Polymer-based scaffolds for soft-tissue engineering

polymers Review Polymer-Based Scaffolds for Soft-Tissue Engineering Victor Perez-Puyana 1, Mercedes Jiménez-Rosado 2, Alberto Romero 1,* and Antonio Guerrero 2,* 1Departamento de Ingeniería Química, Facultad de Química, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] 2Departamento de Ingeniería Química, Escuela Politécnica Superior, Universidad de Sevilla, 41011 Sevilla, Spain; [email protected] *Correspondence: alromer[email protected] (A.R.); aguerrer[email protected] (A.G.); Tel.: +34-9-5455-7179 (A.R.) Received: 16 June 2020; Accepted: 13 July 2020; Published: 15 July 2020   Abstract: Biomaterials have been used since ancient times. However, it was not until the late 1960s when their development prospered, increasing the research on them. In recent years, the study of biomaterials has focused mainly on tissue regeneration, requiring a biomaterial that can support cells during their growth and fulfill the function of the replaced tissue until its regeneration. These materials, called scaffolds, have been developed with a wide variety of materials and processes, with the polymer ones being the most advanced. For this reason, the need arises for a review that compiles the techniques most used in the development of polymer-based scaffolds. This review has focused on three of the most used techniques: freeze-drying, electrospinning and 3D printing, focusing on current and future trends. In addition, the advantages and disadvantages of each of them have been compared. Keywords: freeze-drying; electrospinning; 3D printing; bioactive scaffolds; tissue engineering 1. Introduction A biomaterial is defined as any material used to produce devices that can replace a part or function of an organism in a safe, economical and physically plausible way; although it can also be referred as that material of biological origin or obtained from biological materials [ 1 , 2 ]. Nevertheless, the most accepted definition was given by William in 1987 as “a non-viable material used in a biomedical device intended to interact with biological systems” [3]. Nowadays, applications for the use of biomaterials include aesthetics, trauma damage and congenital or degenerative diseases, with biomaterials becoming a necessity since human beings want to live longer and better [ 4 ]. Currently the average life expectancy in the world is greater than 73 years; it is estimated that more than 900 million people are over 60 years old and by the end of the 21st century, according to the United Nations Organization (UNO), this digit will triple [ 5 ]. The search for improvements in the quality of life and the increase in life expectancy has made it possible for modern society to witness scientific and technological progress in various fields in recent years. The modern field of “biomaterials” is relatively new to be able to compile its history. The first great advances took place at the beginning of the 20th century with the first vanadium steel alloys for fracture fixation or threads capable of being absorbed and degraded by the body. Since the 1960s, the implementation of protocols and statistical analysis of the results, the application of techniques to characterize the structure and surfaces of materials lead to an exponential development of the field of biomaterials with the first definition of biomaterial in 1974 and the creation of the Society for Biomaterials one year later. Since the 1970s, the cooperation of medicine with basic sciences (biology, physics and chemistry) and engineering has been the definitive impulse for the development of biomaterials [6]. Polymers 2020,12, 1566; doi:10.3390/polym12071566 www.mdpi.com/journal/polymers Polymers 2020,12, 1566 2 of 21 Each biomaterial is defined by three factors: characteristics (composition and structure), properties (response of the material in real working conditions) and behavior (response to changes in the environment that surrounds it). The introduction of implants in the human body can activate the immune system, hence the importance of the concept of biocompatibility. Biocompatibility, which is the ability of a material to have an adequate response in a specific application (resistance to blood clotting, bacterial growth and normal healing) with an adequate response from the host (patient). Therefore, biomaterials must be efficient devices that do not generate disorder in the metastable process of the living being or the performance of the device in the inserted environment for an indeterminate period [7]. Biomaterials can be classified in different ways according to the response of the tissue or body to the implant: in case the tissues die, the material is considered toxic, if it forms fibrous tissue, the material is considered inert, if it promotes a link interface, it is considered bioactive and, finally, if it promotes tissue replacement, the material is considered soluble. At the beginning of the use of biomaterials in a more systematic way, in the 1950s, the search focused on bio-inert materials (1st generation). Over time, the search turned to the bioactivity of materials (2nd generation), and more recently, the focus has been on regeneration of true functional tissue (3rd generation), focusing more on the body’s response than the biomaterial’s response, which is to perform the function for which it has been designed with the minimum biological response of the patient. Efforts were devoted to increasing the life of the implant through its interaction with the host tissue interface, then the focus was on the development of biodegradable materials capable of being incorporated or absorbed (after dissolution) by the host tissue, and lately, the concept of biomimetics (4th generation), searching for materials that actively participate in the recovery process, working on the tissue in a specific way, with stimulation at the cell level [8,9]. Biomaterials can also be classified according to their duration, being provisional (temporary use) or definitive (permanent use), as well as their mechanical properties that make them act as hard tissue or soft tissue. However, the best known and most widely used classification, nowadays, is the one that classifies biomaterials according to their origin, as they can be natural or artificial. The latter, depending on the nature of the material, can in turn be classified as metallic, ceramic, polymeric and composite materials [10]. Tissue engineering is an interdisciplinary field that fundamentally requires cultured cell technology. Thus, it utilizes growth factors which allow the precise and continuous control of cell growth conditions and materials engineering for the development of scaffolds (biomaterials) that can imitate the structure of an organ [ 11 , 12 ]. A scaffold is a matrix whose main function is to serve as an anchoring platform for the adhesion of cells and, thus, allow their growth and proliferation, give rigidity to the implanted tissue and provide an empty volume for vascularization [ 13 , 14 ]. However, it must also fulfill other functions such as transporting, storing and releasing active factors, as well as stimulating specific cellular responses, contributing to the structural and mechanical integrity of the treated region. Therefore, characteristics such as external geometry, surface topographic characteristics (roughness or hydrophilic-hydrophobic character) and the scaffold microstructure (pore size, porosity and pores interconnectivity) influence cell-scaffold interactions. Furthermore, the biocompatibility, degradation and mechanical properties of the scaffold play a key role since they affect both the formation of tissue in vitro and its viability and functionality once inserted. Scaffolds must maintain the physical integrity and stability necessary to support the sterilization process and the ability to be stored for a long period of time. Lately, efforts have been made to evaluate the effect of the chemical and structural characteristics of scaffolds on cellular behaviors, that is, on cell adhesion, proliferation, migration and differentiation [15,16]. A suitable raw material to obtain scaffolds with an optimal internal structure has a positive effect on the activity of cells, so it is evident that the selection of a suitable raw material is crucial for an optimal cell growth. In this way, scaffolds can be either natural or synthetic, but it is essential that they exhibit their specific properties throughout the entire runtime [ 17 – 19 ]. Among the most used Polymers 2020,12, 1566 3 of 21 materials we find metallic materials, ceramic materials or polymers. This last type is probably the most versatile group of biomaterials used in the fabrication of biomedical devices. This type of material allows scaffolds to be processed with adequate control of the architectural parameters such as pore size and shape, porosity and pore interconnectivity, wall morphology, and surface area which are key issues for cell seeding, migration, growth, mass transport and tissue formation. The main drawback is the lack of hydrophilicity for cell adhesion, as well as its limited biocompatibility [20,21]. In general, synthetic polymers are divided into two types as biodegradable and non-biodegradable materials. Certainly, the biodegradable ones present the greatest application in tissue engineering. The degradation process of biodegradable polymers is the absorption of water and hydrolysis, as well as the enzymatic cleavage of the polymer chain [ 22 ]. Among the different synthetic polymers, the most used are polyα -hydroxy esters such as polylactic acid (PLA), polyglycolide (PGA) and polycaprolactone (PCL), among others [ 22 – 29 ]. On the other hand, natural polymers (biopolymers) are widely used in tissue engineering due to their structure and properties where they combine low antigenicity and inflammation with high affinity for water and adequate cytotoxic responses, biocompatibility, biodegradability, and muco-adhesiveness. Biocompatibility increases the influence of the cell on the scaffold to regenerate damaged tissue. However, the poor mechanical and structural properties of these polymers prevent their widespread use. The most popular natural polymers are polysaccharides and proteins. Regarding the proteins to be used in scaffolds, it is worth mentioning collagen or gelatin, elastin, fibrin and even silk fibroin. Among the polysaccharides, starch, alginate, chitosan and derivatives of hyaluronic acid stand out [20,30]. There are numerous fabrication techniques for the development of polymer-based scaffolds: the ones based on 3D printing, such as rapid prototyping [ 23 , 31 , 32 ]; solvent-based techniques, such as solvent casting [ 33 ] or phase separation [ 34 ]; and others that are more specific, such as self-assembly [ 35 ]. Among them, one of the most traditionally used techniques is based on the formation of highly porous scaffolds whose microstructure is formed by either nanofibers or interconnected pores [ 36 , 37 ]. However, in the recent years, emerging technologies have acquired relevance in the field due to the possibility to produce highly controlled scaffolds in terms of mechanical and morphological properties. Microfluidics and 3D printing techniques are examples of those emerging techniques that allows obtaining a defined geometry, high control on the uniformity in the pore size and a good interconnectivity [37–44]. Thus, the aim of this work is the description of different processing techniques to develop polymer-based scaffolds. Specifically, a comparison between the traditionally used technique to produce highly porous scaffolds (freeze-drying), the most versatile technique to produce polymer nanofibrous scaffolds from almost any polymer which can be solved or melt (electrospinning) and the most innovative technique to process either acellular or cell-laden scaffolds (3D printing) has been considered as the main topic of this review. Therefore, freeze-drying, electrospinning and 3D printing are described with a general overview of each process together with their current trends. Finally, a comparison of the evolution of the techniques and the future perspectives are included as conclusions. 