1 Introduction
In the world, fracture and bone defect caused by trauma, bone tumor, infection, congenital defects, osteoporosis and so on have seriously endangered human health. Bone repair and bone reconstruction have been a major problem for human development
[1⇓~3]. Although human bone has a certain ability of regeneration and self-repair, that is, after bone tissue is damaged, it can still form new bone through the resorption, secretion of osteogenic matrix and mineralization of osteoclasts and osteoblasts. However, when bone destruction exceeds a specific limit and human bone self-repair can not heal, it is necessary to rely on bone transplantation, including autologous bone transplantation, allograft bone transplantation and artificial bone transplantation. Although autogenous bone and allogeneic bone have certain effects on the treatment of bone defects, there are still problems under the existing medical conditions. Autogenous bone graft has the disadvantages of limited source, limited supply location, secondary trauma, and is not conducive to the repair of large area of bone destruction. Allogeneic bone transplantation has the risk of being infected by viruses, such as AIDS and hepatitis, and is prone to immune rejection, which limits its application
[4][5]. Therefore, how to obtain biomimetic artificial bone repair materials is the key to solve this problem. Electrospun nanofibers have been widely used in bone tissue engineering due to their nanostructure, tunable porosity, high specific surface area, and the ability to closely mimic the structure and biological function of the natural extracellular matrix (ECM) by adjusting the composition of the nanofibers to obtain the desired properties and functions
[6⇓~8].
In this review, we summarize the recent advances in electrospun nanofibers for bone tissue engineering. The principle, influencing parameters, methods and surface modification methods of electrospinning, as well as the application of electrospun nanofibers in bone tissue engineering were reviewed. Finally, the challenges and opportunities for the future development of electrospun nanofibers are summarized.
2 Bone Tissue Engineering
Generally speaking, bone tissue engineering includes three key elements: signaling molecules (bone growth factors and osteoinductive factors), matrix materials and target cells (Fig. 1A)
[9]. Bone tissue engineering refers to the implantation of isolated autologous high-concentration osteoblasts, bone marrow stromal cells or chondrocytes, after in vitro culture and amplification, on biomaterials or extracellular matrices that have good biocompatibility and can be gradually degraded and absorbed by the human body (biological scaffolds can provide three-dimensional growth space for cells.It is advantageous for the cells to obtain sufficient nutrients, gas exchange and waste discharge), and then the cell-carrier material composite is implanted into the bone defect site. With the gradual degradation of biomaterials, target cells are irreversibly differentiated into chondrocytes and osteocytes under the action of osteoinductive factors. Bone growth factors stimulate the mitosis of osteoblasts to form a large number of new bone, so as to achieve the purpose of repairing bone defects. The structure and mechanical properties of bone tissue engineering scaffolds are of great significance for the realization of its role and function as a substitute for bone extracellular matrix. Scaffold architecture directly affects cell behavior (Figure 1B)
[10]. The cells on the micron-sized scaffolds spread out in a manner similar to that on a flat surface. The nanoscale scaffold imitates the physical structure of the extracellular matrix in the human body, and the sufficient surface area of the nanoscale scaffold is beneficial to adsorbing more proteins and providing more adhesion sites for receptors on the cell membrane, and meanwhile, the adsorbed proteins can further change the conformation to expose more adhesion sites, thereby being beneficial to cell adhesion and growth. In addition, bone tissue engineering needs to meet certain mechanical properties. When implanted, the mechanical properties of bone tissue engineering scaffolds must be closely matched with the surrounding host bone tissue and loading conditions to reduce the stress shielding effect. Involving interfacial biomechanics, should allow for interfacial scaffold-bone mechanotransduction to enhance the osseointegration of the scaffold. Finally, as the scaffold degrades, the newly grown bone tissue will begin to support the mechanical load of the bone tissue engineering scaffold
[11].
An ideal bone scaffold should mimic the structure of native ECM and provide a three-dimensional environment for cell adhesion, proliferation, and differentiation
[12]. Natural bone tissue is mainly composed of nano-hydroxyapatite, inorganic minerals and organic matter (type I collagen and non-collagen protein), which are periodically arranged in a regular order to form a continuous inorganic-organic natural nano-composite material with supporting force
[13].
