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FEATURES AND STRATEGIES FOR SCAFFOLD DESIGN AND PRODUCTION FOR TISSUE ENGINEERING
INTRODUCTION WHO Global Observatory on Donation and Transplantation (GODT) provides data that evidence a critical problem about organs donates worldwide; even if the number of solid organ transplants performed achieves over 130,000, it supports only less than ten percent of the global require.11 Zirpe, K. G.; Suryawanshi, P.; Gurav, S.; Deshmukh, A.; Pote, P.; Tungenwar, A.; Malhotra, R.; Indian J. Crit. Care Med. 2020, 24, 804. According to the Brazilian Association of Organ Transplants (ABTO), thousands of patients suffer, per year, from failure or loss of tissues or organs due to disease or accidents. Although the latest survey by the Brazilian Government reveals that the number of organ transplants broke the record, about 27,000 in 2017, there were still more than 32,700 patients waiting for transplantation.22 http://www.brasil.gov.br/noticias/saude/2018/06/doacao-de-orgaos-brasil-salva-numero-recorde-de-vidas, acessado em fevereiro 2022.
Figure 1).4444 O’Brien, F. J.; Mater. Today 2011, 14, 88.,130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1. However, given the complexity of the extracellular matrix and its important functions, it is still a challenging issue to produce fully functioning and suitable scaffolds for tissue generation.4444 O’Brien, F. J.; Mater. Today 2011, 14, 88.,130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1.
Figure 1
Basic conditions for Tissue Engineering. a) Controlled environment provided from Bioreactors, b) Scaffolds properly to support Tridimensional organization of tissue intended, and c) Type cellular to tissue intent
The characteristics required for scaffolds, as well as their composition, will depend on the type of tissue to be cultivated. In addition, factors such as biocompatibility, biodegradability, pore size and their interconnectivity, mechanical properties and functional chemical groups are of paramount importance for the viability of cellular and tissue development.130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1.,131131 Gandaglia, A.; Bagno, A.; Naso, F.; Spina, M.; Gerosa, G.; Eur. J. Cardio-Thoracic Surg. 2011, 39, 523.
The scaffolds can be obtained from biological tissues, by the removal of cells, using physical, chemical and enzymatic methods, followed by washing and sterilization processes. Recently Caires-Junior (2021) investigated the use of by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium to improve liver scaffold recellularization.132132 Caires-Júnior, L. C.; Goulart, E.; Telles-Silva, K. A.; Araujo, B. H. S.; Musso, C. M.; Kobayashi, G.; Oliveira, D.; Assoni, A.; Carvalho, V. M.; Ribeiro-Jr, A. F.; Ishiba, R.; Braga, K. A. O.; Nepomuceno, N.; Caldini, E.; Rangel, T.; Raia, S.; Lelkes, P. I.; Zatz, M.; Mater. Sci. Eng. C 2021, 121, 111862. Decellularized matrices are considered as excellent models for in vitro tissues; however, their use presents certain disadvantages, such as variability of composition and structure, due to the factors involved, ranging from the preparation of the framework to the structural relationship between the decellularized matrices obtained from different individuals, together with the risk of promoting immune rejection.130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1.,133133 Kawecki, M.; ?abu?, W.; Klama-Baryla, A.; Kitala, D.; Kraut, M.; Glik, J.; Misiuga, M.; Nowak, M.; Bielecki, T.; Kasperczyk, A.; J. Biomed. Mater. Res., Part B 2018, 106, 909.
A variety of techniques can be used to obtain scaffolds, such as: hydrogel, leaching of particles, lyophilization, phase separation, gaseous foam formation, 3D printing, self-assembly, electrospinning and centrifugal (or rotary jet) spinning.4242 Dhandayuthapani, B.; Krishnan, U. M.; Sethuraman, S.; J. Biomed. Mater. Res., Part B 2010, 94, 264. Electrospinning is the most reported technique to produce fibrous mats, due to its easy use, relative low cost and great potential. However, centrifugal spinning and airbrushing are emerging as alternative techniques to electrospinning, that show undoubted advantages such as simplicity of execution, high voltage-free operation, besides promoting a high yield production of micro and nanofibers.4242 Dhandayuthapani, B.; Krishnan, U. M.; Sethuraman, S.; J. Biomed. Mater. Res., Part B 2010, 94, 264.
SYNTHESIS AND MANUFACTURING PROCESS OF SCAFFOLD Hydrogels Hydrogels are a special class of polymer matrix, that can be defined as a crosslinked polymer network capable of adsorbing and retaining a large amount of water or fluid within its 3D structure. Such feature is due to the presence of hydrophilic groups, such as, for example, amino, carboxyl and hydroxyl groups, in the polymer chains.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255.,135135 Pal, K.; Banthia, A. K.; Majumdar, D. K.; Des. Monomers Polym. 2009, 12, 197.
Hydrogels can be manufactured by physical and chemical processes, for their use in tissue engineering. In chemically crosslinked gels, the different polymer chains can be linked through covalent bonds, by radical polymerization crosslinking, high radiation energy, chemical reactions of complementary groups (using aldehydes, addition reactions and condensation reactions) and enzymes. Whereas, physical hydrogels are shaped by a crosslinking mechanism involving non-covalent bonds, such as hydrogen bonds, electrostatics interactions, crystallization (homopolymer crystallization and formation of stereocomplexes), protein interactions (using genetically modified proteins and antigen-antibody interactions) and amphiphilic blocks and graft copolymerization.136136 Hennink, W. E.; van Nostrum, C. F.; Adv. Drug Deliv. Rev. 2012, 64, 223.
Hydrogels produced by chemical methods have greater durability compared to those produced by physical methods. However, a specific adjustment is indispensable, since covalent bonds must be established in situ with considerable rapidity to prevent dilution and/or dispersion, but, slow enough to provide injection and minimal heat generation. In the other side, hydrogels produced by physical methods have the advantage of reacting to environmental stimuli, such as temperature or pH, but as drawback, a faster reabsorption/erosion after injection associated to the dynamic equilibrium of the non-covalent crosslinking. The conjunction of chemical and physical crosslinking in a single hydrogel was shown to be an alternative to obtain hydrogels with better biostability, maintaining in breathability.137137 Pakulska, M. M.; Vulic, K.; Tam, R. Y.; Shoichet, M. S.; Adv. Mater. 2015, 27, 5002.Figure 2 shows the crosslinking forms in the construction of hydrogels and their respective advantages.
Figure 2
The crosslinking forms in the production of hydrogels and their respective advantages
For the production of hydrogel scaffolds, techniques such as leaching of particles, solvent casting, lyophilization, phase separation and gas foaming are used to provide scaffold architecture to the hydrogel through the formation of pores.138138 Ambekar, R. S.; Kandasubramanian, B.; Ind. Eng. Chem. Res. 2019, 58, 6163.,139139 Alaribe, F. N.; Manoto, S. L.; Motaung, S. C. K. M.; Biologia (Bratisl) 2016, 71, 353. The agent that promotes pore formation is inserted before the crosslinking process and removed after this step. Hydrogels are also widely used for the construction of scaffolds using 3D printing. The combination of biostability with precise adjustment of the degradation time and mechanical properties makes the hydrogel system attractive for the ECM mimicry.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255. In injectable hydrogels, the crosslinking process could perform in vivo, in the target site. When exposed to mechanical stress, heat or pH, rheological characteristics can be altered, including a shift to a firmer shape when injected in the target site.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255.
Currently, the use of gelatin microgels has been proposed for the construction of an injectable hydrogel. The scaffold tested on a swine cornea tissue was found to be promising for application in regenerative medicine, due to suitable pores, surface migration and cell proliferation.140140 Hou, S.; Lake, R.; Park, S.; Edwards, S.; Jones, C.; Jeong, K. J.; ACS Appl. Bio Mater. 2018, 1, 1430.
Porogen leaching process The leaching process is based on the casting of a polymer solution together with a porogenic agent. Once a dried polymer film is formed, the porogenic agent is then removed by a dissolution process, given raised to a porous membrane. The pores size and density depend directly on the porogen agent size and concentration in the polymer solution, respectively.141141 Patel, H.; Bonde, M.; Srinivasan, G.; Trends Biomater. Artif. Organs 2011, 25, 20. Various hydrosoluble particulates may be used as porogens, such as sodium chloride and sugar microcrystals, for hydrophobic polymer scaffolds fabrications.
Mikos et al. have reported that the pores density affects the cellular adhesion and should be higher than 80%.142142 Mikos, A. G.; Thorsen, A. J.; Czerwonka, L. A.; Bao, Y.; Langer, R.; Winslow, D. N.; Vacanti, J. P.; Polymer (Guildf) 1994, 35, 1068. It is worth to underline that a porous scaffold makes more efficient the incorporation of bioactive molecules (proteins, growth factors, drugs, etc.) and the cells infiltration across the scaffold.
Silva has investigated the morphological properties of PCL and PLLA based dense and porous polymer membranes, produced by a casting process.143143 Silva, F. W. de O.; Dissertação de Mestrado, Universidade Federal do ABC, Brasil, 2015. The polymer pellets were dissolved in chloroform as solvent, under mechanical stirring. Microparticles of sodium chloride (NaCl) were dispersed in the polymeric solutions, with a NaCl/Polymer mass ratio of about 60%. The solvent was then allowed to evaporated overnight at room temperature to obtain a dried polymer film, which was uncast from the container (glass molds) for the porogen removal. The porogen size range was achieved by sieving the NaCl particles between 75 and 150 µm. To leach out the salt, the membranes were completely immersed in individual glass containers filled with 500 mL of deionized water, under magnetic stirring, at room temperature. The water was changed every 8 h. After 5 days of leaching, the salt-free membranes were then dried in a vacuum oven for 2 days and stored in a desiccator under vacuum for posterior use (Figure 3). In Figure 4 is shown the SEM images of the PCL membranes, in top and cross-section views, with and without the leaching process. The authors highlighted the formation of highly density of interconnected porous membrane as a result of the leaching process. Such porous structure was used for the incorporation of an antibiotic drug, hydrochloride tetracycline, for wound dressing application.