2. Freeze-Drying Although other techniques like 3D printing allow obtaining scaffolds with better structural properties, freeze-drying technique allows obtaining 3D porous scaffolds with a global porosity higher than 90% and pore sizes in the range between 20 and 400 µ m [ 45 ]. It is based on the freezing of a polymer solution followed by the sublimation of the solvent, obtaining matrices with an interconnected porous microstructure. The evolution of the technique over the years can be seen in Figure 1. Its origin is found in 1909 when Shackell freeze-dried different biological materials. It is important to mention that it was not until 1927 when the first patent was registered by Tival. Later, in 1934, Flosdorf prepared the first stable structures via freeze-drying. However, it was in 1990 when De Groot et al. developed the first composite based on combinations of polyurethane and poly(l-lactic acid) (PU/PLLA) for tissue Polymers 2020,12, 1566 4 of 21 engineering [ 46 ]. Five years later, the first freeze-dried scaffold was produced [ 47 ]. Since then, the application of this technique in tissue engineering has grown exponentially [37,48]. Polymers 2020, 12, x FOR PEER REVIEW 20 of 20 Figure 1. Timeline of the evolution of freeze-drying over history. 2.1. Technique This technique consists in the formation of a porous structure from a polymer solution by generating the conditions for its thermodynamic destabilization. The solution is separated into a polymer-rich phase and a polymer-depleted phase. The process typically starts with a cooling stage below the freezing point of the solvent, favoring the formation of crystals, until it is completely frozen. The second stage is carried out by means of a solvent removal process by freeze-drying. Thus, the polymer-rich phase tends to form the walls of the scaffold, while the polymer-depleted phase is the one that generates the pores in the internal structure of the acellular scaffold. The main advantage of this process is that the scaffold morphology (pore size and distribution) can be controlled by parameters such as the type of polymer, the concentration of polymer and the freezing temperature since they can regulate the nucleation mechanism and solvent crystal growth. Other additives, such as some surfactants or porogens, can contribute to improving morphological properties. High porosities and interconnectivity can be achieved with this technique, facilitating tissue development. However, the pore size that is usually achieved in this type of matrices is around the lower limit for its application in tissue engineering (TE). Furthermore, despite being a relatively simple technique of growing interest, its use is constrained to a small group of polymers, as well as to the lab-scale. The polymers that have been most used to prepare scaffolds using this technique have been synthetic polymers such as PLA, PGA, or poly(lactic-co-glycolic acid) (PLGA), as well as water-soluble biopolymers such as collagen and chitosan [49]. Considering the formation of hydrogels or sponge-like scaffolds, there are differing processing parameters to take into account. The most important parameters involving the formation of hydrogels are the gelation time, pH and the gelation temperature, among others. In contrast, the formation of sponge-like structures requires the control of other parameters such as the container used, the freezing temperature or cooling rate, and the pH of the solution or the solvent used. 2.2. Current trends Regarding current trends in the fabrication of scaffolds via the freeze-drying method, the main concern is to define the biomaterial microstructure in order to optimize cell adhesion, proliferation and differentiation. As commented before, the process has been developed in two different lines aimed at the formation of hydrogel-like (intermediate product) and sponge-like (final product) biomaterials. The use of this type of biomaterial is very complex due to its poor mechanical resistance that makes it difficult to apply it to bioreactors, which mechanically stimulate cell growth and proliferation. This problem is solvable in two different ways. On the one hand, innovation in the field of hydrogels is found in the use of the nanoparticles that are included in the initial formulation to originate hydrogels. Among the different types of nanoparticles, magnetic nanoparticles that allow the stimulation of the biomaterial through the use of magnets (magnetic field) are especially interesting, thus avoiding the mechanical action that damages the structure of the material. In this way, a synergy occurs between the nanoparticles and the hydrogel, thus enhancing their use in regenerative medicine. The studies of Tanasa et al. (2020) and Shuai et al. (2020) follow this line since they are based on the development of scaffolds stimulated with either a magnetic field or a proper magnetic micro-environment [50,51]. The former Figure 1. Timeline of the evolution of freeze-drying over history. 