Effects of nanofibers properties on cell behavior

Effects of nanofibers properties on cell behavior
Hossein Akbari,¹ Hamid Keshvari,^2,*
1. Amirkabir university of technology (Tehran polytechnic)
2. Amirkabir university of technology (Tehran polytechnic)
Introduction: One of the materials that is currently being researched the most in the medical and tissue engineering fields is nanofibers. They have been put to use for things like cell culture, scaffolds and dressings for tissue engineering, drug delivery systems, and the immobilization of enzymes. The main component of tissue engineering for damaged tissue is scaffolds. They are also necessary for cell survival and function as well as for in vivo and in vitro tissue regeneration. Therefore, important factors in tissue engineering nanofibrous scaffolds include their architecture, pore density and size, morphology, surface adhesion, biocompatibility, and mechanical properties when in contact with body environment [1]–[5]. Polymer nanofibers are synthesized by various methods such as phase separation [6], self-assembly [7] and electrospinning [8]. Electrospinning, however, is a simple, economical and direct method for producing nanoporous scaffolds 63. In the electrospinning method, a high voltage electric field is responsible for the movement of the polymer jet from the tip of the needle that is connected to the source of the polymer solution [9]. With this method, nanoporous scaffolds of polymer biocomposites can be produced with the obtained nanofibers. High surface-to-volume ratio and high porosity are among the advantages of electrospun scaffolds, in which the nanostructure can be changed according to the need by changing parameters such as flow rate, voltage, the distance between the tip of the needle and the collector, and the polymer itself. The porosity and large area of nanofibers lead to more cell proliferation and as a result, it is a promising option for tissue engineering. In addition, these fibrous scaffolds have nanoscale properties and topographies similar to natural extracellular matrix (ECM), which stimulate cell proliferation and differentiation [64]. Cells interact with ECM or biomaterials [10], [11]. The physical characteristics of the substrate of cells are recognized by their surface receptor proteins and are converted into a series of biochemical signals in the cell [12]–[14]. Cell behavior in the case of nanofibers is proportional to some factors, some of which are mentioned in this paper.
Methods: This review article aims to demonstrate the behavior and response of cells towards the different properties that nanofibrous scaffolds can have (like density, fiber diameter, conductivity, mechanical properties, wettability, and alignment of fibers). The objective of reviewing the literature is to summarize some of the effects of nanofibers on cells in one paper. The Google Scholar and Scopus websites were used to find relevant papers. For identifying the papers, several keywords and search terms were used, e.g., cell behavior, migration, viability, adhesion, growth, proliferation, and differentiation, nanofiber diameter, porosity, density, conductivity, stiffness, mechanical properties, alignment, wettability, etc. The variables and outcomes extracted from each paper were nanofiber properties, cell type, and cell behavior. Journals of used papers are indexed under the ISI Web of Science database.
Results: 1- Fiber diameter Nanofiber diameter influences cell response, and this response varies depending on the cell line. In a study by Pelipenko et al. [15], PVA nanofibers with a diameter ranging from 70 to 1120 nm were fabricated, and the behavior of keratinocyts and skin fibroblasts was evaluated on the nanofibers. More so than fibroblasts, keratinocyte size, morphology, and actin organization were influenced by nanofiber thickness. Particularly, keratinocytes grown on nanofibers were smaller and more spherical than control cells, while fibroblasts were barely impacted. They spread across the growth surface and remained essentially unchanged. When keratinocytes were grown on 305 nm thick nanofibers, the cell proliferation as measured by their metabolic activity was at its highest, while fibroblasts grown on analogous nanofibers experienced decreased proliferation. Compared to keratinocytes, fibroblasts showed greater mobility. On nanofibers with a diameter of 300 nm, cell mobility was reduced in both tested cell lines [15]. For implantation and transfer of olfactory ensheathing cells (OECs), silk fibroin nanofibers with diameters of 400 and 1200 nm were prepared by Wu et al [16]. The results showed that 400 nanometer