolithography and micromolding techniques, 3D biocompatible and biodegradable PCL, pillared substrates were produced to replace of the ones realized in silicon. A simple, rapid, and economic method of fabrication, which involved photolithography and micromolding techniques, allowed the production of 3D PCL scaffolds with nanopatterned sidewalls, able to assist adhesion and growth of human neural stem cells .
Cardiovascular disease (CVD) is a term referred to diseases of the heart or blood vessels; blood flow to tissue and organs (brain, heart, kidneys, and so on) can be reduced as the result of thrombosis, atherosclerosis, or coronary heart disease.
The American Heart Association estimates that the 2013 overall rate of death attributable to CVD was about 223 per 100,000 Americans. The death rates were 270 for males and 185 for females . Patients with these conditions and diseases need different treatments, ranging from daily medications to surgical interventions such as stents, pacemakers, angioplasty, or heart transplants.
In recent years, increasing interest has been directed toward the study of the complex interplay of the insoluble ECM adhesion molecules, soluble factors, and hemodynamic factors regulating vascular growth and remodeling . The specialized properties of the myocardium and valvular tissues, including the capability of contraction of cardiac tissue and the deformation of valves, provide obstacles for the fabrication of tissue-engineered implants for cardiac interventions. Tissue-engineered successful vascular grafts should have mechanical strength for the prevention of surface thrombosis, and highly organized structures combining with ECM proteins. It seems that biomaterial surface modification on the nanoscale enhances vascularization in tissue-engineered constructs by influencing cell alignment, adhesion, and differentiation [64, 65, 66]. A study regarding the use of collagen-like synthetic self-assembling nanofiber hydrogels successfully supported the culture of both neonatal rat cardiomyocytes and human embryonic stem cell-derived cardiomyocytes . Tubular collagen scaffolds with nanopatterns were developed; both inside and outside of the tubes, endothelial cells were seeded on the luminal side, while smooth muscle cells were seeded on the outside of the tubes. Following co-culturing in double-sided nanopatterned tubes, it was verified that tensile strength was enhanced while improving cell retention in the lumen under blood flow .
Nanomaterials have also been found to improve cardiomyocyte functions; the addition of carbon nanofibers in PLGA scaffolds promoted cardiomyocyte growth by increasing both the electrical conductivity and tensile strength of the scaffold compared to conventional polymeric materials . One of the major limitations of engineered myocardial patches for heart repair is that insulating polymeric scaffold walls interfere with the transfer of electrical signals between cardiomyocytes.
Recently, when carbon nanotubes (CNTs) platforms were used to culture cardiomyocytes, the growth and electrical activity of the cells were enhanced. Notably, CNTs remarkably accelerated gap junction formation via activation of integrin-mediated FAK/ERK/GATA4 pathway . Furthermore, other group underlines the ability of multiwall carbon nanotubes to promote changes in electrical membrane properties of neonatal rat ventricular myocytes, by proposing them as novel treatments for arrhythmia and conduction disease . By incorporating gold nanowires into alginate scaffolds, it has been possible to bridge the electrically resistant pore walls of alginate and improved electrical coupling between neighboring cardiomyocytes .
3.3.1 Bone TE
Musculoskeletal tissue diseases and traumas, such as intervertebral disk degeneration, tumors, osteoporosis, arthritis, fractures, and tendinopathies, are common clinical situations, as they are also related to the aging of the global population. They usually result in significant loss of bones, muscle, tendons, cartilage, and ligaments. Advances in regenerative therapies depend on the choice of the appropriate material that would provide the right mechanical integrity by tuning cell growth and neotissue formation.
By considering the micrometer length scale, bone can be described as a spongy bone with high porosity or as a lamellar bone with compact cylindrical osteons. On the nanometer level, the collagen fibrils and nanohydroxyapatite are assembled into ordered and aligned patterns. The different tissues of the musculoskeletal system are connected with each other, either hard-to-hard tissue, soft-to-hard tissue, or soft-to-soft tissue. Hence, concerning the nanostructure of a scaffold is crucial when developing a solution for composite tissues, such as an osteochondral graft . The ideal scaffolds, through mimicking the native ECM environments, should provide structural support by imitating the mechanical properties of the tissue, thus allowing nutrients, drugs, and waste transfer through its porous architecture. Many fabrication technologies such self-assembly, rapid prototyping, or multiple methods, offer control over natural or synthetic polymers from nano- to macroscale [74, 75, 76, 77]. Porous chitosan–gelatin composite scaffolds were fabricated using freeze-drying and freeze-gelation methods.
It seems that freeze-gelation process is a promising method for the production of various chitosan-based composite biomaterials and that the freeze-gelation method for hydrogel scaffold fabricated scaffolds is a better method for the growth, proliferation, and viability of nucleus pulposus cells of the human intervertebral disk . Merceron et al. described their method for the 3D biofabrication of a single integrated muscle–tendon unit construct through using multiple synthetic biomaterials and different cell types . Direct 3D printing has been successfully used to fabricate porous ceramic scaffolds with fully interconnected channels directly from hydroxyapatite powder for bone replacement .
The microinjection molding technique has been chosen as an industrially viable process, for the accurate and cost-effective mass production of molded micro-patterns, which influence the osteogenic differentiation of human mesenchymal stem cell, in the absence of growth factors commonly used to induce osteoblast differentiation . Recently, microfabrication techniques have been also used to enhance vascularization in engineered bone substitutes, while micropatterning, microcontact printing, and micromolding have been widely adopted in the development of in vitro microscale vascularized networks .