2.1. Technique This technique consists in the formation of a porous structure from a polymer solution by generating the conditions for its thermodynamic destabilization. The solution is separated into a polymer-rich phase and a polymer-depleted phase. The process typically starts with a cooling stage below the freezing point of the solvent, favoring the formation of crystals, until it is completely frozen. The second stage is carried out by means of a solvent removal process by freeze-drying. Thus, the polymer-rich phase tends to form the walls of the scaffold, while the polymer-depleted phase is the one that generates the pores in the internal structure of the acellular scaffold. The main advantage of this process is that the scaffold morphology (pore size and distribution) can be controlled by parameters such as the type of polymer, the concentration of polymer and the freezing temperature since they can regulate the nucleation mechanism and solvent crystal growth. Other additives, such as some surfactants or porogens, can contribute to improving morphological properties. High porosities and interconnectivity can be achieved with this technique, facilitating tissue development. However, the pore size that is usually achieved in this type of matrices is around the lower limit for its application in tissue engineering (TE). Furthermore, despite being a relatively simple technique of growing interest, its use is constrained to a small group of polymers, as well as to the lab-scale. The polymers that have been most used to prepare scaffolds using this technique have been synthetic polymers such as PLA, PGA, or poly(lactic-co-glycolic acid) (PLGA), as well as water-soluble biopolymers such as collagen and chitosan [49]. Considering the formation of hydrogels or sponge-like scaffolds, there are differing processing parameters to take into account. The most important parameters involving the formation of hydrogels are the gelation time, pH and the gelation temperature, among others. In contrast, the formation of sponge-like structures requires the control of other parameters such as the container used, the freezing temperature or cooling rate, and the pH of the solution or the solvent used. 2.2. Current Trends Regarding current trends in the fabrication of scaffolds via the freeze-drying method, the main concern is to define the biomaterial microstructure in order to optimize cell adhesion, proliferation and differentiation. As commented before, the process has been developed in two different lines aimed at the formation of hydrogel-like (intermediate product) and sponge-like (final product) biomaterials. The use of this type of biomaterial is very complex due to its poor mechanical resistance that makes it difficult to apply it to bioreactors, which mechanically stimulate cell growth and proliferation. This problem is solvable in two different ways. On the one hand, innovation in the field of hydrogels is found in the use of the nanoparticles that are included in the initial formulation to originate hydrogels. Among the different types of nanoparticles, magnetic nanoparticles that allow the stimulation of the biomaterial through the use of magnets (magnetic field) are especially interesting, thus avoiding the mechanical action that damages Polymers 2020,12, 1566 5 of 21 the structure of the material. In this way, a synergy occurs between the nanoparticles and the hydrogel, thus enhancing their use in regenerative medicine. The studies of Tanasa et al. (2020) and Shuai et al. (2020) follow this line since they are based on the development of scaffolds stimulated with either a magnetic field or a proper magnetic micro-environment [ 50 , 51 ]. The former produced silk-fibroin-based scaffolds with preosteoblasts imbibed, whereas the latter used the magnetic micro-environment for bone regeneration. On the other hand, nanoparticles with other objectives have also been studied, e.g., Aradmehr et al. (2020), in their work about the inclusion of silver nanoparticles in scaffolds based on a lignocellulosic film and a chitosan hydrogel [ 52 ]. Another example to point out is the study of Mokhtari et al. (2020), about the combination of gold nanoparticles with chitosan hydrogels in order to reinforce the structure and improve the cytocompatibility [53]. On the other hand, the structure of the sponge-like biomaterials has a high porosity (greater than 80%), making its control and definition essential for potential application in regenerative medicine (specifically in tissue engineering). To achieve it, current studies promote controlling the parameters of the freeze-drying process, specifically the cooling rate of the structure, as it will define the size of crystal formation (and, therefore, the pores of the final scaffold). In this sense, the study of Perez-Puyana et al. (2019) gave a general overview of the effect of each processing parameter on the properties of polymer-based sponge-like scaffolds [ 54 ]. Nevertheless, there are other studies that are based on the effect of a specific parameter. The most studied variable is the freezing temperature, highlighting the studies of Zamanian et al. (2014) or Reys et al. (2017) evaluating the effect of the freezing temperature on gelatin-based and chitosan-based scaffolds, respectively. Both studies obtained better properties for those scaffolds fabricated at a lower temperature [ 55 , 56 ]. On the other hand, Schwarzenbach et al. (2002) reported that the use of hydrophobic containers favors the elimination of aqueous solvents, improving the process [57]. 3. Electrospinning The beginnings of electrospinning go back to the beginning of the 20th century, being patented between 1900 and 1902 by scientists Cooley and Morton (although in those years the technique was not known by the name that is used at the moment). Later, between 1931–1944, Firmhals got 22 patents on the mechanism [ 58 ]. However, it was not until 1964 that Taylor presented his theoretical explanation of the technique, and the known “Taylor’s cone” [ 59 ]. The technique was performed to produce materials with different applications as filters, although this did not receive its name until 1990 (Figure 2). Polymers 2020, 12, x FOR PEER REVIEW 20 of 20 produced silk-fibroin-based scaffolds with preosteoblasts imbibed, whereas the latter used the magnetic micro-environment for bone regeneration. On the other hand, nanoparticles with other objectives have also been studied, e.g., Aradmehr et al. (2020), in their work about the inclusion of silver nanoparticles in scaffolds based on a lignocellulosic film and a chitosan hydrogel [52]. Another example to point out is the study of Mokhtari et al. (2020), about the combination of gold nanoparticles with chitosan hydrogels in order to