Polymer-based platforms by electric field-assisted techniques for tissue engineering and cancer therapy.

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Review

Polymer-based platforms by electric field-assisted techniques for tissue engineering and cancer therapy Expert Rev. Med. Devices 12(1), 113–129 (2015)

Vincenzo Guarino*, Valentina Cirillo, Rosaria Altobelli and Luigi Ambrosio Institute for Polymers, Composites and Biomaterials, National Research Council of Italy, Mostra d’Oltremare, Pad.20, V.le Kennedy 54, 80125 Naples, Italy *Author for correspondence: Tel.: +39 081 242 5944 Fax: +39 081 242 5932 [email protected]; [email protected]

A large variety of processes and tools has been investigated to acquire better knowledge on the natural evolution of healthy or pathological tissues in 3D scaffolds to discover new solutions for tissue engineering and cancer therapy. Among them, electrodynamic techniques allow revisiting old scaffold manufacturing approach by utilizing electrostatic forces as the driving force to assemble fibers and/or particles from an electrically charged solution. By carefully selecting materials and processing conditions, they allow to fine control of characteristic shapes and sizes from micro to sub-micrometric scale and incorporate biopolymers/molecules (e.g., proteins, growth factors) for time- and space-controlled release for use in drug delivery and passive/active targeting. This review focuses on current advances to design micro or nanostructured polymer platforms by electrodynamic techniques, to be used as innovative scaffolds for tissue engineering or as 3D models for preclinical in vitro studies of in vivo tumor growth. KEYWORDS: ECM analog • electrospinning • electrospraying • tissue engineering • tumor drug resistance

Native tissues exhibit inhomogeneous and anisotropic organization, consisting of numerous types of cells and extracellular matrices (ECMs) that are integrated together in defined spatial hierarchies [1]. Among them, ECM is recognized as an articulate assembly of structural and functional biomolecules, that is, collagen, fibronectin and other proteins strictly packed with proteoglycans directly secreted by the local cells, that creates a unique and tissue-specific 3D environment of natural tissues [2]. This peculiar molecular arrangement not only serves as a supporting function but has also a leading role in regulating numerous cellular functions, including cell shape, adhesion, migration, proliferation, polarity, differentiation and apoptosis [3]. Moreover, it concurs to modulate signal transduction mechanisms activated by various bioactive molecules, such as growth factors and cytokines [4]. Indeed, cells require several interactions with ECM components in order informahealthcare.com

10.1586/17434440.2014.953058

to undergo normal morphogenesis in physiological conditions through a fine balance between synthesis and degradation [5]. In pathological conditions, synthesis and/or breakdown of ECM components (i.e., collagen, fibronectin and laminin) with consequent generation of cleavage products (i.e., laminin- or collagen-cleavage products) can contribute to the biological activity, often influencing growth and cancer progression. In both cases, this function is really complex taking into account that cell/ECM interplay is not static but is rather a dynamic event that mainly responds to external stimuli (i.e., biomechanical, chemical, electrical, etc.) [6]. Given the high complexity of these dynamic processes and the multiple roles of the ECM, a great challenge in tissue engineering (TE) and cancer therapy comprises the discovery of new materials and technologies to design bio-inspired platforms able to mimic a 3D instructive microenvironment with specific

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Guarino, Cirillo, Altobelli & Ambrosio

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morphological, biochemical and physical cues (FIGURE 1) able to reproduce all native cell-to-cell and cell-to-ECM interactions occurring during healthy tissue regeneration or tumor progression. In general, this strategy combines cellular elements of living tissues with micro or nanostructured biomaterials to produce living structures of sufficient size and function to improve the quality of life of patients. To properly engineer such complex and multifunctional systems, nanofabrication techniques are powerfully emerging as the best solution to create porous, nanometric-sized fiber substrates with improved surface properties able to influence cell fate, allow regulation of specific protein-expression patterns and encourage cellspecific scaffold remodeling [7,8]. In this regard, the manipulation of electrically charged polymeric fluids by novel electrodynamic technologies (EDTs) currently represents the most interesting strategy to design multicomponent systems with highly tunable chemical and structural features to sagely guide cell response toward different ways, overcoming the body’s innate power of organization and self-repair. Electrodynamic technology

EDT refer to all processes based on the interaction of polymer solutions with electrostatic forces to fabricate fibers or particles of different shapes and sizes from micro to nanometric scale. Morphology and composition of micro/nanostructures can be modulated by an accurate definition of material properties, working conditions and process setups (FIGURE 2), thus giving the opportunity to customize the final properties of the device for specific demands. 114

Electrospinning is the most popular technique for the fabrication of nanofibers due to its simplicity, cost–effectiveness, flexibility, scale-up potential and ability to spin a broad range of polymers [9]. For electrospinning, a strong electrical potential is applied to the polymer liquid (solution or melt) with accumulation of electrical charges on the surface of the liquid droplet at the tip of the capillary. At a critical voltage, the Columbic charge repulsion overcomes the surface tension of the polymer droplet and a charged jet is ejected from the spinneret tip. The jet travels toward a grounded electrode, while the solvent gets evaporated, and the resultant fibers are collected on a grounded target. This process allows obtaining fine and ultrafine fibers with nonwoven and randomly arranged structures with controlled morphology by an accurate setting of materials and process parameters [10]. They include: • parameters strictly inherent to the polymer solution (i.e., molecular weight, concentration, viscosity, dielectric permittivity and surface tension); • parameters strictly inherent to the operational conditions (i.e., feeding rate, tip to collector distance, applied electric field and voltage and tip needle diameter); • parameters inherent to the electrospinning setup (i.e., rotating collectors, drum and auxiliary rings); • environmental parameters (i.e., temperature, humidity and vacuum). Various polymers may be easily processed ranging from natural polymers such as collagen, gelatin, chitosan, silk fibroin and hyaluronic acid to synthetic polymers such as poly(lactic acid), poly(e-caprolactone) (PCL), polydioxanone and copolymers including poly (L-lactide-co-caprolactone) and poly(lacticco-glycolic acid) (PLGA) [11] to fabricate scaffolds with tailored properties for different application in tissue regeneration, drug and gene delivery [12–14]. Electrospraying

Electrospraying is based on atomization of polymer solutions by the application of electrical forces, quite similar to electrospinning. Unlike electrospinning, electrospraying results from the interaction of bulk and surface electrodynamic forces breaking the jet into droplets. Briefly, the liquid flowing out of a capillary nozzle – which is maintained at high electric potential – is forced by electric forces to be dispersed into fine droplets. Due to the surface tension, jet fragments subsequently acquire a spherical shape before being deposited on a grounded plate. During this process, a valuable increase in voltage [15], conductivity [16] and surface tension [17] of the sprayed solution is commonly associated with a decrease in particle diameter. Likewise, an increase of flow rate [18], density [17] and viscosity [19] of the sprayed solution is generally associated with an increase in particle diameter. Moreover, a fine control of solvent evaporation rate may concur with fabrication of particles with spherical morphology and smooth texture [18]. The high tunability of

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the process confers several advantages to electrospraying over traditional mechanical atomization based on drying technologies [20]. Indeed, the size of electrospraying droplets can range from hundreds micrometers down to several tens of nanometer, with respect to particles on micrometric size scale produced by mechanical atomization. Droplet size can be controlled to some extent by the flow rate of the voltage at the capillary nozzle while the electrical charge on particle surface facilitates control of their motion under the electric field forces. This assures a self-dispersion of charged droplets in the space and reducing the coagulation phenomenon, generally due to residual charges on the particles related to the drying effects [21]. Meanwhile, the absence of continuous high-energy shearing force [22] is beneficial to protect highly labile molecules such as proteins or drugs, opening their successful use to different routes in biotechnology as surface coating or drug delivery systems.