Figure 3
Illustration scheme of the leaching process
Figure 4
SEM images of top view of PCL membranes without and with leaching (a) and (c), respectively; and cross-section view of PCL membranes with leaching (b) and (d), respectively143143 Silva, F. W. de O.; Dissertação de Mestrado, Universidade Federal do ABC, Brasil, 2015.
3D Printing By targeting several applications, including tissue engineering, 3D printing is conceived as a splendid innovative manufacturing technology.144144 Jakus, A. E.; Secor, E. B.; Rutz, A. L.; Jordan, S. W.; Hersam, M. C.; Shah, R. N.; ACS Nano 2015, 9, 4636. Overall, 3D printed biomedical devices are built in a layer-by-layer process, involving the following steps: (a) design of a 3D computer model; (b) fractioning of the 3D computer model into a 2D image construction file; (c) manufacturing by a computer-controlled layer-by-layer process and (d) post-processing, such as surface modification for nanoarchitecture.145145 Chia, H. N.; Wu, B. M.; J. Biol. Eng. 2015, 9, 4.
Unlike the traditional 3D printing methods that are employed to print free-cells scaffolds for use in surgeries, bioprinting demands a different approach that is harmonious with the deposition of living cells. During the 3D bioprinting process, cell units and biomaterials are simultaneously released with micrometric precision to form tissue-like structures.146146 Mandrycky, C.; Wang, Z.; Kim, K.; Kim, D.-H.; Biotechnol. Adv. 2016, 34, 422. The three main methods used for 3D biofabrication are inkjet, extrusion and laser-assisted bioprinting. Inkjet-based bioprinting was the first method to emerge, by a simple adaptation of the well-established conventional inkjet printers for the bioink (cells-loaded biomaterial) deposition to build the functional tissue.147147 Zhu, W.; Ma, X.; Gou, M.; Mei, D.; Zhang, K.; Chen, S.; Curr. Opin. Biotechnol. 2016, 40, 103. Extrusion-based bioprinting consists of the ejection and deposition of the bioink from a nozzle by an automated robotic system.148148 Ozbolat, I. T.; Hospodiuk, M.; Biomaterials 2016, 76, 321. Newly Backes used extrusion-based bioprinting to procuce a bioactive composite scaffolds based on the combination of aliphatic polyester and calcium phosphates that showed elevated level of printing accuracy and applicability for bone tissue regeneration.149149 Backes, E. H.; Fernandes, E. M.; Diogo, G. S.; Marques, C. F.; Silva, T. H.; Costa, L. C.; Passador, F. R.; Reis, R. L.; Pessan, L. A.; Mater. Sci. Eng. C 2021, 122, 111928. Light-assisted bioprinting is based on the use of photopolymerization of biomaterials, printing a diversity of cells with good cell viability. There are two light-assisted bioprinting systems: laser-based and digital light processing based (DLP-based) printers.147147 Zhu, W.; Ma, X.; Gou, M.; Mei, D.; Zhang, K.; Chen, S.; Curr. Opin. Biotechnol. 2016, 40, 103.
According to Aguilar,150150 Aguilar, I. N.; Smith, L. J.; Olivos, D. J.; Chu, T.-M. G.; Kacena, M. A.; Wagner, D. R.; Bioprinting 2019, 15, e00048. the Kenzan bioprint is the highest quality technique reported in the literature. The Kenzan matrix is an arrangement of stainless-steel needles 10 mm long and 170 mm in diameter, spaced 400 ?m in a standard 9x9x9 or 26x26x26. First, the vision system in the 3D printer confirms that the set of needles are bent or missing, and only then does the process start. The system also scans the nozzle, which is responsible for removing the tissue constructs from the culture medium to another location. Subsequently, it checks each spheroid to match the diameter, rounding, and location according to the limits specified by the user. Once the spheroid is checked, it is pulled slightly to the tip of the nozzle using the pressure system also in the printer. If the spheroid is no longer seen by the vision system, it is assumed that the spheroid is not in the mouthpiece, which will move to the place in the series of needles, lowering into a preselected needle, impaling the spheroid needle. The entire process is checked after each placement of spheroids using the machine vision system, until the entire structure designed by the software is constructed.
Although it is a simple and apt technique to generate specific structures without the need of scaffolding, it still requires the improvement of some essential points such as optimization of production time, minimization of costs and errors.
Electrospinning Due to the vast interest of the researchers in the technique of electrospinning, the mechanisms and phenomena in the process were well characterized.151151 Barhoum, A.; Rasouli, R.; Yousefzadeh, M.; Rahier, H.; Bechelany, M. Em Handbook of Nanofibers; Barhoum, A., Bechelany, M., Makhlouf, A. S., eds.; Springer International Publishing: Cham, 2018; pp. 1-42.,152152 Merlini, C.; Silveira, A.; Ramôa, S. D. A. S.; Soares, B. G.; Alavarse, A. C.; Bonvent, J.-J.; Barra, G. M. O.; Polym. Test. 2018, 70, 486. The general principle of electrospinning is the use of an electric field to provide a driving force that promotes the elongation of a fiber from a drop of polymer solution or molten polymer. Other techniques applied to electrostatic precipitation and some pesticide sprayers use the principles that strong repulsive electric forces can be used to overcome surface stress forces.153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325.
The typical apparatus for carrying out the technique is composed of a high voltage supplier, a tube system connected to a peristaltic pump or syringe, a spinneret having at its end a capillary and a grounded collector plate, Figure 5.153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325.,154154 Subbiah, T.; Bhat, G. S.; Tock, R. W.; Parameswaran, S.; Ramkumar, S. S.; J. Appl. Polym. Sci. 2005, 96, 557. During the electrospinning process the polymer containing feedstock is pumped into the capillary of the metallic needle, forming a droplet at its end. This droplet is subjected to an electric field by means of a potential difference applied between the needle and the grounded collector, inducing the formation of free charged in the polymer droplet.154154 Subbiah, T.; Bhat, G. S.; Tock, R. W.; Parameswaran, S.; Ramkumar, S. S.; J. Appl. Polym. Sci. 2005, 96, 557. As the electric field intensity increases, the droplet surface at the capillary end elongates to form a conical shape called the Taylor’s Cone, with accumulation of charges induced in this region.155155 Braiek, M.; Rassas, I.; Lagarde, F.; Chateaux, J. F.; Maaref, A.; Jaffrezic-Renault, N.; Science & Engineering of Polymeric Materials, Yasmine Hammamet, Tunisia, 2014. Increasing the field strength further yields a critical value that overcomes the surface tension of the droplet and then a jet of the fluid is ejected from the needle.153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325. Along the flying path of the jet in the direction of the collector, only in the region close to the Taylor’s Cone a stable behavior is observed; after a short distance the jet is subjected to stretching processes and instabilities that promote the evaporation of the solvent and the reduction of the diameter of the jet, ending to a quite dried fibers at the collector.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255.,153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325.
Figure 5
Electrospinning
Due to its ease of operation, electrospinning has been considered as an alternative for large-scale production of nanofibers.156156 Persano, L.; Camposeo, A.; Tekmen, C.; Pisignano, D.; Macromol. Mater. Eng. 2013, 298, 504. However, electrospinning presents some drawbacks that need some attention and that may restrict its application in industrial scale, such as the need for high voltages of 10 to 30 kV,157157 Lu, B.; He, Y.; Duan, H.; Zhang, Y.; Li, X.; Zhu, C.; Xie, E.; Nanoscale 2012, 4, 1003. a suitable choice of the solvent, beside a relatively low yield of about 0.1 g h-1.158158 Lu, Y.; Li, Y.; Zhang, S.; Xu, G.; Fu, K.; Lee, H.; Zhang, X.; Eur. Polym. J. 2013, 49, 3834.
Therefore, strategies for the improvement of the electrospinning technique have been proposed in order to increase its performance using, for example, multichannels or porous tubes to allow the simultaneous ejection of a large number of fibers.157157 Lu, B.; He, Y.; Duan, H.; Zhang, Y.; Li, X.; Zhu, C.; Xie, E.; Nanoscale 2012, 4, 1003.,159159 Lu, B.; Wang, Y.; Liu, Y.; Duan, H.; Zhou, J.; Zhang, Z.; Wang, Y.; Li, X.; Wang, W.; Lan, W.; Xie, E.; Small 2010, 6, 1612. Alavarse used the electrospinning method to produce scaffolds composed of chitosan, PVA and tetracycline hydrochloride (TCH), as fibers that make up or scaffolding with average measurements of about 309 nm. In addition to presenting antibacterial activity, the authors analyzed a cell test by means of a draft test, which demonstrated that the scaffolds had good cytocompatibility. Figure 6 shows how images from the Scanning Electronic Microcopy of PVA, PVA / Chitosan and PVA / Chitosan / TCH scaffolding before and after the crosslinking process with glutaraldehyde (GA).7070 Alavarse, A. C.; de Oliveira Silva, F. W.; Colque, J. T.; da Silva, V. M.; Prieto, T.; Venancio, E. C.; Bonvent, J.-J.; Mater. Sci. Eng. C 2017, 77, 271.
Figure 6
Scanning Electronic Microcopy imagens scaffolds obtained with (A) Pure PVA, (B) PVA/GA, C) PVA/CS, (D) PVA/CS/GA and (E) PVA/CS/TCH and (F) PVA/CS/TCH/GA7070 Alavarse, A. C.; de Oliveira Silva, F. W.; Colque, J. T.; da Silva, V. M.; Prieto, T.; Venancio, E. C.; Bonvent, J.-J.; Mater. Sci. Eng. C 2017, 77, 271.