fibers resulted in more cell adhesion, growth, and migration. As shown in Fig.1, The area of cell spreading on 400 nm silk fibroin fibers was noticeably greater than that on 1200 nm silk fibroin after 4 days. At 7 days, it was found that the OECs grown on the 400 nm TSF fibers and the 1200 nm silk fibroin fibers had significantly different spreading areas. Quantitative analysis also showed that at 4 and 7 days, OECs on 400 nm TSF fibers had significantly longer maximum process length than OECs on 1200 nm fibers. Xie et al. investigated the effects of poly (L-lactic acid) (PLLA) fiber matrices on bone marrow mesenchymal stem cells' (BMSCs') cellular responses, including cell adhesion, migration, proliferation, and osteogenesis [17]. The diameters of the fiber matrices were 600 nm for the nanoscale and 1200 nm for the microscale, respectively. After 24 hours of cell culture, findings showed that nanofibers could influence cell morphology and encourage BMSC migration. Compared to microfibers, PLLA nanofibers had higher osteogenesis and higher cell growth, and cell migration speed (Fig. 2) for BMSCs. Contrary to the results of this study, another study by Badami et al. [18] showed that density of osteoprogenitor cells, cultured on PDLLA, PLLA, PEG-PDLLA and PEG-PLLA fibers (with diameters ranging from 140 nm to 2.1 μm) increased with fiber diameter. Also, a higher aspect ratio and the extension of lamellapodia along individual fibers were seen in cells on 2.1 μm diameter fibers, which is consistent with a contact guidance phenomenon. The adhesion of osteoblast, fibroblast, chondrocyte, and smooth muscle cells on carbon fibers (with a diameter ranging from 60 to 200 nm) was investigated by Price et al., and it was found that compared to larger diameter nanofibers, only osteoblast adhesion improved in smaller diameter ones, and the adhesion of other cells was not affected by the dimensions of electrospun carbon fibers [19]. Glioblastoma cells' migration on uniaxially aligned chitosan-PCL fibers with diameters of 200 nm, 400 nm, and 1.1 µm was observed in the study of Kievit et al. [20]. By measuring the net distance, a cell traveled from its starting point and then plotting the distance against time, effective cell speed was determined. According to Fig. 3, the fastest effective cell speed was seen in nanofibers with diameters of 400 nm. This speed is comparable to that of cells invading along microvessels in vivo. The cells expressed higher levels of invasion-related genes on the aligned 400 nm fibers than they did on the 1.1 µm fibers, suggesting that the fibers' greater curvature encouraged migratory behavior. These results imply that controlling nanofiber diameter offers a promising opportunity to improve tissue scaffold design because cells can distinguish between nanofibers of various sizes and react accordingly. 2- Fibers alignment The orientation of the cells and the tension of the fibers, which are influenced by their geometrical patterns, are the phenomenon of contact guidance [18]. The cells on the nanofibers are affected by this phenomenon in terms of how they behave. In research by Mi et al., random, aligned, and orthogonally polyurethane nanofibers were synthesized that were electrically conductive with CNT and poly(acrylic acid) (PAA) [21]. Results showed that the orientation and migration of 3T3 fibroblast cells matched the orientation of the nanofibers. Randomly oriented PVA nanofibers have been shown by Pelipenko et al. to delay keratinocyte adhesion while improving their strength, significantly changing their morphology, raising their metabolic activity, and restricting their mobility [22]. They have demonstrated that the small interfiber pores prevent whole cells from efficiently penetrating the nanofibrillar network. While cell nuclei remain on the surface of the electrospun scaffold, flexible cell parts can enter the nanofibrillar network. The random orientation of nanofibers, which does not offer consistent pathways for successful cell infiltration, is another factor contributing to poor cell mobility. Consequently, nanofibrillar support with nanosized interfiber pores may be used to promote efficient cell proliferation and quicken the healing of wounds, but not for three-dimensional tissue regeneration. The researchers also demonstrated that aligned nanofibers can successfully control cell migration and proliferation, demonstrating the importance of this property of nanomaterials for the successful regeneration of tissues with a highly organized structure [22]. In another study, random and aligned PANi nanofibers were synthesized and seeded with myoblasts [23]. It was found