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Polymer based platforms by electric field assisted techniques for tissue engineering & cancer therapy

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Starting from previously described basic concepts of EDTs, more complex processes may be implemented to design multicomponent scaffolds in which fibers Coaxial EDT and/or particles of different polymers may be combined to acquire new funcFigure 2. Flow sheet of basic parameters classes to design instructive platforms by electrodynamic technologies: materials and process parameters tionalities from each unit in the same allows for switching between fiber and particle production, whereas structure. Among them, most successful customized setups allows designing more complex devices by dual or coaxial strategies to fabricate multicomponent configuration. scaffolds involve the use of coaxial and dual or co-electrospinning [23,24]. Coaxial electrospinning is an EDT technology for the fabri- viscoelasticity and jet bending phenomena are predominant cation of a novel class of nanofibers with core–shell structure and promote the sole formation of nanofibers. For this advanced process, a wide variety of possibilities for used for targeting and controlling release of molecular species. Similar to electrospinning, coaxial electrospinning employs elec- generating novel nanomaterials have been realized, mainly tric forces on polymer solutions that result in significant based on the encapsulation of drugs or biological agents. This stretching of polymer jets due to a direct pulling and growth of approach allows for a more efficient control of the release rate the electrically driven bending perturbations [25]. At the exit of by shell properties useful in suppressing the initial burst release the core–shell needle, electric Maxwell stresses may generate or protection of biologically active agents in the core from the electrically driven bending instability, over a supercritical elec- effect of harsh solvents present in the outer solution [28] or of tric field value [26], which produces higher jet stretching accom- the electric charges, rapidly escaping to the outer surface at the panied by enormous jet thinning and fast solvent evaporation beginning of core–shell jet formation [29]. More recently, core– that promote faster jet solidification. During the process, core– shell nanofibers have been fabricated by using coaxial electroshell jets under electric forces may also be subjected to the elec- spinning/triaxial electrospinning that offer dual-stage drug trospraying process despite the involvement of hydrodynamic release ascribable to an immediate release of the inner shell folissues that originates completely different products [27]. In lowed by sustained release from the core [30]. Indeed, in pharmaceutical therapy, a sustained release of the the case of electrospraying, no viscoelasticity or jet bending occurs so that the jet may be rapidly atomized into tiny core– remaining dose over a defined period has to be managed to shell droplets, whereas in the case of coaxial electrospinning, avoid repeated administration for the patient’s convenience [31]. informahealthcare.com

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Figure 3. 3D fiber morphology of electrospun scaffolds with selective morphological, biochemical and physical properties. (A) poly(e-caprolactone) fibers with uniaxial alignment (B) bicomponent fibers made of poly(e-caprolactone) with bioactive gelatin cues and (C) poly(vinylidenefluoride) fibers with piezoelectric properties.

The use of carriers with dual-release profiles often represents the best strategy to control the release at two different rates or in two time periods. Nowadays, EDT-based system with dual drug release has been designed by the mutual combination of electrospraying and electrospinning, namely dual or coelectrospinning [32–34]. In this case, it can be performed by the independent pumping of two polymer solutions to simultaneously collect nano- and micro-objects in a unique membrane with controlled drug delivery. Similarly, hydrogels, hydroxyapatite or cells have been also used to postprocess electrospun fibers or particles (i.e., two step or nonsimultaneous dual electrospinning) to form multicomponent scaffolds for different biomedical use [35,36]. Instructive platform for TE Morphological signals

In the natural tissue, ECM provides spatial and mechanical signals to which cells respond, in addition to soluble chemical factors. They mainly depend upon the peculiar organization of collagen fibers that are largely responsible for the structural integrity and porosity, thus playing an important role in controlling cellular responses [37,38]. In the recent years, many attempts have been made to replicate natural ECM by engineered scaffolds with highly hierarchal structure. Recent efforts are focused on the use of electrospinning for creating ECM-like matrices by the assembly of nano- or microscaled continuous fibers with high surface to volume ratio and variable-sized pores. By changing the electrospun fiber size scale, the topographical cues exposed to cells by the electrospun matrix vary, thus significantly influencing cells spreading, migration, proliferation and differentiation [38]. During the deposition, characteristic size of fibers can be controlled by tuning materials properties and process parameters such as solvent type, polymer solution concentration, flow rate, needle size or the distance between the needle and the collector. For instance, Guarino et al. examined the effect on fiber morphology due to interplay between various factors involved in the process, including the dielectric constant and other solution properties (e.g., density, boiling point and 116

solubility) relating to the solvents used. They showed that the coupling of polymer and solvent components can drastically affect the final morphological appearance of electrospun fibers in terms of fiber size scale and beads formation. They suggested that solvents with different permittivity play an active role in polymer chain folding during the fiber deposition, thus affecting the fiber crystallinity. The modulation of fiber diameters can be due also to variation of polymer concentration. In more concentrated solutions, there is an increased viscoelastic force that prevents the jet segment stretching under the effect of the constant Coulomb force, resulting in fibers with larger diameters. This effect, combined with the different polymer–solvent macromolecular interactions, strongly influences the capability of polymer chains to fold and, therefore, affects fiber size and crystallinity [39]. By choosing a specific fiber size scale and mode of assembly of polymer chains (i.e., crystallinity), it has been demonstrated that it is possible to influence the behavior of mesenchymal stem cells (MSCs), affecting the adhesion and/or proliferation kinetics of cells, ultimately determining the course of their differentiation process. Guarino et al. performed a biological study demonstrating that a more drastic increase in cell attachment was detected after 24 h with PCL nanofibers compared with PCL microfibers [39]. Accordingly, PCL electrospun fibers with average fiber diameters ranging from the micro- to nanoscale were assembled to form multilayered platforms [40]. As the fiber diameter increased, the average pore size of the fibers increased, while the porosity remained constant. MSCs showed wider spread morphology on the nanofibers rather than the microfibers, as evidenced by stronger F-actin staining [38]. Indeed, the higher surface to volume ratio in fiber meshes with nanoscale characteristic size has shown selective take-up of proteins relevant for cell attachment, such as fibronectin and vitronectin [41]. Smaller fibers led to a rise in fiber surface area, mechanical strength and enhanced adhesion and proliferation of human umbilical vein endothelial cells. The high surface area of the nanofibers with a small diameter enabled the adhesion, spreading and proliferation of cells because it increased the anchoring area at the outermost surface [42]. Expert Rev. Med. Devices 12(1), (2015)