Solution Blow Spinning (SBS) As seen previously, the electrospinning process is based on an electrical fields generated between the needle and the collector. Thus, often the fiber deposition process can be susceptible to the behavioral conditions of the polymeric solution facing the electric field. If a polymer blend shows phase separation, the jet instability can suffer different conformations and, consequently, the deposition of fibers with variable diameters. In addition to the electric field, a Taylor cone-shaped polymeric jet can be obtained through pressurization by air in the drop of the needle. This process is known as solution blow spinning (SBS) and can be applied to polymer solutions with high or low electrical conductivity. As in the case of electrospinning, a peristaltic pump controls the flow of the polymeric solution located in a syringe. The needle is interconnected to a cylindrical tube with air or gas passage (Figure 7), and the drag force is directed towards the fiber collector. For the polymeric jet to be launched into the collector, the critical force should be greater than the surface tension forces of the solution (in this case, air flow pressurization forces), so that the solvent is evaporated and fibers are deposited.160160 dos Santos, D. M.; Correa, D. S.; Medeiros, E. S.; Oliveira, J. E.; Mattoso, L. H. C.; ACS Appl. Mater. Interfaces 2020, 12, 45673. Thus, the morphology of the fibers obtained also depends on the viscosity of the solution, the distance between the needle and the collector, the gas and solution flow rates.
Figure 7
The formation of Taylor’s cone by SBS process provoked by the high-pressure air flow pivoting in the drop of polymeric solution
PLA fibers formed through SBS process were investigated varying the polymer concentration and equipment setup. As expected, the concentrated PLA solution at 12% resulted in larger diameter fibers (>310 nm), PLA solution at 10% intermediary diameter fibers (174-216 nm) and smaller diameter fibers (110-179 nm) for PLA solution at 8% even when varying other parameters. In concern of the applied air flow rate (40-80 psi), the study shows that the fiber morphology depends when the feed solution rate reaches 50 µL mL-1. The results were supported by statistical analysis of Box-Behnken design, having significant values (p < 0.05) for quadratic and liner model for concentration and air flow rates.161161 da Silva Parize, D. D.; Foschini, M. M.; de Oliveira, J. E.; Klamczynski, A. P.; Glenn, G. M.; Marconcini, J. M.; Mattoso, L. H. C.; J. Mater. Sci. 2016, 51, 4627. Another advantage of the SBS process is the practicality and even the opportunity to synthesize fibers in situ. Gao and collaborators designed an apparatus capable of producing fibers up to 400 mg min-1 of Polyvinyl butyral (PVB). For this, the bottom an aerosol spray (with orifice inner diameter of 2.8 mm) was connected to a dust removing tank (in this case as an air reservoir). The syringe containing the polymeric solution was placed axially to the aerosol (the syringe nozzle also was modified to fit on properly the air flow).162162 Gao, Y.; Xiang, H.-F.; Wang, X.-X.; Yan, K.; Liu, Q.; Li, X.; Liu, R.-Q.; Yu, M.; Long, Y.-Z.; Chem. Eng. J. 2020, 387, 124052. Thus, the apparatus can be transported to other places such as operating rooms, being used as an emergency device (example: bleeding situations, application of fibers with hemostatic feature) or even inserting directly into the target tissue.
Centrifugal spinning Recently, centrifugal spinning has been proposed as one of the most promising alternative technique to overcome the low productivity of the electrospinning.163163 Ren, L.; Kotha, S. P.; Mater. Lett. 2014, 117, 153. Centrifugal spinning is based on the formation of nanometric and submicrometric fibers by centrifugal force, requiring no electric fields neither electrically charged solutions. It is worthy to stress that, although we use the term centrifugal spinning in the present review, this technique is treated through different appellations in the scientific literature, namely Rotary Jet Spinning and ForcespinningTM, introduced by the brand FibeRio® Technology Co.164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97. The term Centrifugal spinning was recently reported in the literature, by Badrossamay165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. and Sarkar.166166 Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K.; Mater. Today 2010, 13, 12. However, the phenomena of fiber formation on a nanometer scale using centrifugal force was unexpectedly discovered by Weitz167167 Weitz, R. T.; Harnau, L.; Rauschenbach, S.; Burghard, M.; Kern, K.; Nano Lett. 2008, 8, 1187. in a coating process with polymethyl methacrylate (PMMA). Using a conventional spin coater apparatus, the authors noted the formation of fibers of diameters up to 25 nm.
The principle of using centrifugal force to promote a motive force that allows the polymeric fluid to overcome the viscous forces and surface stresses involved in centrifugal spinning is already applied in the production of cotton candy164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97. and glass fibers used as thermal insulation in refrigerators and stoves.168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677. Although the principle involved in centrifugal spinning has already been used in other areas, its recent application in the production of polymer nanofibers has boosted its use and the investigation of the influence of the parameters involved in the technique.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257.,168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677. The rising brand involving centrifugal spinning technique can be perceived by the booming of patents filed by important companies such as BASF Aktiengesellschaft, Owens Corning Fiberglas Technology and Akzo Nobel NV.168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677.
Several models of centrifugal spinning equipment have been reported in the literature from commercial equipment.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257.,169169 Silva, M. de L. C. da; Martinez, P. F.; Izeli, N. L.; Silva, I. R.; Vasconcelos, A. F. D.; Cardoso, M. de S.; Stelutti, R. M.; Giese, E. C.; Barbosa, A. de M.; Quim. Nova 2006, 29, 85.,170170 Koo, O. M.; Rubinstein, I.; Onyuksel, H.; Nanomedicine: Nanotechnology, Biology and Medicine 2005, 1, 193. However, some components are required for operation and are common to all centrifugal spinning equipment used as a spinner located in the center of the equipment which contains predetermined radius holes, whereby the polymeric fluid is expelled, a motor which rotates the spinneret, a system coupled to sensors and speed controllers, and a collector where the fibers is deposited, Figure 8.158158 Lu, Y.; Li, Y.; Zhang, S.; Xu, G.; Fu, K.; Lee, H.; Zhang, X.; Eur. Polym. J. 2013, 49, 3834.,166166 Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K.; Mater. Today 2010, 13, 12.,171171 Pradella, J. G. da C.; Relatório Técnico n° 84 396-205 Centro de Tecnologia de de Processos e Produtos, 2006, 1-119.
Figure 8
Centrifugal Spinning Equipment. A) Collector and spinneret. B) Collector with PVA Fibers. C) Spinner and motor
Figure 9
Airbrushing
One of the great concerns during the centrifugal spinning process is the control and the optimization of the process variables, to modulate the characteristics of the final fibrous mat such as fibers diameter and orientation, porosity and homogeneity. In this sense, other components have been added such as heating and temperature control,166166 Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K.; Mater. Today 2010, 13, 12. flexible sheets in the lower region of the nozzles to generate an airflow that prevents the fibers from depositing immediately at the bottom of the collector158158 Lu, Y.; Li, Y.; Zhang, S.; Xu, G.; Fu, K.; Lee, H.; Zhang, X.; Eur. Polym. J. 2013, 49, 3834.,168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677. and mobile collectors, which allow the variation of the distance between the collector and the die.164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97.
During the centrifugal spinning process, the polymeric fluid is fed to the spinneret by a pump or syringe at a predetermined rate. After the feeding, three steps of the process can be considered: (i) the spinneret rotation induces a centrifugal force that, above a given threshold intensity, overcomes the capillary and viscous forces, provoking the ejection of the polymer jet; (ii) the jet is extended towards the collector by the rotation and resistance movement imposed by the viscosity of the fluid, at a molecular level, the polymer chains are stretched and intertwined, promoting their thinning of the fiber, due to the inertia of the rotation the jet travels in an undulatory trajectory and (iii) concomitantly to elongation of the jet the evaporation of the solvent occurs, promoting the solidification of the jet.164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97.,172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369.
Due to the wide range of applications for the polymer nanofibers and the superiority in centrifugal spinning performance in detriment to the electrospinning, there is a great interest to get o better understanding of the fiber’s formation mechanisms and the influence of the different parameters of the process on the final fibrous mat architecture.173173 de Souza, L.; Alavarse, A. C.; da Vinci, M. A.; Bonvent, J.-J.; Fibers Polym. 2021, 22, 942.,174174 Xu, H.; Chen, H.; Li, X.; Liu, C.; Yang, B.; J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 1547. Eventually, defects may occur beside and/or along the fibers such as beads, agglutination and alignment or not that may be unwanted for the final application of the fibers. Therefore, it seems of paramount importance to focus restless efforts to study the best strategies to precisely tune the different involved parameters to match the fibers morphology to a specific application.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257.,172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369.
During the centrifugal spinning process, rotational velocity, viscoelasticity of the polymer, evaporation rate, temperature, spinning radius, collector distance to the spinneret and feed rate of the polymeric fluid are the main parameters that influence the morphology and diameter of the fibers.174174 Xu, H.; Chen, H.; Li, X.; Liu, C.; Yang, B.; J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 1547. De Souza et al. used RJS to produce pullulan and PVA fibers with different diameters, changing the process parameters as collector distance and solution parameters as solvent volatility and polymers composition. Beyond influence, the diameter of the fibers these variables can module diameters distribution and fibers aspects within mats. This work shows that polymer solution proprieties besides concentration and viscosity, as viscoelasticity, influence spinnability, and fibers aspects.173173 de Souza, L.; Alavarse, A. C.; da Vinci, M. A.; Bonvent, J.-J.; Fibers Polym. 2021, 22, 942.
The volatility of the solution is a relevant parameter of the process since a crucial step is the evaporation of the solvent from the polymer jet for the formation of the fiber. Golecki et al.172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369. have reported the hypothesis that the formation of smooth and bead-free fibers is mainly due to the effect of the rapid evaporation of the solvent. The solvent evaporation from the jet is a mass transfer process that can occur in two stages: a predominantly convective and a purely diffusive step. The first step occurs as the jet travels towards the collector and is dependent on the speed of rotation; the second stage occurs by the diffusion of the solvent remaining through the polymer matrix when the fibers are already deposited in the collector. However, as in many cases, after the centrifugal spinning, the mat is removed and submitted to other processes, only the first stage is in generally considered.172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369. For instance, the volatility can be changed by increasing the proportion of chloroform in a PLA solution in order to produce fibers with lower diameter.172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369.