that Young's modulus as well as the tensile strength of aligned fibers are higher than those of random fibers because when the force is applied to the fibers, the tension resulting from the force is equally applied to all fibers. Although the arrangement of fibers did not affect cell proliferation and growth, the growth of cells on random fibers showed flat and multipolar cell morphologies, while in aligned fibers, cells had a bipolar morphology and were attached to individual fibers. According to the results, the arrangement of nanofibers had a significant effect on the growth of myotubes (Fig. 5). While in random nanofibers, only about 10–20% of myotubes were aligned with the Y axis. The length of myotubes was also dependent on the conductivity (the amount of polyaniline in the polymer) and the arrangement of the nanofibers. It was also found that the alignment improved the differentiation of myoblast cells into myotubes (Fig. 11). In an interesting study, the behavior of C12 cells on polystyrene nanofibers with different arrangements (single fiber, two parallel fibers, and crossed fibers) and on a flat surface was investigated [24]. A comparison was made of the shapes of the cells planted on a flat surface, a single fiber, two parallel fibers, and crossed fibers, which resulted in cells with flat, spindle, parallel, and polygonal shapes, respectively (Fig. 6). Then, the migration speed of cells with different shapes was measured: The polygonal cells present at the intersection of fibers had the lowest migration speed. Spindle cells are located on a fiber and tend to be stretched in its direction, and since they have two focal adhesions and can migrate only in the axis of their fiber, they are suitable for researching the effect of fiber diameter on cell adhesion and migration [24]. So, the different geometries of nanofibers affect the migration speed of cells. Due to contact guidance, cells migrate along aligned nanofibers in a linear direction that corresponds to the direction of fiber orientation, increasing the speed of migration. When 3T3 fibroblasts were cultured on thermoplastic polyurethane nanofibers, for example, the migration speed of the cells cultured on uniaxially aligned nanofibers was roughly twice that of the cells cultured on random nanofibers for a similar nanofiber diameter [21]. On mats made of electrospun PLA and PCL nanofibers, respectively, astrocytes and L929 cells were found to exhibit comparable behaviors [25], [26]. Aligned nanofibers have been shown to speed up the migration of stem cells. For a given fiber diameter, human neural progenitor cells and mesenchymal stem cells (MSCs) both demonstrated faster migration rates on uniaxially aligned nanofibers than in the case of random nanofibers [27], [28]. To study how the cytoskeleton changes during cell migration, Dai et al. devised a simple fabrication method using nanofibers with different topographies that mimicked the alignment of extracellular nanofibers [29]. For the purpose of time-lapse imaging analysis, they used a breast carcinoma cell line. They discovered that biointerface anisotropy modified cell morphology and mediated the migration pattern. Cells on anisotropic nanofibers exhibited an extending spindle shape morphologically. The topographic pattern on the biointerface was patterned by the migration trajectories. Besides, aligned nanofibers induced a caterpillar-like model of migration (Fig. 7) through the protrusion-retraction cycle, which was indicated by periodic variation of aspect ratio and velocity of cells. The biointerface anisotropy triggered vimentin filaments and microtubule networks preferentially oriented along the alignment of nanofibers. And the velocity of cell mobility enhanced by vimentin, β-catenin or CDC42 knockdown was significantly enhanced on aligned nanofibers. Thus, they implied that biointerface anisotropy modulated the migration of breast cancer cells and was associated with the reorganization of the cytoskeleton [29]. 