An interesting issue is the mimicking of anisotropic properties of highly oriented tissues including nerves, muscles or tendons. In this direction, electrospinning setup can be properly defined to achieve micro or nanofibers with different spatial orientation. Fibers can be oriented in one direction by deposition onto a mandrel rotating at a relatively high speed or onto the edge of a spinning disk. These nanofibers, which can be arranged in parallel (FIGURE 3A), represent a simple, scalable and straightforward strategy to induce cell alignment and cell growth directionality upon cell seeding. It was reported that cells cultured on such scaffolds recognized the underlying geometry and thereby aligned themselves along the long axis of the fibers [43–46]. These aligned nanofibers are particularly relevant to the regeneration of tissue such as skeletal muscle, ligaments, nerves and blood vessels. Aligned electrospun fibers 400–600 nm in diameter have been shown to orient dorsal root ganglia (DRG) alignment and Schwann cells migration in vitro [47]. The size of electrospun fibers can influence alignment, growth potential and differentiation. For example, electrospun micro and nanoscale poly (L-lactic acid) fibers were equally capable of orienting neural stem cells soma and processes though the nanoscale fibers regardless of orientation increased neural stem cells differentiation over the microscale fibers (average diameters 250 nm and 1.25 mm) [48]. Interestingly, neurite length increased preferentially on nanoscale aligned fibers as confirmed by DRG culture on poly-L-lactate fibers with different alignment degree [49]. On aligned fibers, neurites sprouted radially but turned to align to fibers upon contact, and neurite length increased in the case of higher fiber alignment [50]. Aviss et al. even investigated the in vitro response of C2C12 myoblasts onto aligned PLGA electrospun nanofibers [51]. SEM and fluorescence staining results showed that C2C12 cultured on the aligned electrospun PLGA nanofibers elongated along the parallel axis of the electrospun fibers. Also, the expression of myosin heavy chain, which is a marker of skeletal muscle differentiation, was higher on the cells cultured on the aligned PLGA nanofibers compared with those cultured on the randomly arranged nanofibers. These results indicate that aligned electrospun nanofibers provide contact guidance for myoblast adhesion, elongation and differentiation. By following a bio-mimetic approach, Cirillo et al. produced fully aligned bicomponent fibers as potential bioactive substrate for nerve regeneration. It has been demonstrated that full alignment of fibers positively influences in vitro response of human MSCs (hMSC) and rat pheochromocytoma (PC12) cells in a neurogenic way. Immunostaining images show that in random fibrous meshes, the presence of kinks, for example, fiber crossing points, acts as topological defects, interfering with proper neurite outgrowth. On the contrary, fully aligned fibers without kinks offer a more efficient contact guidance to direct the orientation of nerve cells along the fibers compared to randomly organized ones, promoting a high elongation of neurites at 7 days and the formation of bipolar extensions. So, these results corroborated that the topological cue of full alignment of fibers elicits a favorable environment for nerve regeneration [52]. informahealthcare.com

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Biochemical signals

In the native tissue, ECM polysaccharides and proteins surrounding cells act as structural framework and modulate the right supply of ions and molecules, thus allowing for cell survival [37]. Growth factors and signaling molecules of ECM are responsible for pattern formation, morphogenesis, apoptosis and cells phenotype acquisition and maintenance through interactions with receptors on the surfaces of cells. Hence, several processes such as wound healing, inflammation, formation of granulation tissue and remodeling are all mediated by cell– ECM interaction [53]. It is noteworthy that cell cross-talking is complex and dynamic. Cells are attracted and adhere to the ECM mainly due to hydrophobic interactions that stimulate transmembrane proteins, known as integrins, on the cell surface to bind specific small peptide fragment sequences on the ECM. These connections enable cells to bind to the ECM through focal adhesions and promote direct cross-talking between them, supporting cells to differentiate, secrete and absorb matrix and transmit signals [54]. The proper design of scaffolds made of synthetic polymers guarantees tunable chemical and mechanical properties but a poor bio-functionality that drastically limit the communication mechanisms between cells [38]. An ideal TE scaffold has to replicate the ECM architecture and also reproduce the many functions that ECM elicits [53]. In this context, electrospinning process allows for the manipulation of natural and synthetic polymers for the fabrication of more efficient ECM analogs. The custom design of functional electrospun fibers with defined chemical and biological properties is crucial to regulate cellular behavior on the scaffolds and to achieve cell interactions similar to those in native ECM [38]. Since ECM of most hard and soft tissues is mainly composed by collagen fibers assembly, first studies have been focused on the electrospinning of proteins such as collagen [54] or its denatured forms, that is, gelatin. However, electrospun collagenous materials often failed in terms of mechanical and structural stability upon hydration so that cross-linking strategies based on glutaraldehyde vapors, formaldehyde and epoxy compounds have been considered to increase the strength of electrospun collagen, with negative effects on cell response in vivo [53,55]. Hence, a mixture of synthetic and natural polymers has been successfully used to overcome the limitations of monocomponent systems (FIGURE 3B). For instance, it has been shown that collagen type I blended with poly (L-lactic acid)-co-poly(e-caprolactone) (70:30) enhanced viability, spreading and attachment of human coronary artery endothelial cells preserving their phenotype [56]. Alvarez-Perez et al. demonstrated the integration of natural polymers such as gelatin into PCL nanofibers act as biological cue for promoting nerve repair and improving the biointeraction of PC12 nerve cells, compared to PCL alone nanofibers. In this case, bioactivity was due to the synergistic contribution of scaffold material topography – nanoscale fiber diameter – and biochemical signals offered by structural proteins [57], while the presence of PCL in the fibers assures more extended times of protein degradation [58]. 117


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Alternatively, elastin has been used in combination with other polymers to form electrospun fibers. It is a key structural protein found in the native ECM of connective tissues and it concurs to modulate the cellular physiology, affecting signaling, chemotaxis, proliferation and protease release of fibroblasts (FBs) and chondrocytes as well as monocytes, macrophages, neutrophils and lymphocytes [53,59]. Nivison-Smith et al. proposed to process tropoelastin based on the repeated elastomeric peptide sequence of natural elastin – in 1,1,1,3,3,3, hexafluoropropanol to form synthetic elastin microfiber network [60]. Human dermal FBs, human umbilical vein endothelial cells and human coronary artery smooth muscle cells were seeded on these scaffolds and were found to attach and grow on the seeded surface. To further improve biological response, collagen was included into elastin electrospun fibers to combine beneficial properties of both proteins for a better mimicking of physical, mechanical and biological cues of native skin dermis [61]. Other studies investigated the use of fibrinogen, a glycoprotein that plays a major role in homeostasis. McManus et al. studied the interaction of neonatal rat cardiac FBs with fibrinogen electrospun fibers. Cell culture demonstrated that FBs readily migrate into and remodel electrospun fibrinogen scaffolds with deposition of native collagen [62]. As other natural polymers, electrospun fibrinogen is not stable to act as a mechanical support on its own for long periods of time [53,63]. To improve the mechanical response, fibrinogen fibers were cross-linked with genipin [64] or combined with polydioxanone to create bicomponent fibers [65]. More recently, relevant studies have been performed on silk fibroin, extracted from silkworm cocoons for their interesting properties in terms of natural strength, biocompatibility, slow degradation rate, good water vapor and oxygen permeability and minimal inflammatory response, which makes possible its use in several applications [66,67]. Since numerous efforts to create the ideal ECM analog structure beginning from monocomponent fibers, more recent studies are also underlining the opportunity to process natural polymers natively found in the ECM, including chitosan and its derivative and hyaluronic acid as polysaccharides, collagen and gelatin as protein in combination with synthetic polymers, in order to better control fiber architecture on a nanoscale, showing promising results at this time. Physical signals