reinforce the structure and improve the cytocompatibility [53]. On the other hand, the structure of the sponge-like biomaterials has a high porosity (greater than 80%), making its control and definition essential for potential application in regenerative medicine (specifically in tissue engineering). To achieve it, current studies promote controlling the parameters of the freeze-drying process, specifically the cooling rate of the structure, as it will define the size of crystal formation (and, therefore, the pores of the final scaffold). In this sense, the study of Perez-Puyana et al. (2019) gave a general overview of the effect of each processing parameter on the properties of polymer-based sponge-like scaffolds [54]. Nevertheless, there are other studies that are based on the effect of a specific parameter. The most studied variable is the freezing temperature, highlighting the studies of Zamanian et al. (2014) or Reys et al. (2017) evaluating the effect of the freezing temperature on gelatin-based and chitosan-based scaffolds, respectively. Both studies obtained better properties for those scaffolds fabricated at a lower temperature [55,56]. On the other hand, Schwarzenbach et al. (2002) reported that the use of hydrophobic containers favors the elimination of aqueous solvents, improving the process [57]. 3. Electrospinning The beginnings of electrospinning go back to the beginning of the 20th century, being patented between 1900 and 1902 by scientists Cooley and Morton (although in those years the technique was not known by the name that is used at the moment). Later, between 1931–1944, Firmhals got 22 patents on the mechanism [58]. However, it was not until 1964 that Taylor presented his theoretical explanation of the technique, and the known “Taylor’s cone” [59]. The technique was performed to produce materials with different applications as filters, although this did not receive its name until 1990 (Figure 2). Figure 2. Timeline of the evolution of “electrospinning” over history. 3.1. Technique Electrospinning is a technique that allows the formation of continuous ultrafine membranes formed by polymeric fibers. The process consists of the application of a high voltage to induce the evaporation of the solvent of a polymeric solution, leading to the formation of micrometric and nanometric fibers on a metallic surface called the collector [60]. In this technique, a high-potential electric field (around 10–30 kV) is applied between a metallic syringe needle, through which a polymer solution emerges with a controlled flow rate, and a metal grounded collector (Figure 3). The high electrical potential generates strong repulsions on the surface of the polymer solution, which constitutes the main driving force of the process, producing, first, a deformation at the outlet of the capillary needle in the form of a conical droplet (Taylor cone), followed by a sudden projection of a liquid jet, from the tip of the cone in the direction of the collector. Figure 2. Timeline of the evolution of “electrospinning” over history. 3.1. Technique Electrospinning is a technique that allows the formation of continuous ultrafine membranes formed by polymeric fibers. The process consists of the application of a high voltage to induce the evaporation of the solvent of a polymeric solution, leading to the formation of micrometric and nanometric fibers on a metallic surface called the collector [60]. In this technique, a high-potential electric field (around 10–30 kV) is applied between a metallic syringe needle, through which a polymer solution emerges with a controlled flow rate, and a metal grounded collector (Figure 3). The high electrical potential generates strong repulsions on the surface of the polymer solution, which constitutes the main driving force of the process, producing, first, a Polymers 2020,12, 1566 6 of 21 deformation at the outlet of the capillary needle in the form of a conical droplet (Taylor cone), followed by a sudden projection of a liquid jet, from the tip of the cone in the direction of the collector. Polymers 2020, 12, x FOR PEER REVIEW 20 of 20 Figure 3. Schematic illustration of the electrospinning process and the fibrous structure achieved. Subsequently, bending instability of the jet takes place, causing the formation of large loops parallelly oriented to the collector in the whipping jet zone. This zone is the one that presents the greatest contribution to the generation of nanofibers, as it leads to a severe elongation that produces fiber thinning at stretching ratios in the order of thousands [61]. Simultaneously, the solvent evaporation occurs, giving rise to the solidification of polymeric fibers, which are continuously deposited on the target collector in that it forms a nanofiber membrane, with a typical random orientation. There is good interconnectivity between the pores of the membrane, although the pore sizes achieved are usually smaller than those obtained with other techniques. The morphology of the membranes depends on many variables that can be grouped into three types: (i) properties of the solution (concentration and nature of the polymer, dielectric properties of the solvent, viscosity, conductivity and surface tension); (ii) processing parameters (voltage, flowrate, capillary dimensions, needle to collector distance and polarity); (iii) environmental factors (relative humidity and temperature). Electrospinning technique allows to obtain fibers with diameters between 2 nm and several µm with a wide variety of either synthetic polymers or biopolymers [62]. The optimization of the above variables is essential to achieve more uniform fibers with diameters at the nanoscale. In general, it is a processing technique that has acquired great relevance, in a wide variety of applications and disciplines, including TE. Thus, this technique presents a great potential for the manufacture of functional artificial tissue, thanks to electrospun polymer matrices’ ability to mimic native extracellular