3- Fibers density Many studies have been conducted to construct scaffolds with larger interfibrillar porosity, or lower density, to allow our nanofibrous structure to provide a 3D environment instead of 2D itself. Compared to the usual 2D scaffolds, 3D scaffolds have more internal surface area and pore size and thus improve cell infiltration [30]. In a study by Huang et al., a platform made of electrospun nanofibers that had been carefully aligned and densely packed was created to prevent cell migration [31]. An inverse relationship between the cell migration rate and nanofiber density was observed when cells were cultured on nanofibers of various fiber densities (Fig. 8). This was attributed to the formation of focal adhesions. While focal adhesions in the dense fiber mats were large, aligned with the nanofibers, and dispersed throughout the cells, those in the sparse fiber matrix were small [31]. According to Wang et al., fibers with a large diameter were packed more tightly than those with an intermediate or small diameter [32]. They revealed a direct correlation between fiber density and cell migration, in contrast to Huang's study results. The Schwann cells migrated the most widely on the large PLA fibers and the shortest distances on the small nanofibers on uniaxially aligned PLA fibers with different diameters (large, 1325 ± 383 nm; intermediate, 759 ± 179 nm; and small, 293 ± 65 nm). The dense fibers served as barriers to stop the Schwann cells from crossing onto them. Since there was a large distance between each fiber on the intermediate and small fibers, these fibers were unable to give off enough topographical cues to direct the migration of Schwann cells. Berti et al., synthesized bacterial cellulose nanofibers with low and high densities (porous and entangled, respectively) and evaluated the viability of human umbilical vein endothelial cells (HUVECs) on them. Results showed that in a period of 20 days, more cells were viable on porous (less dense) nanofibers (Fig. 9) [33]. The high density of nanofibers may also inhibit the diffusion of growth factors in tissue engineering applications [34]. Chen et al. used PCL nanofiber scaffolds with different densities to investigate the relationship between nanofiber density and osteogenic differentiation [35]. The ability of hBMSCs to differentiate into osteoblasts was assessed after 14 days using osteogenic marker gene expression and after 50 days using calcium mineralization, demonstrating improved osteogenic differentiation with a rise in nanofiber density. Wang et al. achieved different densities of bacterial cellulose nanofiber by changing the bacterial density during the biosynthesis of cellulose [36]. According to their results, a scaffold with a higher bacterial cellulose nanofiber density may encourage the proliferation of adipose-derived stem cells (ADSCs). It's interesting to note that ADSCs seeded in scaffolds with higher bacterial cellulose nanofiber densities displayed more spherical and smaller morphology, suggesting the potential preservation of ADSC phenotype. Rnjak-Kovacina et al. synthesized low- and high-porosity synthetic human elastin scaffolds. Both types of scaffolds displayed Young’s moduli comparable to those of native elastin. Primary dermal fibroblasts could attach, spread, and proliferate on scaffolds with low and high porosities, but only scaffolds with high porosities allowed for active cell infiltration and migration [37]. 4- Electrical conductivity of fibers Electrical stimulation is beneficial for tissue engineering scaffolds because it regulates cell adhesion, migration, proliferation and differentiation. It also increases DNA synthesis, collagen and protein formation for cardiac [38], [39], nerve [40], and muscle [41] regeneration and wound repair [42]. Conductivity can be improved with the help of conductive fillers (graphene, carbon nanotubes, etc.), conductive polymers (polythiophene, polypyrrole (PPy), polyaniline (PANi)) and conductive metals (silver and gold nanoparticles) [43], [44]. Electrical stimulation has a significant impact on the regeneration of tissues like nerve and myocardium because these tissues naturally transmit electrochemical signals throughout the entire tissue [45]. Therefore, it's crucial to use materials with electrical conductivity to repair these tissues' damage. It has been suggested that electrically responsive cells, such as nerve and cardiac cells, could multiply more rapidly when contained within conductive polymer nanofibers. After 8 days of cell culture, neural stem cells on PLLA/PANi scaffolds proliferated more than those on PLLA nanofibers [46]. Even with a 30% increase in the PANi component of the composite, PANi was incorporated into PLCL scaffolds to improve myoblast proliferation [47]. Similar to this, H9c2 rat embryonic heart cell proliferation was greater on 15% and 30% PANi-gelatin composite nanofibers than on either tissue culture polystyrene or gelatin scaffolds [48]. However, a high concentration of conductive polymers in nanofibers might prevent cell growth. According to studies, when the concentration of PPy was 15%, the proliferation of cardiomyocytes on PPy/PCL/gelatin nano-fibers increased, whereas when the PPy concentration was 30%, the cell proliferation was inhibited [49]. A scaffold with cellulose nanofibers modified with polythiophene and polypyrrole derivatives was fabricated by Zha et al [50]. Compared to unmodified and non-conductive nanofibers, these conductive nanofibers showed more adhesion and proliferation of PC12 nerve cells. Also, cell viability was higher on modified conductive polymers (Fig. 10). In the research conducted by Ku et al., the effect of the conductivity of nanofibers on cell differentiation was investigated, and it was found that by increasing the percentage of PANi conductive polymer in nanofibers, cell differentiation of myoblasts increased by 1.3 to 1.6 times (Fig. 5 and 11) [23]. 