Several tissues, such as nerves or myocardium muscles, are responsive to external electrical forces, which may produce pulsatile stimuli to trigger specific cell event on supporting their natural function [68]. Recently, polymers with intrinsic conductive properties have been studied in relation to their incorporation into bioactive scaffolds for use in TE [68,69]. Their electrical and optical properties similar to those of metals and inorganic semiconductors, combined with conventional properties of common polymers, such as ease of synthesis and flexibility in processing, are attractive to support the native functionality (i.e., cardiac beating) of cells in several applications. 118

From chemical point of view, intrinsic conductive properties have an unstable backbone, resulting from the formation of alternate single and double bonds along with the monomer units during polymerization. The delocalized p bonding electrons, produced across the conjugated backbone, offer an electrical pathway for mobile charge carriers that are introduced through doping. Consequently, the electronic properties, as well as many other physicochemical properties, are due to the structure of the polymer backbone and the nature and concentration of the dopant ion [70]. Conducting polymers such as polypyrrole, polythiophene and polyaniline (PANi) have widely been utilized in the microelectronics industry [71] as bio-actuators [68,72], neural probes [68,73,74] and biosensors [75,76]. Among them, PANi has received increasing attention due to its environmental stability, controllable electrical conductivity and interesting redox properties. In the emeraldine oxidation state, PANi can be reversibly switched between electrically insulating and conducting forms according to its proton doping state [71]. These features and the excellent processability have substantially enhanced the potential applications of aniline polymers for their use in several application fields [77]. Borriello et al. proposed PANi/PCL electrospun fibers as an electroactive, biodegradable and biocompatible scaffold to potentially support the regeneration of the myocardium. After 3 and 5 days, the survival rate of cardiomyocyte-like cells seeded onto PCL/PANi samples was significantly higher than that on the PCL surface, thus demonstrating the effect of conductive signal of PANi on supporting the cell proliferation. PANi/PCL electrospun membranes with controlled fiber texture further concurs to create an electrically conductive environment able to stimulate the cell differentiation to cardiomyocytes [71]. Bioelectricity plays an integral role in maintaining biological functions such as signaling of the nervous system. Moreover, recent studies on the electrical stimulation of cells cultured on conducting polymeric substrates have shown that electrical stimulation is an effective cue for stimulating cell proliferation and differentiation, supporting nerve regeneration [78]. Tissue engineered constructs seeded with nerve stem cells, once electrically stimulated, promote neurites outgrowth representing the first step of peripheral nerve regeneration. Polypyrrole was one of the first conducting polymers studied for its effect on mammalian cells. To date, polypyrrole has been reported to support cell adhesion and growth of a number of different cell types, including endothelial cells, PC12 cells, neurons and support cells (i.e., glia, FBs) associated with DRG, primary neurons, keratinocytes and hMSC [68]. Based on electro-responsive polymers, even electrospun fibers with piezoelectric properties are gaining a recent interest in biomedical field because they can induce a transient change in surface charge without the need of additional energy sources or electrodes, promoting higher levels of neuronal differentiation and neurites outgrowth of mouse neuroblastoma cells [79]. Piezoelectricity consists in the generation of surface charges related to dipole crystal orientation into the polymer chains in the presence of small mechanical deformations [70]. At rest, dipole Expert Rev. Med. Devices 12(1), (2015)



structures in piezoelectric polymers are well-organized and no net charge is produced. When a mechanical force is applied, the shifting or rotation of the dipole crystal results in the change of polarization density, hence, causing the transient change of electric charge. Upon the removal of mechanical force, the dipole crystal returns to its original space. Poly(vinylidenefluoride) (PVDF) is a synthetic, thermoplastic, fluoropolymer. It is typically 50–70% crystalline with five distinct crystal polymorphs named a, b, g, d and e [80] depending on the chain conformations as trans (T) or gauche (G) linkages. These different forms are fundamental to the unique properties of PVDF and among the five polymorphs, the b-phase is the most interesting due to its piezo-, pyro- and ferroelectric properties, which can be attributed to the all trans conformation of the polymer chains [80]. During the electrospinning process, PVDF solutions amplify their piezoelectric character due to the rapid orientation of dipoles under the effect of applied high voltage [81]. This promotes the formation of fibers on the nanometric scale (FIGURE 3C) due to the strong stretching of the polymer solution under the electric forces. Taking into account the assessment of piezoelectric materials in several biomedical applications for bone, muscle and perhaps the epidermis [82], the fabrication of electrically charged platforms by electrospinning seems to be promising for several uses also in modern TE. Indeed, the piezoelectric property may be induced in these fibers in vitro and in vivo via minute deformations of the fibers due to cell attachment and migration, which has been shown in other non-neural cell types on collagen fiber matrices [83]. Piezoelectric activity in these fibers may also be activated in vivo due to bulk deformations from the cerebrospinal fluid circulation or in the peripheral nervous system, in response to changes in neighboring anatomic structures. Polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) is a copolymer with a permanent piezoelectric nature, thanks to the steric hindrance of TrFE group, which forces PVDF in all-trans configuration similar to b phase. DRGs and human neonatal porcine sertoli cells have been shown to extend neurites along aligned PVDF-TrFE fibers. Weber et al. suggested that the dipole structure may result in selective protein adsorption from the serum for favorable cell attachment [82], enhanced by the nanoscale dimension of the electrospun fibers, which show an increased surface area compared with a flat tissue culture plate surface [82,84]. Other researchers examined PVDF-TrFE films and tubes for nerve growth and showed promising results owing to the intrinsic piezoelectric property of the material both in vitro and in vivo [82]. All these studies indicate that PVDF-based electrospun fibers are promising as electroresponsive platform and able to guide the biological fate of cells by the mutual effects of microscopic and macroscopic properties of fibers, which support the native ECM functions. 3D models for cancer therapy

Although much effort has been devoted to study in vivo cancer evolution, the long duration of cancer development restricts the informahealthcare.com