3.3.2 Cartilage TE
Nanotechnology for cartilage TE application has recently seen great improvements. Cartilage is an inhomogeneous, anisotropic, porous-viscoelastic material composed of a low percentage of chondrocytes embedded in a dense nanostructured ECM network of elastin fibers, collagen fibers, and proteoglycans . Compared with bone, it is a flexible connective tissue that does not require post implantation vascularization. Micro- and nanofabrication techniques as micromachining, photolithography, rapid prototyping, electrospinning, fiber bonding, electrospinning, electrostatic spray deposition, plasma deposition, and molecular self-assembly have been investigated to mimic the microscale porosity and the complex organization of native tissues . Electrospinning represents a successful technological tool for bone tissue engineering allowing the reparation of thin or larger defects through electrospun sheets that may be layered or rolled for the restoration of the defeats tissues . Using a crosslinking method, novel chitosan artificial cartilage tissue has been obtained .
It should be reviewed, in this contest, the uses of particular cell-encapsulating hydrogel, such as gelatin-methacryloyl (gelMA), as a base material for cartilage tissue engineering solution . The concentration of this polymer, its UV exposure time, and its thermal gelation before UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co-deposition with thermoplastics allows gelMA to be used widely in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties. In stiffer (~30 kPa) photocrosslinked gelMA, and gelMA/hyaluronic acid hydrogels, chondrogenic redifferentiation occurs, both in vitro  and in vivo .
3.3.3 Muscle TE
Since the muscle is a particular tissue able to produce force and motion under the direction of the nerve, engineered muscle tissues should mimic the native structure with regards to densely packed and uniformly aligned myofibers throughout the tissue volume . Skeletal muscle TE offers its solutions for the treatment of post-traumatic damages, pathological conditions (diseases like Duchenne muscular dystrophy and spinal muscular atrophy), and post-surgery tissue ablation [89, 90].
When engineering a skeletal muscle, one of the key points is to obtain the muscle fiber formation by aligning the cells. Micro- and nanofabrication techniques enhance the possibility to create this kind of tissue. Many techniques such as photo and soft lithography, electrospinning, hot embossing, and solvent casting have been used to realize an environment able to induce cell alignment [91, 92, 93, 94, 95]. Since nanotopography greatly influences cell contact guidance, nanofeatured materials can interact with cells by mimicking the ECM natural environment. It seems that the use of biocompatible surfaces characterized by nanometric structures (pillars, pits, nodes, and nano-islands) promotes cell adhesion, whereas increases in the size of these features, decreases it . It has been shown that also the symmetry of these features, as well as the surface’s roughness, affects cell adhesion on the substrate [97, 98, 99]. Electrospinning has been used to fabricate aligned nanofiber scaffolds that induced myoblasts alignment, as the structure of those fibers resembles the one of native collagen fibrils characterizing the ECM [94, 100]. In order to improve cells infiltration into the fibers network, direct electrospinning of a 3D aligned nanofibrous tube has been realized, promoting cell alignment and myotube formation .
3.4 Skin TE
Skin loss occurs for surgical interventions, thermal, mechanical and physical–chemical trauma, chronic wounds, and also genetic disorders; it can be divided into epidermal, superficial partial thickness, deep partial thickness, and full thickness based on the depth of the injury [102, 103, 104]. It becomes urgent to replace the damage via grafts, as it serves to protect from water loss and the risk of pathogens intrusions. Skin grafts can assist the wound-healing process by restoring the physiological functions at that site . Bioengineered skin substitutes emerged over the past 60 years . They were initially applied to replace autograft, allograft, and xenograft in burn applications, but they later found even wider application in the treatment of chronic venous and diabetic ulcers.
Tissue scaffolds realized using natural and synthetical materials are able to provide a physical barrier, by covering the wound, and thus to stimulate the re-epithelialization process. Among natural polymers collagen, fibrin, chitosan, gelatin, and elastin have been developed as biological skin substitutes for wound healing [107, 108, 109, 110, 111]. Several collagen-based dressings in the form of a gel, sheet, lattice, or sponge are commercially available and are successful used as temporary covering for ulcers and burns .
Many TE solutions use synthetic materials that are able to degrade in the place of the implant by allowing host tissue ingrowth while avoiding the drawbacks associated with permanent synthetic material implantation. PCL is one of these materials and is currently utilized for some medical applications including adhesion barrier and wound dressing . Its biocompatibility, mechanical strength, high elasticity, and degradation time issue the popularity of this polymer in TE contest. The strict combination of appropriate biomaterials and techniques can reduce the risk of failure in skin graft; electrospinning can produce new biocompatible and biodegradable scaffolds with a porosity structure that can mimic the one of the native dermis.
Native tissues are highly organized at the micro- and nanoscale and mimicking natural tissue structure is the most important target for TE. The control of the mechanisms underlying how cells sense nanoscale patterns could enhance scientists’ ability to manipulate cells behavior by allowing successful development of new methods to grow artificial tissues/organs and replace the counter parts in vivo. The actual strategy applied to the biocompatible and biodegradable nanostructured biomaterials design, thanks to the identification of the suitable grade of porosity, effectively contributes to the fabrication of promising multi- component and multi-functional materials for different TE applications. To conclude, in light of the contents summarized in this review, we underline how the integration of medicine and nanotechnology, with regard to microfabrication and nanolithographic techniques, are deeply impacting modern TE solutions.
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