5- Wettability Greater surface wettability encourages cell attachment, spreading, focal contact formation, and metabolic activity [51]–[54]. Surface wettability also influences cell behavior. It is well known that the wettability and chemical composition of a surface also have a significant impact on cell adhesion and protein adsorption onto a substrate [55]. Due to the type of chemical bonds (covalent and ionic) holding them together, high-energy surfaces typically have higher wettability. Thus, one of the most crucial prerequisite parameters associated with cell-biomaterial interfacial interactions is wettability, which is defined by the presence of chemical groups on a material's surface [56]. For example, polyurethane nanofibers with a smaller diameter showed more hydrophilicity, which is because when the fibers are thinner, more hydrophilic functional groups are exposed to the surrounding environment. It is also said that thinner fibers have smaller pores, which causes more water absorption through capillarity [57]. In addition, thicker fibers can sometimes have large voids that trap air and increase hydrophobicity [58]. Because absorption and permeation properties are crucial for maintaining cell integrity and ensuring access to blood and nutrients during cell growth and proliferation, the capacity to absorb water is a crucial parameter for tissue engineering scaffolds [59]. Since the majority of the biomolecules required for repair are hydrophilic, increased water absorption improves tissue repair while also speeding up the rate at which the scaffold degrades. According to the literature, rat-isolated hepatocytes interact with wettable membranes more effectively than they do with non-wettable ones [60]. In another study, Lee et al. [61] prepared low-density polyethylene (PE) sheets with a wettability gradient. The interaction of various cell types (Chinese hamster ovary, fibroblast, and endothelial cells) was examined using the prepared wettability gradient surfaces. It was found that the positions of the wettability gradient surface with moderate hydrophilicity had greater cell adhesion, spread, and growth than the positions with greater hydrophobicity or hydrophilia. Regardless of the cell types used, the maximum cell adhesion and growth occurred at water contact angles of about 55°. The viability of primary neurons is also influenced by wettability, with more hydrophilic surfaces producing better viability [62]. Increased wettability does not always benefit cells; for example, motor neurons' survival on etched fibers was decreased after plasma etching increased the wettability of nanofibers [63]. 6- Mechanical properties of fibers Research has shown that mechanical properties affect cell behavior. For example, osteogenesis in stem cells can be achieved by increasing the strength and stiffness of the matrix [64], while chondrogenesis is stimulated by the softness of the substrate [65]. Tissue modulus changes both during development and in various diseases in vivo, ranging from 0.5 kPa (adipose tissue) to 20 MPa (bone). Numerous studies [66]–[70] have shown how substrate modulus controls cell motility. The nanofiber modulus can also control how cells migrate when nanofibers are used as substrates. In one study, co-axial electrospinning was used to create fibrous mats with various surface moduli [71]. Gelatin, poly(ethersulfone), poly(dimethylsiloxane), and PCL were used as the core and sheath of the composite, respectively, to modulate the fiber moduli and preserve surface chemistry. Fig. 11 depicts how quickly a single glioblastoma cell migrates across various fibrous mats. Cell migration was fastest on nanofibers with an intermediate modulus (11 µm/h for PCL nanofibers with an 8 MPa modulus), while slower migration rates were seen on nanofibers with low and high moduli (i.e., 3.5 and 6.3 µm/h for PES-PCL and PDMS-PCL, respectively, with both 30 MPa moduli). The "catch-bond formation" mechanism [72], a cellular sensing process by which larger traction forces generated