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feasibility and reproducibility of cancer studies in situ. So, there is an urgent need to define in vitro cancer models that are efficient in mimicking the microenvironment in the presence of resident cancer cells and tumor development. In the past, many in vitro cancer models have proved effective in studying cancer pathogenesis, anticancer mechanism and drug testing [85–88]. Among them, most quantitative studies have focused on the behavior of isolated cancer cells, neglecting the collective behavior between cells and between cells and the stroma or the matrices. However, a comprehensive knowledge of invasion would require understanding collective behavior, as well as the transition from the single to the collective state and vice versa [89]. In this context, the use of a 3D cancer model along with microarray technologies could accelerate study on different causes and modes of cancer cell motility in the metastasis process, as a 3D environment resembles the in vivo situation of cancer cell invasion [85,90]. Possible mechanisms of cell death could also be revealed in an in vitro model, particularly to identify how cells alter its apoptotic behavior when reacting to exogenous stimuli produced by surrounding tissue architecture [85,91]. From this point of view, in vitro physiological models have been variously applied to discover cell–drug interactions, evaluate target organ toxicity arising from exposure to drugs or other external agents and study cancer metastasis in a disease model [92]. Moreover, the definition of novel in vitro model also contributes to the reduced use of animal models to test drug toxicity and tumors ingrowth in vivo. The current state-of-the-art shows that most cancer and tumor biological studies are mainly conduit by 2D cell models (i.e., 2D monolayer cell models cultured on tissue culture plates). However, the interactions between cells–cells and cells– ECMs in true physiological tissues are difficult to mimic with 2D models due to insufficient structural, mechanical and biochemical cues. In most cases, 2D models show several drawbacks related to the appropriate transport of fluids and molecules, which negatively influence the natural cell crosstalking in the tumor microenvironment [93]. This is corroborated by many studies that underlined a different behavior of cells in a 3D model in terms of cell function [85], differentiation [85,94], drug metabolism [85,95], gene expression and protein synthesis [85,96], morphology [85,97], proliferation [85] and viability [85,97]. Compared to 2D-cultured tumor cells, cells from a 3D culture display a decreased sensitivity to apoptosis and to cytostatic or cytotoxic effects of chemotherapeutic agents [98–102], showing a more accurate control of tumor cells activity in vitro. For these reasons, 3D in vitro tissue models are increasingly proposed in biomedical engineering research, TE, disease study and new drug discovery. Fundamental design principles for creating 3D in vitro tissue models from the bioengineering perspectives include matrix design, ECM replication and the control of molecular gradients and chemical signaling [103,104]. Based on these principles, an ideal 3D in vitro cancer model should present the following characteristics: reflect the invasive behavior of tumor reproducibly, be capable of mimicking the 119

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tumor–stromal cell interaction and enable the investigation of unknown tumor–stromal feedback mechanisms [85].


An ECM analog for tumors

In the past decade, novel materials and technologies have enabled fundamental improvements in applied cancer researches, opening a new vision of the problem of tumor invasion and metastasis. They mainly include the ability to influence the controlled but complex in vitro culture conditions and to model and predict complex processes in vivo, by integrating advanced clinical outcomes with novel engineering approaches based on scaffold technologies [89]. The first step toward properly designing a model culture system for tumor cells consists of the reconstruction of the characteristic 3D architecture of the native tissue to successfully investigate the pathobiology of the disease [105]. In particular, tumor invasion is a dynamic, complex and multistep process that has a central role in metastasis. As a common in vivo tissue of the human body, a tumor is composed of two main constituents: cells, which regulate body processes and perform normal maintenance and wound healing, and the ECM. The chemical and structural complexity of the tumor ECM dynamically influences both phenotypic and other cellular behavior by providing indirect and direct signaling cues that are generally different with respect to the healthy tissue [106]. Thus, in order to achieve a control of tumor progression, it is extremely necessary to ex novo build an ECM-like analog that reproduces more closely in vivo environment (i.e., chemical composition, morphology and surface functional groups) [106] and changes itself coherently with pathological tissue and more promptly than traditional scaffold for TE. Micro and nanostructured scaffolds used in TE have to act as temporary templates to mimic the extracellular matrix prior to the regeneration of biologically functional tissue [107]. Conversely, scaffolds for cancer therapy have to collect the right combination of chemical, physical and biological properties in order to correctly drive cell activities such as adhesion, invasion, proliferation and differentiation according to the in vivo tumor progression. In this context, engineered scaffolds have been recently introduced to reproduce the physical/structural properties of ECM and to influence the release mechanisms of chemotherapeutic drugs. They are generally made of synthetic polymers with well-recognized biocompatibility and allow creating a support matrix for in vitro cell culture as well as in vivo tumor growth [85,108]. Micro/nanofabrication techniques such as EDTs may be successfully used to design new platforms with chemical and physical features able to in vitro reproduce all the main functionalities that tumor microenvironment exerts in vivo. Indeed, the well-known ability of nanofibers to mimic fibrillar structure of native ECM may be properly adapted to reproduce the tumor microenvironment, mimicking major components of the pathological milieu from both structural and functional point of view. For instance, Hartman et al. proposed the fabrication of electrospun fibers to study the in vitro progress of epithelial 120

tumor [109]. They verified that the 3D network obtained from a randomly organized matrix made of collagen type I really resembles the native tissue architecture, thus representing an interesting model to study cell phenotype in relation to the peculiar matrix organization. Alternatively, other studies also investigated two-component fibers by combining natural polymers (i.e., collagen) to synthetic polymers (i.e., PCL) in order to improve the physical properties of the scaffold (i.e., stiffness) influencing viability, proliferation, adhesion and infiltration of tumor cells in vitro [110]. Saha et al. also explored PCL electrospun fiber mats with different orientations to study the ability of structural organization and patterning to affect cell crosstalking in the case of breast cancer. They demonstrate that cells showed elongated spindle-like morphology in the presence of aligned fibers, whereas they maintained a mostly flat stellar shape in the case of random fiber organization. Different gene upregulation – TGF-b-1 along with other mesenchymal biomarkers – suggests a different mechanism of epithelial-mesenchymal transitions in response to the surrounding scaffold architecture, thus confirming the direct role of morphological and topographical cues of tumor microenvironment on cancer progression [111]. Similar outcomes have been reported by Agudelo-Garcia et al. in the analysis of migration inhibitors of glioma cells cultured on submicron-sized PCL fibers, where it is demonstrated that migration of glioma cells through nanofiber scaffolds reproduces not only the morphology but also characteristic molecular features of 3D migration providing a large pattern of gene expression as a function of fiber alignment [112]. These experimental evidences are also supported by similar studies of Girard and colleagues who developed nanofibers based on a mixture of PLGA and a block copolymer of poly(lactic acid) and monomethoxypolyethylene glycol. In this case, cancer cells formed tight irregular aggregates that are morphologically similar to in vivo tumors. Moreover, due to the peculiar fibers topography and polymer net charge, cells in 3D culture also show a higher resistance to anticancer drugs than the same tumor cells grown as monolayers, thus suggesting their use in testing chemotherapeutic drugs for personalized cancer treatment [113]. Drug delivery systems for tumors