Badrossamay165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. has reported the influence of the rotation speed in the process. They showed that the diameter of PVA fibers dropped from 1143 ± 50 nm to 424 ± 41 nm, by increasing the rotation from 4000 rpm to 12000 rpm.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. Ren and Kotha175175 Ren, L.; Pandit, V.; Elkin, J.; Denman, T.; Cooper, J. A.; Kotha, S. P.; Nanoscale 2013, 5, 2337. showed a similar behavior for BaTiO3 fibers, achieving a mean diameter of 1497 nm and 788 nm, using a speed of 7000 rpm and 9000 rpm, respectively, evidencing an inversely relation between the speed of rotation and the mean diameter of the fibers.
The rheological properties of the polymeric fluid are of great importance for the centrifugal jet spinning process, influencing the formation or not, as well as the morphology of fibers.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. The viscosity can be understood as the degree of interlacing of the polymer chains, promoting greater resistance to shear stresses. When the viscosity value of the polymeric fluid is below a critical value, the degree of entanglement between the polymer chains is not sufficient to provide resistance for the jet stretching, provoking the jet rupture and the formation of beads.175175 Ren, L.; Pandit, V.; Elkin, J.; Denman, T.; Cooper, J. A.; Kotha, S. P.; Nanoscale 2013, 5, 2337. By increasing the polymer concentration, the viscosity is enhanced, allowing an overlapping of the polymer chains to takes place, and leading to more rigid chains conformations, favorable for the spinning capacity of the solution; however, higher rotation speeds are required for the fiber’s formation.176176 Hammami, M. A.; Krifa, M.; Harzallah, O.; J. Text. Inst. 2014, 105, 637.
Vida et al.177177 Vida, T. A.; Motta, A. C.; Santos Jr., A. R.; Cardoso, G. B. C.; Brito, C. C. de; Zavaglia, C. A. de C.; Mater. Res. 2018, 20, 910. showed, by comparing fibrous scaffolds produced by centrifugal spinning and electrospinning, that the latter technique gives rise to thinner fibers; however, both techniques allow suitable biocompatibility and cell viability, for tissue engineering. Furthermore, the authors emphasize that the worrisome drawback associated to the use of fibrous scaffolds in tissue engineering is the control of the architecture obtained by these processes.177177 Vida, T. A.; Motta, A. C.; Santos Jr., A. R.; Cardoso, G. B. C.; Brito, C. C. de; Zavaglia, C. A. de C.; Mater. Res. 2018, 20, 910.
Airbrushing Airbrushing or Blow Spinning just like Centrifugal Spinning is an alternative technique for the production of polymeric fibers. The technique is based on the use of pressurized air to promote the extrusion and traction of a polymeric solution that will give rise to the fibers.178178 Tutak, W.; Gelven, G.; Markle, C.; Palmer, X.; J. Appl. Polym. Sci. 2015, 132.,179179 Vasireddi, R.; Kruse, J.; Vakili, M.; Kulkarni, S.; Keller, T. F.; Monteiro, D. C. F.; Trebbin, M.; Sci. Rep. 2019, 9, 14297. This technique requires a simple apparatus such as an air compressor, hoses and a pump for the polymeric solution; despite being a simple equipment, there are commercially available airbrush apparatus.179179 Vasireddi, R.; Kruse, J.; Vakili, M.; Kulkarni, S.; Keller, T. F.; Monteiro, D. C. F.; Trebbin, M.; Sci. Rep. 2019, 9, 14297.,180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951.
During the fiber manufacturing process, two fluid streams concentrate, the polymer solution in the center and the surrounding air jet.178178 Tutak, W.; Gelven, G.; Markle, C.; Palmer, X.; J. Appl. Polym. Sci. 2015, 132. The flow pressurized air promotes the displacement of the polymeric solution producing a polymeric jet, the airflow also provides the rapid solvent evaporation present in the jet, leading then to the formation of the polymeric fiber. The use of heated air further favors the drying process of the polymeric jet.180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951.
Among the advantages of the technique can be mentioned productivity up to ten times higher than electrospinning, without the need for an electric field or solvents with specific dielectric constants. In addition, as the air flow remains around the polymeric solution, the solution does not come into contact with the nozzle of the equipment, which minimizes the occurrence of clogging of the equipment nozzle, which can occur more frequently in electrospinning and centrifugal spinning.179179 Vasireddi, R.; Kruse, J.; Vakili, M.; Kulkarni, S.; Keller, T. F.; Monteiro, D. C. F.; Trebbin, M.; Sci. Rep. 2019, 9, 14297.,180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951. Another advantage of the technique is the possibility of depositing the fibers directly on a desired surface, be it flat or with different topographies.180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951. Airbrushing has shown promise for the production of scaffolds for tissue engineering.181181 Abdal-ha, A.; Hamlet, S.; Ivanovski, S.; Biofabrication 2018, 11, 015006.
182 Cui, T.; Yu, J.; Li, Q.; Wang, C.; Chen, S.; Li, W.; Wang, G.; Adv. Mater. 2020, 32, 2000982.-183183 Cerna Nahuis, L. E.; Alvim Valente, C.; de Freitas Oliveira, D.; de Souza Basso, N. R.; Antonio Malmonge, J.; Macromol. Symp. 2019, 383, 1800030.
The Table 1 shows examples of scaffolds produced of different polymers and by different techniques for applications in Tissue Engineering.
Thumbnail Table 1
Scaffolds of different polymers produced by different techniques
COMBINATION OF DIFFERENT SCAFFOLD-FORMING PROCESSES Wei et al. have investigated the combination of 3D printing and particulate leaching techniques to produce alginate scaffolds with both controlled macro and micropores. The authors claimed that the combination of these two techniques allow the obtention of versatile scaffolds for different tissues with suitable properties using alginate-based bioink.254254 Wei, X.; Luo, Y.; Huang, P.; Polym. Bull. 2019, 76, 6077.
Yan et al. also argue that the combination of different processes overcomes some limitations of single isolated processes, for scaffolds manufacturing. The authors proposed the integration of 3D printing, as macro-structure forming process, and near-field electro-spinning (NFES) process, as micro-structure forming process. The experimental results show that the two processes could be switched to get a multi-scale scaffold of 3D printed of gelatin and chitosan together with electrospun chitosan and PVA.255255 Yan, F.; Liu, Y.; Chen, H.; Zhang, F.; Zheng, L.; Hu, Q.; AIP Adv. 2014, 4, 031321.
Mayer et al. have investigated the release profile of two pain relief drugs (diclofenac sodium - DCS and lidocaine -LID), incorporated in both a 3D printed and electrospun scaffolds, for wound dressing applications. The authors proposed, on the basics of their experimental results, an integrated bi-layered scaffold, based on a top 3D printed layer loaded with DCS, and an inner layer filled with LID in contact with the wound. Such combination could allow an immediate pain relief by the LID release, followed by a prolonged DCS release, until the wound dressing removal after 2 days of application.256256 Maver, T.; Smrke, D. M.; Kure?i?, M.; Gradišnik, L.; Maver, U.; Kleinschek, K. S.; J. Sol-Gel Sci. Technol. 2018, 88, 33.
Techniques to produce fibrous scaffolds shows difficult to control of the architecture obtained, this limitation may possibly be overcome by the method, reported recently by Wang et al.,257257 Wang, L.; Wang, B.; Ahmad, Z.; Li, J.-S.; Chang, M.-W.; Drug Deliv. Transl. Res. 2019, 9, 204. for nanofibers production based on the combination of electrospinning and centrifugal spinning. In this innovative strategy, the centrifugal spinning was used to simultaneously generate fibers of two distinct polymers, and the electrospinning to induce an alignment of the collected fibers.
It seems that the combination of different processes for scaffolds manufacturing could overcome the draw backs of each isolated process, for specific tissue engineering applications and is pointing, seemingly, to interesting future scaffolds design with tunable features. The Table 2 shows the pros and cons of different scaffold-processes related on this review.
Thumbnail Table 2
Comparison of diferent scaffold-forming process
CONCLUSIONS Based on previous reported works, important aspects of the scaffold design have been highlighted in this review. We have focused on important features for the development of a 3D framework that mimics the extracellular matrix in both the morphological and functional character, imposed by the in vivo environment for cells adhesion and proliferation. To face this challenge, innovative strategies for the scaffold fabrication have discussed. Among the different techniques, polymeric fibrous mats production plays an important role for tissue engineering applications. To sum up, considering the diversity of materials that can be used for scaffold fabrication, we gave a special emphasis on the interactions between the scaffold on the biological environment, that are essential for cell adhesion, growth, and proliferation. For broad clinical applications, the large production scale with competitive cost may be achieved considering both the nanostructured material functionality and innovative strategies based, for instance, on the combination of two or more fabrication techniques.
LIST OF ABREVIATIONS
Figure 1).4444 O’Brien, F. J.; Mater. Today 2011, 14, 88.,130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1. However, given the complexity of the extracellular matrix and its important functions, it is still a challenging issue to produce fully functioning and suitable scaffolds for tissue generation.4444 O’Brien, F. J.; Mater. Today 2011, 14, 88.,130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1.
Figure 1
Basic conditions for Tissue Engineering. a) Controlled environment provided from Bioreactors, b) Scaffolds properly to support Tridimensional organization of tissue intended, and c) Type cellular to tissue intent
The characteristics required for scaffolds, as well as their composition, will depend on the type of tissue to be cultivated. In addition, factors such as biocompatibility, biodegradability, pore size and their interconnectivity, mechanical properties and functional chemical groups are of paramount importance for the viability of cellular and tissue development.130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1.,131131 Gandaglia, A.; Bagno, A.; Naso, F.; Spina, M.; Gerosa, G.; Eur. J. Cardio-Thoracic Surg. 2011, 39, 523.