matrix (ECM), through its micro and nanofiber structure with interconnected pores. The effect of each parameter on the morphology of the fibers is summarized in Table 1. This technique manufactures acellular scaffolds. However, several studies are demonstrating the ability of these electrospun matrices to adhere to a wide variety of cell types, allowing their proliferation and tissue development. To date, studies have been conducted, in the field of TE, with scaffolds of electrospun matrices with functionality for the generation of epithelial or muscle tissues, bones, cartilages and blood vessels. The most commonly used synthetic polymers in tissue engineering, due to their good electrospinning behavior, their good ability to mimic ECM and their good cytocompatibility characteristics and biodegradability have been PLA, PGA and PCL or their copolymers (e.g., PLGA, PLLA or PCLL) [63–65]. Among naturally occurring polymers, the most commonly used in electrospinning processes for TE applications have been some proteins such as collagen (or gelatin), often in combination with another polymer (e.g., PCL), elastin and fibrinogen or silk fibroin. Some polysaccharides (chitosan, alginate or hyaluronic acid) have also been used, with excellent results in terms of morphological and functional properties [63–67]. Figure 3. Schematic illustration of the electrospinning process and the fibrous structure achieved. Subsequently, bending instability of the jet takes place, causing the formation of large loops parallelly oriented to the collector in the whipping jet zone. This zone is the one that presents the greatest contribution to the generation of nanofibers, as it leads to a severe elongation that produces fiber thinning at stretching ratios in the order of thousands [ 61 ]. Simultaneously, the solvent evaporation occurs, giving rise to the solidification of polymeric fibers, which are continuously deposited on the target collector in that it forms a nanofiber membrane, with a typical random orientation. There is good interconnectivity between the pores of the membrane, although the pore sizes achieved are usually smaller than those obtained with other techniques. The morphology of the membranes depends on many variables that can be grouped into three types: (i) properties of the solution (concentration and nature of the polymer, dielectric properties of the solvent, viscosity, conductivity and surface tension); (ii) processing parameters (voltage, flowrate, capillary dimensions, needle to collector distance and polarity); (iii) environmental factors (relative humidity and temperature). Electrospinning technique allows to obtain fibers with diameters between 2 nm and several µ m with a wide variety of either synthetic polymers or biopolymers [ 62 ]. The optimization of the above variables is essential to achieve more uniform fibers with diameters at the nanoscale. In general, it is a processing technique that has acquired great relevance, in a wide variety of applications and disciplines, including TE. Thus, this technique presents a great potential for the manufacture of functional artificial tissue, thanks to electrospun polymer matrices’ ability to mimic native extracellular matrix (ECM), through its micro and nanofiber structure with interconnected pores. The effect of each parameter on the morphology of the fibers is summarized in Table 1. This technique manufactures acellular scaffolds. However, several studies are demonstrating the ability of these electrospun matrices to adhere to a wide variety of cell types, allowing their proliferation and tissue development. To date, studies have been conducted, in the field of TE, with scaffolds of electrospun matrices with functionality for the generation of epithelial or muscle tissues, bones, cartilages and blood vessels. The most commonly used synthetic polymers in tissue engineering, due to their good electrospinning behavior, their good ability to mimic ECM and their good cytocompatibility characteristics and biodegradability have been PLA, PGA and PCL or their copolymers (e.g., PLGA, PLLA or PCLL) [ 63 – 65 ]. Among naturally occurring polymers, the most commonly used in electrospinning processes for TE applications have been some proteins such as collagen (or gelatin), often in combination with another polymer (e.g., PCL), elastin and fibrinogen or silk fibroin. Some polysaccharides (chitosan, alginate or hyaluronic acid) have also been used, with excellent results in terms of morphological and functional properties [63–67]. Polymers 2020,12, 1566 7 of 21 Table 1. Summary of the effects produced by the most important variables of the electrospinning process on the morphology of the fibers formed. Parameters General Effects on the Morphology of Fiber Mats References Composition parameters Concentration Fiber diameter increases with the polymer concentration [68,69] A decrease in concentration leads to formation of beads Viscosity There is direct relationship with concentration that leads to similar effects [70,71] An increase in viscosity may prevent a stable flow through the nozzle Molecular weight of polymer Similar effect than viscosity that grows with increasing molecular weight [72,73] An increase may prevent the occurrence of beads Conductivity An increase favors the formation of uniform fibers free of beads [74–76] It also favors a reduction in size (with some exceptions) Surface tension There is not a general trend between surface tension and fiber morphology [70] Volatility A low volatility level may impair solvent removal [77,78] A high volatility level may lead to ribbon-like and porous fibers Dielectric constant of solvent High values of the solvent dielectric constant favor electrospinning [79,80] A secondary solvent may be added to increase the dielectric constant Processing parameters Flow rate Low flow rates give rise to small fiber diameters [81,82] High flow rates may prevent full solvent removal before the target Voltage There is no clear correlation