A relevant failing point in cancer therapy still concerns the ineffectiveness of current therapeutic strategies of tumor treatment. To date, a wide range of cancer therapeutic agents and techniques, such as metabolism revision, interaction with microtubule degradation to improve mitotic arrest, reduction of cell motility and interruption of intercellular signal transmission, have been developed to effectively treat various cancers [13,114,115]. However, systemic administration (both orally and intravenously) of anticancer drugs is often associated with several drawbacks, such as poor solubility and instability of the drug in the biological environment, low concentration of the drug around the tumor site, low efficacy for solid tumors, undesired side effects on healthy tissues such as neutropenia or cardiomyopathy and high rate of elimination by the Expert Rev. Med. Devices 12(1), (2015)



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reticulo-endothelial system [114,116,117]. Formulations based on liposomes, micelles, hydrogels and nanoparticles have been explored to deal with these problems, leading to more efficient, safe, viable, sustained and even-targeted drug delivery systems for cancer chemotherapy. But the common polymeric drug delivery systems like nano- or microspheres, liposomes and hydrogels often shows sharp burst drug release and sometimes low drug-loading efficiency because of the hydrophobic nature of the drug [114,116,118]. EDTs-based devices have demonstrated their efficacy Absorption Entrapment Intrafiber Interfiber with respect to traditional techniques (i.e., emulsion, drying) to develop drug delivery Figure 4. Schematic picture of different mechanism of drug encapsulation in vehicles since they have a unique functionelectrodynamic technologies platforms. ality and inherent morphological characteristics at submicrometric scale [119]. In comparison with emulsion techniques, they avoid high shearing multiple scale or retarded release mechanisms, EDT conceives forces related to the use of stirring or sonication methodologies more complex systems with interfibers or intrafibers encapsulaor problems due to the removal of residual products (i.e., surfac- tion of drug based on less time-consuming release strategy. In tant), preserving the chance to obtain high encapsulation effi- the former case, drugs may be included by the design of core– shell structures that drastically reduce the presence of starting ciency and high loading level [120]. The large variety of EDT techniques (i.e., electrospraying, burst effect – that is, near zero-order release kinetics – so avoidelectrospinning, coaxial electrospinning/spraying, dual electro- ing undesired loss of encapsulation efficiency and loading spinning, spraying or spraying/spinning) and the flexibility of capacity [127]. In the latter one, different drug encapsulated its material processing conditions allow fabricating various mul- polymeric systems, that is, nanoparticles, nanofibers, may be tiple composition systems with different structural architectures combined, chemically bound or simply adsorbed [128], to better and different ways of administration [121]. In EDT devices, control encapsulation and release times to achieve a more unidrug release mechanisms may be associated to two different form dispersion of drug with high loading capacity and miniphysical mechanisms involving polymer degradation and/or mal drug loss [119]. All the reported strategies enable designing drug delivery complex diffusion pathways along mesospaces into the fibers and/or micro or submicrospaces within nanofiber mesh. So, devices suitable for cancer therapy. For instance, antineoplastic drug release profiles can be properly set by the definition of agent delivery systems based on nanofibers are successfully used various parameters referred to polymer formulation (e.g., one for the treatment of malignant gliomas (a type of brain or more components with different relative ratios), their spatial tumor) [129]. Otherwise, EDTs-based drug-loaded systems have distribution (e.g., blends, surface coating) and, most impor- been variously used for the treatment of tumors due to their tantly, different drug encapsulation methods that concur to great capability in facilitating both passive and active drug tarmore finely control release kinetics. Accordingly, several strate- geting [114]. For instance, paclitaxel-loaded PLGA microspheres gies based on physical or chemical methods may be recognized have been entrapped in atomized alginate hydrogels to extend as summarized in FIGURE 4. The simplest mechanism is based on the release times over 60 days for a sustained release at nearthe adsorption of drug onto the electrospun fibers by exposing zero-order kinetics with low initial burst release [114,130]. Alterthem to a drug solution. In this case, the adsorbed drug is natively, micro/nanoparticles have been safely and precisely often loosely attached, promoting the formation of release pro- inserted by stereotaxy for targeting into the brain [114,131] or files with a prominent burst that often do not meet the specific used as coating for local tumor chemotherapy by single-/multirequirement for long drug delivery applications, but is generally layered stenting [114,132]. The main advantage of electrosprayed referred to fast release applications. Alternatively, drugs may be particles in drug-delivery applications is their more efficient dissolved or suspended in polymer solution to generate loaded uptake by cells for internalization due to the surface charges of fibers by specific evaporation of solvents controlled by tailored particles and influencing the tumor cell response by transferring process conditions prior to reach the solidification [122–125]. It bioactive agents across the cell membrane [114,133,134]. may occur by the support of chemical or ionic cross-linking of polymer into a liquid bath containing the binder, which allows Expert commentary for the entrapment of drug contextually to the formation of In the various and heterogeneous scenario of scaffold technolothe fiber/droplet [126]. In the case of application involving gies discovered in the last 20 years, EDTs certainly represent informahealthcare.com

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the most promising technologies to produce micro and/or nanotextured scaffolds comprising micro/nanofibers and/or micro/nanoparticles for extensive use in TE, drug delivery and cancer therapy. Overcoming the limitations of traditional approaches based on templating and rapid prototyping techniques, EDTs allow manipulating materials properties down to the nanoscale, which is the most realistic scale to mimic the architecture of extracellular matrix of natural tissues [135]. For instance, electrospun fibers are assembled similarly to native collagen fibrils, which represent the real structural scaffold of all natural tissues from softer (i.e., skin, cornea and ligaments) to hardest (i.e., bone) ones. For this reason, in the past two decades, new technological variants to the basic principle of EDTs have been implemented to fabricate engineered tissues that not only resemble the native tissues but also confer innovative properties to support the main biological functions of cells during the regeneration. For instance, the high surface to volume ratio of fibers or particles at the nanometric scale may be highly useful to improve drug loading, while full pore interconnection along fiber meshes assure the presence of free percolative pathways to support nutrient and waste exchange during tissue regeneration [136]. Moreover, fiber/particle diameter scaling from micro to nanometric scale is easy to obtain by properly controlling the synergistic contribution of a large number of variables relating to polymer solution properties and process parameters, which ultimately control the final fiber characteristics [137]. Likewise, it is also possible to finely control characteristic size distribution (i.e., diameters), thus enabling the production of strictly narrow fiber distribution or quasimonodisperse particles [22,119]. Submicrometric size of fiber and/or particles may importantly affect cell materials interaction mechanisms and, ultimately, the fine control of size distribution may lead to different biological responses [138,139]. Several groups are including topographical patterns within 3D porous scaffolds [140] or combining topographical and chemical cues [141], thus taking advantage of the effect of cell material interaction in the fabrication of novel tissues. In fact, an emerging issue is the control of cell material interaction at microand nanoscale through the realization of elementary units including cells and material tassels as starting point for the expansion of 3D more complex structures. In this direction, the realization of ‘bio-inspired’ scaffolds based on the assembling of (micro or nano) building blocks are powerfully emerging due to their ability to better mimic the natural tissue generation process [142,143]. Similarly, EDTs allow for the scaffold fabrication by the assembly of elementary tassels, namely fibers or particles, eventually endowed with molecular signals, which can be spatially organized to form hierarchically complex structures, able to recapitulate the functional properties of natural complex structures. In this case, EDT units may be designed by including complex information coded in their physical and chemical structures, which influence most of the cell activities [144]. For instance, the characteristic size scale of fibers may influence the adhesion and proliferation mechanisms of hMSC. In the case of submicrometric fibers, high specific 122