The scaffolds can be obtained from biological tissues, by the removal of cells, using physical, chemical and enzymatic methods, followed by washing and sterilization processes. Recently Caires-Junior (2021) investigated the use of by pre-coating decellularized tissue scaffolds with HepG2-conditioned medium to improve liver scaffold recellularization.132132 Caires-Júnior, L. C.; Goulart, E.; Telles-Silva, K. A.; Araujo, B. H. S.; Musso, C. M.; Kobayashi, G.; Oliveira, D.; Assoni, A.; Carvalho, V. M.; Ribeiro-Jr, A. F.; Ishiba, R.; Braga, K. A. O.; Nepomuceno, N.; Caldini, E.; Rangel, T.; Raia, S.; Lelkes, P. I.; Zatz, M.; Mater. Sci. Eng. C 2021, 121, 111862. Decellularized matrices are considered as excellent models for in vitro tissues; however, their use presents certain disadvantages, such as variability of composition and structure, due to the factors involved, ranging from the preparation of the framework to the structural relationship between the decellularized matrices obtained from different individuals, together with the risk of promoting immune rejection.130130 Ahmed, S.; Chauhan, V. M.; Ghaemmaghami, A. M.; Aylott, J. W.; Biotechnol. Lett. 2019, 41, 1.,133133 Kawecki, M.; ?abu?, W.; Klama-Baryla, A.; Kitala, D.; Kraut, M.; Glik, J.; Misiuga, M.; Nowak, M.; Bielecki, T.; Kasperczyk, A.; J. Biomed. Mater. Res., Part B 2018, 106, 909.
A variety of techniques can be used to obtain scaffolds, such as: hydrogel, leaching of particles, lyophilization, phase separation, gaseous foam formation, 3D printing, self-assembly, electrospinning and centrifugal (or rotary jet) spinning.4242 Dhandayuthapani, B.; Krishnan, U. M.; Sethuraman, S.; J. Biomed. Mater. Res., Part B 2010, 94, 264. Electrospinning is the most reported technique to produce fibrous mats, due to its easy use, relative low cost and great potential. However, centrifugal spinning and airbrushing are emerging as alternative techniques to electrospinning, that show undoubted advantages such as simplicity of execution, high voltage-free operation, besides promoting a high yield production of micro and nanofibers.4242 Dhandayuthapani, B.; Krishnan, U. M.; Sethuraman, S.; J. Biomed. Mater. Res., Part B 2010, 94, 264.
SYNTHESIS AND MANUFACTURING PROCESS OF SCAFFOLD Hydrogels Hydrogels are a special class of polymer matrix, that can be defined as a crosslinked polymer network capable of adsorbing and retaining a large amount of water or fluid within its 3D structure. Such feature is due to the presence of hydrophilic groups, such as, for example, amino, carboxyl and hydroxyl groups, in the polymer chains.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255.,135135 Pal, K.; Banthia, A. K.; Majumdar, D. K.; Des. Monomers Polym. 2009, 12, 197.
Hydrogels can be manufactured by physical and chemical processes, for their use in tissue engineering. In chemically crosslinked gels, the different polymer chains can be linked through covalent bonds, by radical polymerization crosslinking, high radiation energy, chemical reactions of complementary groups (using aldehydes, addition reactions and condensation reactions) and enzymes. Whereas, physical hydrogels are shaped by a crosslinking mechanism involving non-covalent bonds, such as hydrogen bonds, electrostatics interactions, crystallization (homopolymer crystallization and formation of stereocomplexes), protein interactions (using genetically modified proteins and antigen-antibody interactions) and amphiphilic blocks and graft copolymerization.136136 Hennink, W. E.; van Nostrum, C. F.; Adv. Drug Deliv. Rev. 2012, 64, 223.
Hydrogels produced by chemical methods have greater durability compared to those produced by physical methods. However, a specific adjustment is indispensable, since covalent bonds must be established in situ with considerable rapidity to prevent dilution and/or dispersion, but, slow enough to provide injection and minimal heat generation. In the other side, hydrogels produced by physical methods have the advantage of reacting to environmental stimuli, such as temperature or pH, but as drawback, a faster reabsorption/erosion after injection associated to the dynamic equilibrium of the non-covalent crosslinking. The conjunction of chemical and physical crosslinking in a single hydrogel was shown to be an alternative to obtain hydrogels with better biostability, maintaining in breathability.137137 Pakulska, M. M.; Vulic, K.; Tam, R. Y.; Shoichet, M. S.; Adv. Mater. 2015, 27, 5002.Figure 2 shows the crosslinking forms in the construction of hydrogels and their respective advantages.
Figure 2
The crosslinking forms in the production of hydrogels and their respective advantages
For the production of hydrogel scaffolds, techniques such as leaching of particles, solvent casting, lyophilization, phase separation and gas foaming are used to provide scaffold architecture to the hydrogel through the formation of pores.138138 Ambekar, R. S.; Kandasubramanian, B.; Ind. Eng. Chem. Res. 2019, 58, 6163.,139139 Alaribe, F. N.; Manoto, S. L.; Motaung, S. C. K. M.; Biologia (Bratisl) 2016, 71, 353. The agent that promotes pore formation is inserted before the crosslinking process and removed after this step. Hydrogels are also widely used for the construction of scaffolds using 3D printing. The combination of biostability with precise adjustment of the degradation time and mechanical properties makes the hydrogel system attractive for the ECM mimicry.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255. In injectable hydrogels, the crosslinking process could perform in vivo, in the target site. When exposed to mechanical stress, heat or pH, rheological characteristics can be altered, including a shift to a firmer shape when injected in the target site.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255.
Currently, the use of gelatin microgels has been proposed for the construction of an injectable hydrogel. The scaffold tested on a swine cornea tissue was found to be promising for application in regenerative medicine, due to suitable pores, surface migration and cell proliferation.140140 Hou, S.; Lake, R.; Park, S.; Edwards, S.; Jones, C.; Jeong, K. J.; ACS Appl. Bio Mater. 2018, 1, 1430.
Porogen leaching process The leaching process is based on the casting of a polymer solution together with a porogenic agent. Once a dried polymer film is formed, the porogenic agent is then removed by a dissolution process, given raised to a porous membrane. The pores size and density depend directly on the porogen agent size and concentration in the polymer solution, respectively.141141 Patel, H.; Bonde, M.; Srinivasan, G.; Trends Biomater. Artif. Organs 2011, 25, 20. Various hydrosoluble particulates may be used as porogens, such as sodium chloride and sugar microcrystals, for hydrophobic polymer scaffolds fabrications.
Mikos et al. have reported that the pores density affects the cellular adhesion and should be higher than 80%.142142 Mikos, A. G.; Thorsen, A. J.; Czerwonka, L. A.; Bao, Y.; Langer, R.; Winslow, D. N.; Vacanti, J. P.; Polymer (Guildf) 1994, 35, 1068. It is worth to underline that a porous scaffold makes more efficient the incorporation of bioactive molecules (proteins, growth factors, drugs, etc.) and the cells infiltration across the scaffold.
Silva has investigated the morphological properties of PCL and PLLA based dense and porous polymer membranes, produced by a casting process.143143 Silva, F. W. de O.; Dissertação de Mestrado, Universidade Federal do ABC, Brasil, 2015. The polymer pellets were dissolved in chloroform as solvent, under mechanical stirring. Microparticles of sodium chloride (NaCl) were dispersed in the polymeric solutions, with a NaCl/Polymer mass ratio of about 60%. The solvent was then allowed to evaporated overnight at room temperature to obtain a dried polymer film, which was uncast from the container (glass molds) for the porogen removal. The porogen size range was achieved by sieving the NaCl particles between 75 and 150 µm. To leach out the salt, the membranes were completely immersed in individual glass containers filled with 500 mL of deionized water, under magnetic stirring, at room temperature. The water was changed every 8 h. After 5 days of leaching, the salt-free membranes were then dried in a vacuum oven for 2 days and stored in a desiccator under vacuum for posterior use (Figure 3). In Figure 4 is shown the SEM images of the PCL membranes, in top and cross-section views, with and without the leaching process. The authors highlighted the formation of highly density of interconnected porous membrane as a result of the leaching process. Such porous structure was used for the incorporation of an antibiotic drug, hydrochloride tetracycline, for wound dressing application.
Figure 3
Illustration scheme of the leaching process
Figure 4
SEM images of top view of PCL membranes without and with leaching (a) and (c), respectively; and cross-section view of PCL membranes with leaching (b) and (d), respectively143143 Silva, F. W. de O.; Dissertação de Mestrado, Universidade Federal do ABC, Brasil, 2015.
3D Printing By targeting several applications, including tissue engineering, 3D printing is conceived as a splendid innovative manufacturing technology.144144 Jakus, A. E.; Secor, E. B.; Rutz, A. L.; Jordan, S. W.; Hersam, M. C.; Shah, R. N.; ACS Nano 2015, 9, 4636. Overall, 3D printed biomedical devices are built in a layer-by-layer process, involving the following steps: (a) design of a 3D computer model; (b) fractioning of the 3D computer model into a 2D image construction file; (c) manufacturing by a computer-controlled layer-by-layer process and (d) post-processing, such as surface modification for nanoarchitecture.145145 Chia, H. N.; Wu, B. M.; J. Biol. Eng. 2015, 9, 4.