between voltage and diameter of fibers [75,83,84] High voltage values may lead to formation of beads along the fibers Very high voltages may lead to formation of ultrathin secondary filaments Nozzle–collector distance A minimum distance is required to produce solvent-free fibers [85,86] Too long or too short distances may lead to formation of beads Type of nozzle Coaxial nozzles may be used to produce hollow fibers [41,87] Multiple nozzles are used to increase the production scale of fiber mats Collector Metallic collectors lead to smooth fibers and porous collectors to porous fibers [41,88] Rotatory drum collectors may be used to control fiber alignment in the mat Environmental Temperature A rise in temperature reduces the viscosity with its corresponding effects [69,85,89] Tend to reduce fiber size and may lead to bead formation at high concentration It may also extend the polymer concentration window for electrospinning Relative humidity (RH) Low RH values anticipate evaporation and solidification, increasing fiber size [90–92] High RH causes water condensation on the filaments and polymer precipitates. This effect leads to thick and porous fibers, even preventing their formation. Polymers 2020,12, 1566 8 of 21 All the progress achieved with the electrospinning process has been driven mainly by the great versatility of the process, by its low cost and by a great efficiency in obtaining membranes formed by micro and nanofibers with good pore interconnectivity. However, it also involves significant limitations, such as the time required to obtain suitable thickness membranes or problems to the cytotoxicity of solvent residues, the slack of polymer biocompatibility, or the smaller pore sizes obtained as compared to other techniques. These sizes may be suitable for some applications such as blood vessel generation, but they can result in some constrains for cell migration through the scaffold structure, as is the case for the development of bone or cartilaginous tissue. 3.2. Current Trends In the last years, among the variety of polymers that can be used to obtain electrospun fiber mats, biopolymers (either protein or polysaccharide-based) are demanding more attention in biomedical applications. As more knowledge becomes available, it can be foreseen that these biopolymers will likely play an important role in the development of biomaterials with sustainability applications in tissue engineering. The electrospinning technique is changing that, as with sponge-like biomaterials, implies a modification of its microstructure. Indeed, this modulation has been carried out by modifying the process conditions during the biomaterial formation process. Specifically, there are more and more studies that adjust the speed of rotation of the collector to achieve a certain orientation of the fibers, thus we control their microstructure and, therefore, two key aspects such as porosity and mechanical properties. On the other hand, coaxial electrospinning led to core–shell fibers and their ability to preserve the bioactivity of incorporated-sensitive biomolecules (such as drug, protein, and growth factor) and to subsequently control biomolecule release to the targeted microenvironments to achieve therapeutic effects. Such qualities are highly favorable for tissue engineering and drug delivery, and these features are not able to be offered by monolithic fibers [ 93 ]. Apart from that, melt electrospinning is another technique based on electrospinning, which allows the production of fibrous structures from polymer melts in a similar way to solution electrospinning and is applicable to different materials including ceramics [94]. However, there is a growing trend that combines the electrospinning technique with other techniques to fabricate a scaffold with combined properties. In this sense, in a similar way to the previous technique, electrospinning has also been combined with the nanoparticles field to drive up the properties of the membranes produced, e.g., the survey driven by Manjumeena et al. (2015) to obtain PVA fibers with gold nanoparticles with potential anticancer properties [ 95 ]. On the other hand, there are some studies devoted to the combination of electrospinning and 3D printing to produce a hybrid scaffold. Naghieh et al. (2017) presented pioneering research to create a composite based on the combination of poly(lactic acid), gelatin and forsterite [ 28 ]. In addition, Rajzer et al. (2018) combined gelatin and PLLA to produce scaffolds to promote nasal cartilages and subchondral bone reconstruction [ 96 ]. More recent is the work of Chen et al. (2020), in which the combination of these techniques was applied to cartilage regeneration [97]. Recently, electrospinning has been combined with another molecular printing technique allowing the modulation of the surface of the biomaterial by including small proteins and peptides, favoring surface-cell interaction. This technique is called molecular imprinting and is based on the study of surface-peptides interaction to promote cell adhesion and proliferation over the surface of the scaffold [ 98 ]. Especially relevant are the studies of Chronakis et al. (2006), in which the combination of electrospinning and molecular imprinting [ 99 ]. Other approaches were produced combining electrospun nanofibers and the molecular imprinting technique [100,101]. 4. 