surface area is generally better recognized by cells, being more reactive to the adhesion ligands [39]. Meanwhile, the presence of highly crystalline surfaces concurs to promote a more slow proliferation of cells [145]. In summary, thanks to the proper setting of polymer solution properties (i.e., polymer concentration, solvent permittivity), it is possible to modify, in turn, macroscopic (i.e., the extent of fiber curvature) and microscopic (i.e., degree of crystallinity) properties of fibers that differently affect the cell response [39]. More recently, other studies also demonstrated the capability of electrospun/electrospray systems with nanometric sizes to influence complex cellular functions, including oxygen metabolism, mainly responsible of the way by which cells acquire energy from the environment, and internalization mechanisms mediated by cellular membrane. It has been reported that material nature and topography might influence oxygen consumption rates probably as function of integrinmediated recognition mechanisms [146]. Accordingly, it has been demonstrated that electrospun fiber sizes with submicrometric size scale may directly support oxygen metabolism of hMSC cells, suggesting a morphologically driven control of cell biosynthetic activity for a tunable regeneration of natural tissue that is directly guided by the electrospun scaffold properties [147]. Despite a large number of in vitro studies reported in literature, more attention must be focused on the successful in vivo outcomes by using electrospun platforms. They underline how in vivo response is affected by several factors related to the peculiar features of electrospun implants (i.e., thickness, stiffness, etc). For instance, to achieve a successful in vivo application of electrospun conduits as blood vessels, they should first resist a thrombotic response. Failure to resist thrombosis can result in acute graft occlusion or thromboembolism as demonstrated by Hashi et al. with poly(lactic acid) electrospun vascular grafts [148]. Overcoming the confined vision of using scaffolds only to regenerate ex novo tissues, electrospun fibers are currently investigating 3D models to assess tumor cell biology in vitro. This approach is particularly interesting because it allows crossing the intrinsic limitations of 2D culture systems for mechanistic studies of standard chemotherapies and/or biologically targeted therapies toward a more efficient preclinical investigation of tumors in vivo. In vitro strategies to study cellular behavior in complex environments have been historically plagued by the use of flat substrates that are far from replicating in vivo conditions and show a lack of quantitative tools to study the mechanical and biochemical feedback between cells and the surrounding matrix. Conversely, using in vivo study leads to highly qualitative understanding of the biology, but also to a poor control of key cellular or extracellular regulators of invasion [149]. Nevertheless, this emerging approach is rather complex because it requires to adapt the multiple variables typically inherent in scaffold design for TE to the features of 3D culture models that better satisfy the mechanisms of tumor ingrowth, still partially unknown. Hence, it is required that synthetic scaffolds might reproduce ECM-like elements with peculiar biochemical and biophysical cues able to support changes Expert Rev. Med. Devices 12(1), (2015)



occurring to cellular phenotype and molecular and gene pathways, providing an experimental model that truly reflects native functions of in vivo environment. Several studies have widely underlined differences in signaling pathways in 3D tumor models based on the use of spheroid culture systems [150]. However, spheroids are nonadhesion-mediated systems, which mean that they prohibit cellular attachment on them, only inducing cells to autonomously aggregate until they form a 3D tumor-like tissue. Alternatively, multicomponent gels enriched with aspecific molecular signals such as Matrigel have been largely used in the place of scaffolds to provide a dense media with morphological (i.e., porosity) and physical (i.e., stiffness) properties compatible with those of tumor microenvironment. However, data taken from 3D culture in Matrigel are frequently contrasting due to tremendous heterogeneity of local composition of the gel, thus making it impossible to compare results over different days and between different research groups [151]. Contrariwise, EDTs offer the unique and highly reproducible set of techniques to control the tumor architecture to directly affect basic cell-to-cell and cell-to-ECM cross-talking. Besides, electrospun fibers reveal a number of characteristics that more closely resemble those observed in vivo, reflecting the 3D tumor architecture and ECM properties, including stiffness and the in vivo tumor milieu [152]. Electrospun fibers with tunable mechanical stiffness can be fabricated by setting suitable materials and process conditions in order to regulate tumor progression by altering focal adhesion formation and integrin signaling, strictly dependent upon matrix mechanical properties [153]. A fine control of fiber mesh size at the molecular scale not only contributes to restoration of structurally complex in vivo-like cell to cell contacts, but also allows influencing the cell resistance to conventional chemotherapeutic drugs (i.e., doxorubicin) homogeneously dispersed in the culture medium, so reducing the side effects of cell death related to local drug overdosing [154]. In this direction, electrospinning and other EDT techniques are particularly suitable for a more controlled and highly reproducible release profile of chemotherapeutic drugs, thanks to a fine optimization of fiber/particle sizes (e.g., monodispersity); furthermore, they guarantee a more easy device customization as function of the clinical use. The high process versatility in terms of polymers and solvents to be used in combination with therapeutic molecules assures a more accurate prevention of molecular degradation/denaturation with respect to traditional encapsulation techniques. The efficacy of EDT techniques is due to the incorporation of therapeutic molecules into the polymer solution prior to the fiber/particle processing, only applying external forces generated by electric fields, which do not basically alter the chemistry of molecular factors, thus offering numerous advantages with respect to other traditional techniques. No use of high temperature or further drying steps is required since fibers or particles may be instantaneously dried by natural convection during the deposition. This assures also the encapsulation of a large variety of therapeutic molecules such as growth factors [155,156], antibiotics and anticancer agents [157]. The relevant shortcoming of direct incorporation of growth factors into EDT scaffolds due to informahealthcare.com

Review

drawbacks in terms of low reproducibility and insufficient control of drug release profiles is ascribable to the frequent tendency of drug aggregation in the solution, which limits distribution homogeneity of molecular factors [158]. For instance, the use of polar molecules such as NGF may negatively affect mesh properties and loading efficiency ascribable to amplified instabilities phenomena occurring between biomolecules and polymer chains with high charge densities [159]. Hence, relevant efforts are underway to find a new solution in the design of EDT devices – that is, core–shell systems or nanoparticle endowed fibers – able to create controlled kinetics of drug release tailored for the specific application. Five-year view