Unlike the traditional 3D printing methods that are employed to print free-cells scaffolds for use in surgeries, bioprinting demands a different approach that is harmonious with the deposition of living cells. During the 3D bioprinting process, cell units and biomaterials are simultaneously released with micrometric precision to form tissue-like structures.146146 Mandrycky, C.; Wang, Z.; Kim, K.; Kim, D.-H.; Biotechnol. Adv. 2016, 34, 422. The three main methods used for 3D biofabrication are inkjet, extrusion and laser-assisted bioprinting. Inkjet-based bioprinting was the first method to emerge, by a simple adaptation of the well-established conventional inkjet printers for the bioink (cells-loaded biomaterial) deposition to build the functional tissue.147147 Zhu, W.; Ma, X.; Gou, M.; Mei, D.; Zhang, K.; Chen, S.; Curr. Opin. Biotechnol. 2016, 40, 103. Extrusion-based bioprinting consists of the ejection and deposition of the bioink from a nozzle by an automated robotic system.148148 Ozbolat, I. T.; Hospodiuk, M.; Biomaterials 2016, 76, 321. Newly Backes used extrusion-based bioprinting to procuce a bioactive composite scaffolds based on the combination of aliphatic polyester and calcium phosphates that showed elevated level of printing accuracy and applicability for bone tissue regeneration.149149 Backes, E. H.; Fernandes, E. M.; Diogo, G. S.; Marques, C. F.; Silva, T. H.; Costa, L. C.; Passador, F. R.; Reis, R. L.; Pessan, L. A.; Mater. Sci. Eng. C 2021, 122, 111928. Light-assisted bioprinting is based on the use of photopolymerization of biomaterials, printing a diversity of cells with good cell viability. There are two light-assisted bioprinting systems: laser-based and digital light processing based (DLP-based) printers.147147 Zhu, W.; Ma, X.; Gou, M.; Mei, D.; Zhang, K.; Chen, S.; Curr. Opin. Biotechnol. 2016, 40, 103.
According to Aguilar,150150 Aguilar, I. N.; Smith, L. J.; Olivos, D. J.; Chu, T.-M. G.; Kacena, M. A.; Wagner, D. R.; Bioprinting 2019, 15, e00048. the Kenzan bioprint is the highest quality technique reported in the literature. The Kenzan matrix is an arrangement of stainless-steel needles 10 mm long and 170 mm in diameter, spaced 400 ?m in a standard 9x9x9 or 26x26x26. First, the vision system in the 3D printer confirms that the set of needles are bent or missing, and only then does the process start. The system also scans the nozzle, which is responsible for removing the tissue constructs from the culture medium to another location. Subsequently, it checks each spheroid to match the diameter, rounding, and location according to the limits specified by the user. Once the spheroid is checked, it is pulled slightly to the tip of the nozzle using the pressure system also in the printer. If the spheroid is no longer seen by the vision system, it is assumed that the spheroid is not in the mouthpiece, which will move to the place in the series of needles, lowering into a preselected needle, impaling the spheroid needle. The entire process is checked after each placement of spheroids using the machine vision system, until the entire structure designed by the software is constructed.
Although it is a simple and apt technique to generate specific structures without the need of scaffolding, it still requires the improvement of some essential points such as optimization of production time, minimization of costs and errors.
Electrospinning Due to the vast interest of the researchers in the technique of electrospinning, the mechanisms and phenomena in the process were well characterized.151151 Barhoum, A.; Rasouli, R.; Yousefzadeh, M.; Rahier, H.; Bechelany, M. Em Handbook of Nanofibers; Barhoum, A., Bechelany, M., Makhlouf, A. S., eds.; Springer International Publishing: Cham, 2018; pp. 1-42.,152152 Merlini, C.; Silveira, A.; Ramôa, S. D. A. S.; Soares, B. G.; Alavarse, A. C.; Bonvent, J.-J.; Barra, G. M. O.; Polym. Test. 2018, 70, 486. The general principle of electrospinning is the use of an electric field to provide a driving force that promotes the elongation of a fiber from a drop of polymer solution or molten polymer. Other techniques applied to electrostatic precipitation and some pesticide sprayers use the principles that strong repulsive electric forces can be used to overcome surface stress forces.153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325.
The typical apparatus for carrying out the technique is composed of a high voltage supplier, a tube system connected to a peristaltic pump or syringe, a spinneret having at its end a capillary and a grounded collector plate, Figure 5.153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325.,154154 Subbiah, T.; Bhat, G. S.; Tock, R. W.; Parameswaran, S.; Ramkumar, S. S.; J. Appl. Polym. Sci. 2005, 96, 557. During the electrospinning process the polymer containing feedstock is pumped into the capillary of the metallic needle, forming a droplet at its end. This droplet is subjected to an electric field by means of a potential difference applied between the needle and the grounded collector, inducing the formation of free charged in the polymer droplet.154154 Subbiah, T.; Bhat, G. S.; Tock, R. W.; Parameswaran, S.; Ramkumar, S. S.; J. Appl. Polym. Sci. 2005, 96, 557. As the electric field intensity increases, the droplet surface at the capillary end elongates to form a conical shape called the Taylor’s Cone, with accumulation of charges induced in this region.155155 Braiek, M.; Rassas, I.; Lagarde, F.; Chateaux, J. F.; Maaref, A.; Jaffrezic-Renault, N.; Science & Engineering of Polymeric Materials, Yasmine Hammamet, Tunisia, 2014. Increasing the field strength further yields a critical value that overcomes the surface tension of the droplet and then a jet of the fluid is ejected from the needle.153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325. Along the flying path of the jet in the direction of the collector, only in the region close to the Taylor’s Cone a stable behavior is observed; after a short distance the jet is subjected to stretching processes and instabilities that promote the evaporation of the solvent and the reduction of the diameter of the jet, ending to a quite dried fibers at the collector.134134 Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.; Chem. Soc. Rev. 2017, 46, 6255.,153153 Bhardwaj, N.; Kundu, S. C.; Biotechnol. Adv. 2010, 28, 325.
Figure 5
Electrospinning
Due to its ease of operation, electrospinning has been considered as an alternative for large-scale production of nanofibers.156156 Persano, L.; Camposeo, A.; Tekmen, C.; Pisignano, D.; Macromol. Mater. Eng. 2013, 298, 504. However, electrospinning presents some drawbacks that need some attention and that may restrict its application in industrial scale, such as the need for high voltages of 10 to 30 kV,157157 Lu, B.; He, Y.; Duan, H.; Zhang, Y.; Li, X.; Zhu, C.; Xie, E.; Nanoscale 2012, 4, 1003. a suitable choice of the solvent, beside a relatively low yield of about 0.1 g h-1.158158 Lu, Y.; Li, Y.; Zhang, S.; Xu, G.; Fu, K.; Lee, H.; Zhang, X.; Eur. Polym. J. 2013, 49, 3834.
Therefore, strategies for the improvement of the electrospinning technique have been proposed in order to increase its performance using, for example, multichannels or porous tubes to allow the simultaneous ejection of a large number of fibers.157157 Lu, B.; He, Y.; Duan, H.; Zhang, Y.; Li, X.; Zhu, C.; Xie, E.; Nanoscale 2012, 4, 1003.,159159 Lu, B.; Wang, Y.; Liu, Y.; Duan, H.; Zhou, J.; Zhang, Z.; Wang, Y.; Li, X.; Wang, W.; Lan, W.; Xie, E.; Small 2010, 6, 1612. Alavarse used the electrospinning method to produce scaffolds composed of chitosan, PVA and tetracycline hydrochloride (TCH), as fibers that make up or scaffolding with average measurements of about 309 nm. In addition to presenting antibacterial activity, the authors analyzed a cell test by means of a draft test, which demonstrated that the scaffolds had good cytocompatibility. Figure 6 shows how images from the Scanning Electronic Microcopy of PVA, PVA / Chitosan and PVA / Chitosan / TCH scaffolding before and after the crosslinking process with glutaraldehyde (GA).7070 Alavarse, A. C.; de Oliveira Silva, F. W.; Colque, J. T.; da Silva, V. M.; Prieto, T.; Venancio, E. C.; Bonvent, J.-J.; Mater. Sci. Eng. C 2017, 77, 271.
Figure 6
Scanning Electronic Microcopy imagens scaffolds obtained with (A) Pure PVA, (B) PVA/GA, C) PVA/CS, (D) PVA/CS/GA and (E) PVA/CS/TCH and (F) PVA/CS/TCH/GA7070 Alavarse, A. C.; de Oliveira Silva, F. W.; Colque, J. T.; da Silva, V. M.; Prieto, T.; Venancio, E. C.; Bonvent, J.-J.; Mater. Sci. Eng. C 2017, 77, 271.
Solution Blow Spinning (SBS) As seen previously, the electrospinning process is based on an electrical fields generated between the needle and the collector. Thus, often the fiber deposition process can be susceptible to the behavioral conditions of the polymeric solution facing the electric field. If a polymer blend shows phase separation, the jet instability can suffer different conformations and, consequently, the deposition of fibers with variable diameters. In addition to the electric field, a Taylor cone-shaped polymeric jet can be obtained through pressurization by air in the drop of the needle. This process is known as solution blow spinning (SBS) and can be applied to polymer solutions with high or low electrical conductivity. As in the case of electrospinning, a peristaltic pump controls the flow of the polymeric solution located in a syringe. The needle is interconnected to a cylindrical tube with air or gas passage (Figure 7), and the drag force is directed towards the fiber collector. For the polymeric jet to be launched into the collector, the critical force should be greater than the surface tension forces of the solution (in this case, air flow pressurization forces), so that the solvent is evaporated and fibers are deposited.160160 dos Santos, D. M.; Correa, D. S.; Medeiros, E. S.; Oliveira, J. E.; Mattoso, L. H. C.; ACS Appl. Mater. Interfaces 2020, 12, 45673. Thus, the morphology of the fibers obtained also depends on the viscosity of the solution, the distance between the needle and the collector, the gas and solution flow rates.