3D Printing A timeline with the evolution of the 3D printing technique is shown in Figure 4. The first documented use of 3D printing dates back to 1981. Then, the first stereolithographic process was Polymers 2020,12, 1566 9 of 21 patented in 1984 by three French researchers [ 102 ]. However, the 1990s supposed the true growth of the technique with the development of the 3D printers of industrial grade in 1990, and 1993 was the year in which Solidscape developed their dot-on-dot 3D printing technique. This technique integrated the use of polymer-jet fabrication with high precision models, opening new application horizons for the technique like regenerative medicine [ 103 ]. The first biomedical use of 3D printing dates back to 1999, and since then, the number of studies relating 3D printing and biomedicine have shown an exponential growth. The first biomedical milestone was in 2002, when a miniature of a kidney with the same features was 3D printed. Other important dates to mention are 2009, when Organovo produced the first 3D printed blood vessel, and 2016 for the fabrication of the first 3D printed bone [104]. Polymers 2020, 12, x FOR PEER REVIEW 20 of 20 horizons for the technique like regenerative medicine [103]. The first biomedical use of 3D printing dates back to 1999, and since then, the number of studies relating 3D printing and biomedicine have shown an exponential growth. The first biomedical milestone was in 2002, when a miniature of a kidney with the same features was 3D printed. Other important dates to mention are 2009, when Organovo produced the first 3D printed blood vessel, and 2016 for the fabrication of the first 3D printed bone [104]. Figure 4. Timeline of the evolution of “3D printing” over history. 4.1. Technique Most of today’s production of scaffolds for tissue engineering uses the conventional techniques described above. However, these techniques may impose some limitations on the design of the scaffold, providing only relative control over its morphology. Therefore, there is still a growing demand in TE for the development of techniques that allow to achieve a high degree of control over the morphological, biological and mechanical properties of scaffolds, with the aim of mimicking the macro and microstructure of the different types of tissues to be replaced or regenerated. Moreover, the progress in this field is favoring an increasingly specific demand, which converges towards the application of a personalized medicine in TE. Thus, several techniques have been recently developed that allow the manufacture of scaffolds with precise spatial control of their structure, from designs mimicking images of specific functional tissues. There is still some confusion with the term 3D printing, which has been used either to describe a specific technique (e.g., 3D printing using polymeric powder as a raw material) or to encompass a set of them. This set of techniques has been denoted by different authors as rapid prototyping or as additive manufacturing 3D printing. It consists of a group of techniques that can directly generate structures, layer-by-layer, from models obtained using computer-aided design (CAD) techniques. Brief descriptions of the 3D printing technologies of greatest application potential in the field of tissue engineering are given as follows: Stereolithography (SLA) 3D Printing This technique is basically used to manufacture solid 3D objects by consecutively printing thin liquid layers of UV curable material. After the addition of each layer, a spatially controlled photopolymerization stage is applied. The process is repeated layer by layer until the 3D scaffold is fully formed. The uncured polymer is then removed, keeping intact the structure of the designed model, which undergoes a new curing stage to reinforce the 3D structure. The resolution obtained with this technique is higher than others of additive manufacturing. Moreover, the fact that the resin used is liquid facilitates the removal of the excess material, although it also imposes certain restrictions on the design of the stereolithography equipment. However, the biggest challenge for its application to TE is the difficulty of finding suitable reactive resins that do not present cytotoxicity problems [105]. A variant of this technique, which uses light photopolymerization is digital light processing (DLP). Fused Deposition Modeling (FDM) FDM is a 3D printing technology based on the extrusion of molten polymers. As illustrated in Figure 5, the polymer thread is loaded from a spool and fed to a extrusion head where it is heated Figure 4. Timeline of the evolution of “3D printing” over history. 4.1. Technique Most of today’s production of scaffolds for tissue engineering uses the conventional techniques described above. However, these techniques may impose some limitations on the design of the scaffold, providing only relative control over its morphology. Therefore, there is still a growing demand in TE for the development of techniques that allow to achieve a high degree of control over the morphological, biological and mechanical properties of scaffolds, with the aim of mimicking the macro and microstructure of the different types of tissues to be replaced or regenerated. Moreover, the progress in this field is favoring an increasingly specific demand, which converges towards the application of a personalized medicine in TE. Thus, several techniques have been recently developed that allow the manufacture of scaffolds with precise spatial control of their structure, from designs mimicking images of specific functional tissues. There is still some confusion with the term 3D printing, which has been used either to describe a specific technique (e.g., 3D printing using polymeric powder as a raw material) or to encompass a set of them. This set of techniques has been denoted by different authors as rapid prototyping or as additive manufacturing 3D printing. It consists of a group of techniques that can directly generate structures, layer-by-layer, from models obtained using computer-aided design (CAD) techniques. Brief descriptions of the 3D printing technologies of greatest application potential in the field of tissue engineering are given as follows: 4.1.1. Stereolithography (SLA) 3D Printing This technique is basically used to manufacture solid 3D objects by consecutively printing thin liquid layers of UV curable material. After the addition of each layer, a spatially controlled photopolymerization stage is applied. The process is repeated layer by layer until the 3D scaffold is fully formed. 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