by cells can be evoked to encourage their migration, is thought to be the cause of cells' sensitivity to the fiber modulus. Park et al. showed that transforming growth factor β (TGF-β) can promote mesenchymal stem cells (MSC) differentiation into either smooth muscle cells (SMCs) or chondrogenic cells. They showed that the stiffness of cell adhesion substrates modulated these differential effects. MSCs on soft substrates had less spreading, fewer stress fibers, and a lower proliferation rate than MSCs on stiff substrates. MSCs on stiff substrates had higher expression of SMC markers α-actin and calponin-1; in contrast, MSCs on soft substrates had a higher expression of the chondrogenic marker collagen-II and the adipogenic marker lipoprotein lipase (LPL) [73]. According to experimental findings, thinner nanofibers were mechanically stronger. This is because thinner nanofibers underwent greater tensile deformation during nanofiber formation, which led to the arrangement of more polymer chains along the fiber and ultimately increased its strength [74]. Most of the time, when fibers' diameters are decreased, their densities rise, which can also aid in enhancing their mechanical properties [75]. A 3D polyethylene-glycol-dimethacrylate nanofiber hydrogel matrix with tunable elasticity was created by Wingate et al., using electrospinning and photopolymerization techniques for use as a cellular substrate [76]. Similar to the in vivo elasticity of the intima basement membrane and media layer, compression testing revealed that the elastic modulus of the hydrated 3D matrices ranged from 2 to 15 kPa. Compared to MSC seeded on soft matrices (2-5 kPa), those on rigid matrices (8-15 kPa) displayed a growth in cell area. Additionally, the elasticity of the matrix helped the cells express various phenotypes that are unique to the vascular system with high differentiation efficiency. Within 24 hours, approximately 95% of MSC seeded on matrices with an elasticity of 3 kPa demonstrated Flk-1 endothelial markers, whereas only 20% of MSC seeded on matrices with an elasticity >8 kPa did so. On the other hand, less than ~10% of MSC seeded on matrices with elasticity 5 kPa showed a-actin markers within 24 hours, compared to ~80% of MSC seeded on matrices with elasticity >8 kPa. A potent tool for vascular tissue regeneration could be the ability to control MSC differentiation into endothelial or smooth muscle-like cells solely based on the local elasticity of the substrate. On the behavior of embryonic mesenchymal progenitor cells, Nam et al. specifically looked at how stiff the scaffolding is [77]. Core-shell electrospinning was used to create mechanically distinct scaffolds with identical microstructures and surface chemistry. Core-shell PES-PCL fibers had a modulus of 30.6 MPa, which was more than four times greater than the modulus of pure PCL (7.1 MPa). The results of the differentiation of progenitor cells into chondrogenic and osteogenic tissues on each scaffold show that the lower modulus PCL fibers offered more favorable microenvironments for chondrogenesis, as shown by a notable up-regulation of chondrocytic Sox9, collagen type II, and aggrecan gene expression as well as chondrocyte-specific extracellular matrix glycosaminoglycan production. By encouraging the expression of the osteogenic Runx2, alkaline phosphatase, and osteocalcin genes as well as alkaline phosphatase activity, the stiffer core-shell PES-PCL fibers supported enhanced osteogenesis. The results show that stem cell differentiation may be significantly regulated by the microstructural stiffness or modules of a scaffold and the pliability of each of the fibers.
Conclusion: As the potential range of tissue engineering continues to grow, the appropriate scaffolding choice is necessary to create tightly defined artificial microenvironments for each target organ. Due to their ability to mimic extracellular matrix and their tailorable properties, nanofibers are one of the most commonly used materials for tissue engineering applications. These tailorable morphological, physical, and mechanical properties have different effects on the behavior of different cell lines. Thus, to fabricate the proper scaffold for each tissue target, it is essential to understand the behavior of cells towards them. With regard to the application of nanofibers in the body, an optimal and synergistic relationship between nanofiber characteristics should be discovered because cellular behavior does not always have a direct relationship with each physical, mechanical, or morphological property of nanofibers.
Keywords: nanofibers, scaffold, cell behavior, cell response