Figure 7
The formation of Taylor’s cone by SBS process provoked by the high-pressure air flow pivoting in the drop of polymeric solution
PLA fibers formed through SBS process were investigated varying the polymer concentration and equipment setup. As expected, the concentrated PLA solution at 12% resulted in larger diameter fibers (>310 nm), PLA solution at 10% intermediary diameter fibers (174-216 nm) and smaller diameter fibers (110-179 nm) for PLA solution at 8% even when varying other parameters. In concern of the applied air flow rate (40-80 psi), the study shows that the fiber morphology depends when the feed solution rate reaches 50 µL mL-1. The results were supported by statistical analysis of Box-Behnken design, having significant values (p < 0.05) for quadratic and liner model for concentration and air flow rates.161161 da Silva Parize, D. D.; Foschini, M. M.; de Oliveira, J. E.; Klamczynski, A. P.; Glenn, G. M.; Marconcini, J. M.; Mattoso, L. H. C.; J. Mater. Sci. 2016, 51, 4627. Another advantage of the SBS process is the practicality and even the opportunity to synthesize fibers in situ. Gao and collaborators designed an apparatus capable of producing fibers up to 400 mg min-1 of Polyvinyl butyral (PVB). For this, the bottom an aerosol spray (with orifice inner diameter of 2.8 mm) was connected to a dust removing tank (in this case as an air reservoir). The syringe containing the polymeric solution was placed axially to the aerosol (the syringe nozzle also was modified to fit on properly the air flow).162162 Gao, Y.; Xiang, H.-F.; Wang, X.-X.; Yan, K.; Liu, Q.; Li, X.; Liu, R.-Q.; Yu, M.; Long, Y.-Z.; Chem. Eng. J. 2020, 387, 124052. Thus, the apparatus can be transported to other places such as operating rooms, being used as an emergency device (example: bleeding situations, application of fibers with hemostatic feature) or even inserting directly into the target tissue.
Centrifugal spinning Recently, centrifugal spinning has been proposed as one of the most promising alternative technique to overcome the low productivity of the electrospinning.163163 Ren, L.; Kotha, S. P.; Mater. Lett. 2014, 117, 153. Centrifugal spinning is based on the formation of nanometric and submicrometric fibers by centrifugal force, requiring no electric fields neither electrically charged solutions. It is worthy to stress that, although we use the term centrifugal spinning in the present review, this technique is treated through different appellations in the scientific literature, namely Rotary Jet Spinning and ForcespinningTM, introduced by the brand FibeRio® Technology Co.164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97. The term Centrifugal spinning was recently reported in the literature, by Badrossamay165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. and Sarkar.166166 Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K.; Mater. Today 2010, 13, 12. However, the phenomena of fiber formation on a nanometer scale using centrifugal force was unexpectedly discovered by Weitz167167 Weitz, R. T.; Harnau, L.; Rauschenbach, S.; Burghard, M.; Kern, K.; Nano Lett. 2008, 8, 1187. in a coating process with polymethyl methacrylate (PMMA). Using a conventional spin coater apparatus, the authors noted the formation of fibers of diameters up to 25 nm.
The principle of using centrifugal force to promote a motive force that allows the polymeric fluid to overcome the viscous forces and surface stresses involved in centrifugal spinning is already applied in the production of cotton candy164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97. and glass fibers used as thermal insulation in refrigerators and stoves.168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677. Although the principle involved in centrifugal spinning has already been used in other areas, its recent application in the production of polymer nanofibers has boosted its use and the investigation of the influence of the parameters involved in the technique.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257.,168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677. The rising brand involving centrifugal spinning technique can be perceived by the booming of patents filed by important companies such as BASF Aktiengesellschaft, Owens Corning Fiberglas Technology and Akzo Nobel NV.168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677.
Several models of centrifugal spinning equipment have been reported in the literature from commercial equipment.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257.,169169 Silva, M. de L. C. da; Martinez, P. F.; Izeli, N. L.; Silva, I. R.; Vasconcelos, A. F. D.; Cardoso, M. de S.; Stelutti, R. M.; Giese, E. C.; Barbosa, A. de M.; Quim. Nova 2006, 29, 85.,170170 Koo, O. M.; Rubinstein, I.; Onyuksel, H.; Nanomedicine: Nanotechnology, Biology and Medicine 2005, 1, 193. However, some components are required for operation and are common to all centrifugal spinning equipment used as a spinner located in the center of the equipment which contains predetermined radius holes, whereby the polymeric fluid is expelled, a motor which rotates the spinneret, a system coupled to sensors and speed controllers, and a collector where the fibers is deposited, Figure 8.158158 Lu, Y.; Li, Y.; Zhang, S.; Xu, G.; Fu, K.; Lee, H.; Zhang, X.; Eur. Polym. J. 2013, 49, 3834.,166166 Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K.; Mater. Today 2010, 13, 12.,171171 Pradella, J. G. da C.; Relatório Técnico n° 84 396-205 Centro de Tecnologia de de Processos e Produtos, 2006, 1-119.
Figure 8
Centrifugal Spinning Equipment. A) Collector and spinneret. B) Collector with PVA Fibers. C) Spinner and motor
Figure 9
Airbrushing
One of the great concerns during the centrifugal spinning process is the control and the optimization of the process variables, to modulate the characteristics of the final fibrous mat such as fibers diameter and orientation, porosity and homogeneity. In this sense, other components have been added such as heating and temperature control,166166 Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K.; Mater. Today 2010, 13, 12. flexible sheets in the lower region of the nozzles to generate an airflow that prevents the fibers from depositing immediately at the bottom of the collector158158 Lu, Y.; Li, Y.; Zhang, S.; Xu, G.; Fu, K.; Lee, H.; Zhang, X.; Eur. Polym. J. 2013, 49, 3834.,168168 Zhang, X.; Lu, Y.; Polym. Rev. 2014, 54, 677. and mobile collectors, which allow the variation of the distance between the collector and the die.164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97.
During the centrifugal spinning process, the polymeric fluid is fed to the spinneret by a pump or syringe at a predetermined rate. After the feeding, three steps of the process can be considered: (i) the spinneret rotation induces a centrifugal force that, above a given threshold intensity, overcomes the capillary and viscous forces, provoking the ejection of the polymer jet; (ii) the jet is extended towards the collector by the rotation and resistance movement imposed by the viscosity of the fluid, at a molecular level, the polymer chains are stretched and intertwined, promoting their thinning of the fiber, due to the inertia of the rotation the jet travels in an undulatory trajectory and (iii) concomitantly to elongation of the jet the evaporation of the solvent occurs, promoting the solidification of the jet.164164 Rogalski, J. J.; Bastiaansen, C. W. M.; Peijs, T.; Nanocomposites 2017, 3, 97.,172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369.
Due to the wide range of applications for the polymer nanofibers and the superiority in centrifugal spinning performance in detriment to the electrospinning, there is a great interest to get o better understanding of the fiber’s formation mechanisms and the influence of the different parameters of the process on the final fibrous mat architecture.173173 de Souza, L.; Alavarse, A. C.; da Vinci, M. A.; Bonvent, J.-J.; Fibers Polym. 2021, 22, 942.,174174 Xu, H.; Chen, H.; Li, X.; Liu, C.; Yang, B.; J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 1547. Eventually, defects may occur beside and/or along the fibers such as beads, agglutination and alignment or not that may be unwanted for the final application of the fibers. Therefore, it seems of paramount importance to focus restless efforts to study the best strategies to precisely tune the different involved parameters to match the fibers morphology to a specific application.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257.,172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369.
During the centrifugal spinning process, rotational velocity, viscoelasticity of the polymer, evaporation rate, temperature, spinning radius, collector distance to the spinneret and feed rate of the polymeric fluid are the main parameters that influence the morphology and diameter of the fibers.174174 Xu, H.; Chen, H.; Li, X.; Liu, C.; Yang, B.; J. Polym. Sci., Part B: Polym. Phys. 2014, 52, 1547. De Souza et al. used RJS to produce pullulan and PVA fibers with different diameters, changing the process parameters as collector distance and solution parameters as solvent volatility and polymers composition. Beyond influence, the diameter of the fibers these variables can module diameters distribution and fibers aspects within mats. This work shows that polymer solution proprieties besides concentration and viscosity, as viscoelasticity, influence spinnability, and fibers aspects.173173 de Souza, L.; Alavarse, A. C.; da Vinci, M. A.; Bonvent, J.-J.; Fibers Polym. 2021, 22, 942.
The volatility of the solution is a relevant parameter of the process since a crucial step is the evaporation of the solvent from the polymer jet for the formation of the fiber. Golecki et al.172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369. have reported the hypothesis that the formation of smooth and bead-free fibers is mainly due to the effect of the rapid evaporation of the solvent. The solvent evaporation from the jet is a mass transfer process that can occur in two stages: a predominantly convective and a purely diffusive step. The first step occurs as the jet travels towards the collector and is dependent on the speed of rotation; the second stage occurs by the diffusion of the solvent remaining through the polymer matrix when the fibers are already deposited in the collector. However, as in many cases, after the centrifugal spinning, the mat is removed and submitted to other processes, only the first stage is in generally considered.172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369. For instance, the volatility can be changed by increasing the proportion of chloroform in a PLA solution in order to produce fibers with lower diameter.172172 Golecki, H. M.; Yuan, H.; Glavin, C.; Potter, B.; Badrossamay, M. R.; Goss, J. A.; Phillips, M. D.; Parker, K. K.; Langmuir 2014, 30, 13369.
Badrossamay165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. has reported the influence of the rotation speed in the process. They showed that the diameter of PVA fibers dropped from 1143 ± 50 nm to 424 ± 41 nm, by increasing the rotation from 4000 rpm to 12000 rpm.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. Ren and Kotha175175 Ren, L.; Pandit, V.; Elkin, J.; Denman, T.; Cooper, J. A.; Kotha, S. P.; Nanoscale 2013, 5, 2337. showed a similar behavior for BaTiO3 fibers, achieving a mean diameter of 1497 nm and 788 nm, using a speed of 7000 rpm and 9000 rpm, respectively, evidencing an inversely relation between the speed of rotation and the mean diameter of the fibers.