A key goal in developing biomimetic matrices is to engineer a 3D matrix able to morphologically and chemically mimic natural tissues. Despite substantial and progressive innovation in biomaterials that have been synthesized to investigate the response of healthy or tumor cells behavior, several major challenges still remain. First of all, more knowledge is required to understand how novel biomaterials influence the cellular mechanical and biochemical processes simultaneously. Biomaterials may be also degraded by cells activities, thereby further influencing the mechanical properties and remodeling of the extracellular matrix analog. Recent advances in biomaterial science and technologies suggest the use of multicomponent or hybrid systems in which natural biomaterials are used alongside synthetic ones in order to develop active sites [95] able to reproduce a nutrient-rich microenvironment in which cells might behave in response of structural and biochemical cues as in native-like 3D context [160]. They generally involve the use of post-treatments to increase surface properties (i.e., hydrophilicity) in order to improve the cell–material interfaces [161]. In this direction, EDTs-produced fibers or particles may be widely modified by various 3D surface treatments to add other valuable features of ECM, including plasma treatments, wet chemical method or surface graft polymerization [162]. Alternatively, different methods to functionalize biomaterial surface involve the use of proteins or peptides that show advantageous cost/ benefit ratio maintaining key biological functionality [163]. For example, surface-modified nanofibrous scaffold by conjugated polymers (i.e., heparin, polysaccharides) is a valid solution to efficiently mimic native tissue composition due to their ability to efficiently interact with bioactive molecules (i.e., DNA, RNA and growth factors) via negatively charged groups on its backbone [164,165]. Similar approaches are being proposed successfully in cancer therapy where the control of morphology at the nanoscale concurs to better model the full range of microenvironmental cues naturally elicited by 3D cell–cell and cell–extracellular matrix interactions. In particular, Fong et al. demonstrated the ability of electrospun PCL fibers to strongly influence growth and survival of bone sarcoma by restoring structurally complex cell-to-cell contacts similarly to in vivo model. In this case, cells strictly embedded in the 3D fiber network may be also protected by an excessive exposure to 123


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chemotherapeutics, acting as a barrier to molecular transport to the surrounding cells [152]. The greatest impact of EDTs is mainly referred to design multifunctional devices able to model, not only morphology, but also protein expression and drug pharmacokinetic profiles in in vivo human tumors. Recent advances in protein engineering and materials science are opening new possibilities to optimize novel nanoscale targeting approaches to overcome relevant shortcoming of current tumor therapies such as the reduced efficacy of conventional chemotherapeutic drugs in the tumor milieu [166]. Nanoparticles and/or fibers processed by EDTs may assure a more efficient targeted delivery of drug molecules to diseased areas, offering many advantages such as protection from degradation, selective and improved absorption into a selected tissue and control of the pharmacokinetic and drug tissue distribution. Given the optimization capacity of EDTs processes, novel solution to design 3D models with growing complexity are emerging, which include nonspherical shape [167], co-cultures and/or nontumorigenic cells for short-term and long-term investigations. They will enable investigation of the influence of recently discovered mechanisms (i.e., autophagy) strongly implicated in the positive outcome of tumor response [168]. Previous studies demonstrated that autophagy modulation mediated by nanoparticles may act as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anticancer agent [169]. Likewise, EDTs have been recently applied to gene therapies to

fight the tumor pathology by upregulation or downregulation of target genes [170]. The success of gene therapy applied to EDTs mainly depends on the efficient delivery of nucleic acids into the cells [171]. With respect to conventional vectors (i.e., viruses) having short half-lives due to nonimmunogenic recognition of foreign nucleic acids, EDTs-based devices allow circumventing these problems by allowing the design of nucleic acids grafted systems made of ionic polymers able to interact with cells by a selective degradation of binding sites [172]. All these recent studies suggest that EDTs may epitomize in the near future interesting tools to engineer nanostructured platforms in order to study in vitro as in vivo cancer and to clarify the role of some important issues such as oxygen consumption, nutrient and molecular transport on the tumor phenotype and drug efficiency. Financial & competing interests disclosure

This study was financially supported by REPAIR (PON01-02342), MERIT (FIRB-RBNE08HM7T), POLIFARMA (PON02_3203241) and NEWTON (FIRB-RBAP11BYNP). Scanning Electron Microscopy was supported by the Transmission and Scanning Electron Microscopy Labs (LAMEST) of the National Research Council. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Key issues • There is a tremendous need for new 3D cellular models enabling a thorough understanding of biological processes at the base of healthy and pathological tissue and organ development. • The scientific community pointed out their next-generation guidelines, underlining the necessity of complex in vitro biomimetic models able to finely predict biological mechanisms occurring in vivo. • Instructive-platform inspired tissue engineering approaches promise new relevant discoveries to learn about fundamental cell–biomaterial and cell–cell cross-talking elicited during regenerative processes. • Electrodynamics are processing techniques with incomparable versatility and reproducibility, able to mimic the innate complexity of tissue microenvironment by a sage manipulation of several features (e.g., morphology, mechanical properties, degradation, oxygen and molecular transport). • Electrodynamic technologies platforms currently represent valid tools not only for temporary mimicking structural and biochemical properties of native extracellular matrix during the tissue regeneration, but, most interestingly, for the investigation of tumor development and resistance to current chemotherapeutic therapies.

material: structure and function. Acta Biomater 2009;5(1):1-13

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129

Repair and tissue engineering techniques for articular cartilage.

Electroactive Tissue Scaffolds with Aligned Pores as Instructive Platforms for Biomimetic Tissue Engineering.

Abstract 88: microvascular integration into versatile tissue engineering platforms.

Going with the flow: microfluidic platforms in vascular tissue engineering.

Click hydrogels, microgels and nanogels: emerging platforms for drug delivery and tissue engineering.

Sterilization techniques for biodegradable scaffolds in tissue engineering applications.

Ischemic preconditioning for cell-based therapy and tissue engineering.

Osteochondral tissue engineering with biphasic scaffold: current strategies and techniques.

Engineering alternative isobutanol production platforms.

TISSUE ENGINEERING PERFUSABLE CANCER MODELS.

Non-viral gene therapy for bone tissue engineering.

Engineering virus-like particles as vaccine platforms.

Ultrasound Imaging Techniques for Spatiotemporal Characterization of Composition, Microstructure, and Mechanical Properties in Tissue Engineering.

Tissue engineering a surrogate niche for metastatic cancer cells.

Improved fabrication of melt electrospun tissue engineering scaffolds using direct writing and advanced electric field control.

Cardiac tissue engineering and regeneration using cell-based therapy.

Rapid manufacturing techniques for the tissue engineering of human heart valves.

[Stem cell therapy and tissue engineering in regenerative urology].

Tissue Engineering for Pediatric Applications.

3D Printing for Tissue Engineering.

Advanced T-Cell Engineering for Cancer Therapy (ATECT).

Cell and tissue engineering for liver disease.

Manufacturing Techniques and Surface Engineering of Polymer Based Nanoparticles for Targeted Drug Delivery to Cancer.

Bone tissue engineering for dentistry and orthopaedics.