The rheological properties of the polymeric fluid are of great importance for the centrifugal jet spinning process, influencing the formation or not, as well as the morphology of fibers.165165 Badrossamay, M. R.; McIlwee, H. A.; Goss, J. A.; Parker, K. K.; Nano Lett. 2010, 10, 2257. The viscosity can be understood as the degree of interlacing of the polymer chains, promoting greater resistance to shear stresses. When the viscosity value of the polymeric fluid is below a critical value, the degree of entanglement between the polymer chains is not sufficient to provide resistance for the jet stretching, provoking the jet rupture and the formation of beads.175175 Ren, L.; Pandit, V.; Elkin, J.; Denman, T.; Cooper, J. A.; Kotha, S. P.; Nanoscale 2013, 5, 2337. By increasing the polymer concentration, the viscosity is enhanced, allowing an overlapping of the polymer chains to takes place, and leading to more rigid chains conformations, favorable for the spinning capacity of the solution; however, higher rotation speeds are required for the fiber’s formation.176176 Hammami, M. A.; Krifa, M.; Harzallah, O.; J. Text. Inst. 2014, 105, 637.
Vida et al.177177 Vida, T. A.; Motta, A. C.; Santos Jr., A. R.; Cardoso, G. B. C.; Brito, C. C. de; Zavaglia, C. A. de C.; Mater. Res. 2018, 20, 910. showed, by comparing fibrous scaffolds produced by centrifugal spinning and electrospinning, that the latter technique gives rise to thinner fibers; however, both techniques allow suitable biocompatibility and cell viability, for tissue engineering. Furthermore, the authors emphasize that the worrisome drawback associated to the use of fibrous scaffolds in tissue engineering is the control of the architecture obtained by these processes.177177 Vida, T. A.; Motta, A. C.; Santos Jr., A. R.; Cardoso, G. B. C.; Brito, C. C. de; Zavaglia, C. A. de C.; Mater. Res. 2018, 20, 910.
Airbrushing Airbrushing or Blow Spinning just like Centrifugal Spinning is an alternative technique for the production of polymeric fibers. The technique is based on the use of pressurized air to promote the extrusion and traction of a polymeric solution that will give rise to the fibers.178178 Tutak, W.; Gelven, G.; Markle, C.; Palmer, X.; J. Appl. Polym. Sci. 2015, 132.,179179 Vasireddi, R.; Kruse, J.; Vakili, M.; Kulkarni, S.; Keller, T. F.; Monteiro, D. C. F.; Trebbin, M.; Sci. Rep. 2019, 9, 14297. This technique requires a simple apparatus such as an air compressor, hoses and a pump for the polymeric solution; despite being a simple equipment, there are commercially available airbrush apparatus.179179 Vasireddi, R.; Kruse, J.; Vakili, M.; Kulkarni, S.; Keller, T. F.; Monteiro, D. C. F.; Trebbin, M.; Sci. Rep. 2019, 9, 14297.,180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951.
During the fiber manufacturing process, two fluid streams concentrate, the polymer solution in the center and the surrounding air jet.178178 Tutak, W.; Gelven, G.; Markle, C.; Palmer, X.; J. Appl. Polym. Sci. 2015, 132. The flow pressurized air promotes the displacement of the polymeric solution producing a polymeric jet, the airflow also provides the rapid solvent evaporation present in the jet, leading then to the formation of the polymeric fiber. The use of heated air further favors the drying process of the polymeric jet.180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951.
Among the advantages of the technique can be mentioned productivity up to ten times higher than electrospinning, without the need for an electric field or solvents with specific dielectric constants. In addition, as the air flow remains around the polymeric solution, the solution does not come into contact with the nozzle of the equipment, which minimizes the occurrence of clogging of the equipment nozzle, which can occur more frequently in electrospinning and centrifugal spinning.179179 Vasireddi, R.; Kruse, J.; Vakili, M.; Kulkarni, S.; Keller, T. F.; Monteiro, D. C. F.; Trebbin, M.; Sci. Rep. 2019, 9, 14297.,180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951. Another advantage of the technique is the possibility of depositing the fibers directly on a desired surface, be it flat or with different topographies.180180 Daristotle, J. L.; Behrens, A. M.; Sandler, A. D.; Kofinas, P.; ACS Appl. Mater. Interfaces 2016, 8, 34951. Airbrushing has shown promise for the production of scaffolds for tissue engineering.181181 Abdal-ha, A.; Hamlet, S.; Ivanovski, S.; Biofabrication 2018, 11, 015006.
182 Cui, T.; Yu, J.; Li, Q.; Wang, C.; Chen, S.; Li, W.; Wang, G.; Adv. Mater. 2020, 32, 2000982.-183183 Cerna Nahuis, L. E.; Alvim Valente, C.; de Freitas Oliveira, D.; de Souza Basso, N. R.; Antonio Malmonge, J.; Macromol. Symp. 2019, 383, 1800030.
The Table 1 shows examples of scaffolds produced of different polymers and by different techniques for applications in Tissue Engineering.
Thumbnail Table 1
Scaffolds of different polymers produced by different techniques
COMBINATION OF DIFFERENT SCAFFOLD-FORMING PROCESSES Wei et al. have investigated the combination of 3D printing and particulate leaching techniques to produce alginate scaffolds with both controlled macro and micropores. The authors claimed that the combination of these two techniques allow the obtention of versatile scaffolds for different tissues with suitable properties using alginate-based bioink.254254 Wei, X.; Luo, Y.; Huang, P.; Polym. Bull. 2019, 76, 6077.
Yan et al. also argue that the combination of different processes overcomes some limitations of single isolated processes, for scaffolds manufacturing. The authors proposed the integration of 3D printing, as macro-structure forming process, and near-field electro-spinning (NFES) process, as micro-structure forming process. The experimental results show that the two processes could be switched to get a multi-scale scaffold of 3D printed of gelatin and chitosan together with electrospun chitosan and PVA.255255 Yan, F.; Liu, Y.; Chen, H.; Zhang, F.; Zheng, L.; Hu, Q.; AIP Adv. 2014, 4, 031321.
Mayer et al. have investigated the release profile of two pain relief drugs (diclofenac sodium - DCS and lidocaine -LID), incorporated in both a 3D printed and electrospun scaffolds, for wound dressing applications. The authors proposed, on the basics of their experimental results, an integrated bi-layered scaffold, based on a top 3D printed layer loaded with DCS, and an inner layer filled with LID in contact with the wound. Such combination could allow an immediate pain relief by the LID release, followed by a prolonged DCS release, until the wound dressing removal after 2 days of application.256256 Maver, T.; Smrke, D. M.; Kure?i?, M.; Gradišnik, L.; Maver, U.; Kleinschek, K. S.; J. Sol-Gel Sci. Technol. 2018, 88, 33.
Techniques to produce fibrous scaffolds shows difficult to control of the architecture obtained, this limitation may possibly be overcome by the method, reported recently by Wang et al.,257257 Wang, L.; Wang, B.; Ahmad, Z.; Li, J.-S.; Chang, M.-W.; Drug Deliv. Transl. Res. 2019, 9, 204. for nanofibers production based on the combination of electrospinning and centrifugal spinning. In this innovative strategy, the centrifugal spinning was used to simultaneously generate fibers of two distinct polymers, and the electrospinning to induce an alignment of the collected fibers.
It seems that the combination of different processes for scaffolds manufacturing could overcome the draw backs of each isolated process, for specific tissue engineering applications and is pointing, seemingly, to interesting future scaffolds design with tunable features. The Table 2 shows the pros and cons of different scaffold-processes related on this review.
Thumbnail Table 2
Comparison of diferent scaffold-forming process
CONCLUSIONS Based on previous reported works, important aspects of the scaffold design have been highlighted in this review. We have focused on important features for the development of a 3D framework that mimics the extracellular matrix in both the morphological and functional character, imposed by the in vivo environment for cells adhesion and proliferation. To face this challenge, innovative strategies for the scaffold fabrication have discussed. Among the different techniques, polymeric fibrous mats production plays an important role for tissue engineering applications. To sum up, considering the diversity of materials that can be used for scaffold fabrication, we gave a special emphasis on the interactions between the scaffold on the biological environment, that are essential for cell adhesion, growth, and proliferation. For broad clinical applications, the large production scale with competitive cost may be achieved considering both the nanostructured material functionality and innovative strategies based, for instance, on the combination of two or more fabrication techniques.
LIST OF ABREVIATIONS
- WHO World Health Organization
- GODT Global Observatory on Donation and Transplantation
- 3D Three-Dimensional
- 2D Two-Dimensional
- PMMA Poly (methyl methacrylate)
- FDA Food and Drug Administration
- PLA Polylactic acid
- PGA Polyglycolic acid their
- PLGA Polylactide-co-glycolide
- PCL Poly (?-caprolactone)
- PVL Poly(?-valerolactone)
- PEG Polyethylene glycol
- PEO Polyethylene oxide
- PU Polyurethane
- PVA Poly (vinyl alcohol)
- ECM Extracellular matrix
- Tg Glass transition temperature
- HGF Hepatocyte growth factor
- IL6 Interleukin-6
- IL8 Interleukin-8
- FGF2 Fibroblast Growth Factor 2
- TNFa Tumor Necrosis Factor Alpha
- TGFa Transforming Growth Factor Alpha
- TGF-p1 Transforming Growth Factor-p1
- VEGF Vascular endothelial growth fator
- CXCL1 C-X-C Motif Chemokine Ligand 1
- CXCL2 C-X-C Motif Chemokine Ligand 2
- CXCL3 C-X-C Motif Chemokine Ligand 3
- CCL20 Chemokine (C-C motif) ligand 20
- RJS Rotary Jet Spinning
- SBS Solution Blow Spinning
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 870292.