Skeleton is divided into seven hierarchical structures, primary structure-hydroxyapatite, collagen and its matrix components, secondary structure-collagen fibers, tertiary structure-collagen fiber bundles (mineralized collagen fibers), quaternary structure-arrangement of collagen fiber bundles, fifth structure-osteon and trabecular bone, sixth structure-cancellous bone and compact bone, and seventh structure-bone
[14,15]. ECM is similar to bone tissue engineering scaffolds, which are self-assembled into fibers by cross-linked collagen fibers, and then assembled into fibrous sheets or bundles. The compact bone is mainly distributed in the diaphysis of long bones and is composed of the Haversian system of cylindrical structure
[16].
In recent years, according to the type of scaffold material and the repair purpose to be achieved, various methods for preparing tissue engineering scaffolds have been developed, such as bioassembly, 3D printing technology and electrospinning (Fig. 2). Besides metals and bioceramics, a variety of polymers can be used in bone tissue engineering. Natural polymers (gelatin (Gel), collagen, chitosan (CS), hyaluronic acid) and synthetic polymers (polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL) and polymethylmethacrylate (PMMA)) have been successfully used to reconstruct bone defects (Fig. 3). Compared with inorganic and metallic materials, polymer fibers have better biocompatibility, more controllable degradation rate and better flexibility. The polymer fibers can be tailored to specific needs and do not elicit immune or foreign body reactions
[17]. Fig. 3A shows the use of polymer fibers for bone, cartilage, and osteochondral regeneration
[18]. Shi et al. proposed a strategy based on dynamic metal-ligand coordination chemistry to assemble SF-based hydrogels based on silk fibroin ultrafine fibers (MSF) and polysaccharide binders under physiological conditions (Fig. 3B)
[19]. The self-healing and photopolymerizable SF-based hydrogel developed has good injection performance and can fill irregularly shaped tissue defects without causing gel fragmentation. Lin et al. prepared alginate bioink (ALG/ε-PL) modified by ε-polylysine, and prepared a scaffold with adjustable surface charge, controllable degradation behavior and stable mechanical properties by 3D printing (Fig. 3C). Biofunctionalization of the Alg/ε-PL scaffold with chondroitin sulfate or vascular endothelial growth factor and co-culture with human bone marrow mesenchymal stem cells indicated that the scaffold promoted proliferation and chondrogenic differentiation of human bone marrow mesenchymal stem cells
[20].
Zhang et al. prepared a bioceramic (BRT-H) scaffold stacked by hollow tube elements using coaxial 3D printing technology
[21]. The scaffold can not only promote the ingrowth of blood vessels, but also promote the transmission of stem cells and growth factors, which is more conducive to the repair of large bone defects (Fig. 3D). Huang et al. synthesized eggshell particles coated with MgO nanoparticles (CaCO
3/MgO nanocomposite), and prepared a biomimetic active scaffold by chemically cross-linking CaCO
3/MgO nanocomposite, carboxymethyl chitosan (CMC), and bone morphogenetic protein 2 (BMP2) (Fig. 3e). The composite scaffold shows high mechanical properties, remarkable mineralization ability and good bone repair effect
[22].
In view of the fibrous nature of osteocyte extracellular matrix, the introduction of fibers into bone tissue engineering biomaterials provides an idea for the bionic design of biomaterials with bone-mimicking function.
3 Electrospinning
3.1 Electrospinning principle
Electrospinning is a special fiber manufacturing technology, which makes charged polymer solution or melt spray in a strong electric field to obtain nano-scale fibers. As shown in Figure 4A, the typical experimental device required for electrospinning is an electrospinning machine that includes four parts: a high-voltage power supply, a syringe pump for controlling polymer extrusion, a jet device filled with spinning solution, a spinneret (usually a blunt medical injection needle), and a collection device composed of a metal plate or aluminum foil
[24][25].
In the electrospinning process, when the viscoelastic fluid is pushed out of the spinneret, the surface tension will promote it to form suspended spherical droplets. Under the induction of electric field, the surface tension of polymer droplets is opposite to the electric field force. Because of the high voltage applied to the spinneret, the surface of the droplet is charged with the same charge. When the voltage is slowly increased to the point where the electrostatic repulsion is strong enough, it counteracts the surface tension and deforms the spherical droplet at the nozzle into a Taylor cone (as the electric force increases, the droplet is gradually elongated, and when the value of the applied electric force is equal to the surface tension of the droplet, the droplet forms a cone with a vertex angle of 49.3 °, which is named "Taylor cone"). Figure 4 B shows the evolution of a PEO pendant droplet from a spherical shape to a conical shape and then ejecting a jet
[26]. When the electric field intensity increases to a certain critical value, the surface tension of the liquid is completely overcome by the electric field force, so that the liquid is ejected from the Taylor cone.
After the start of spinning, the liquid jet consists of two typical stages (fig. 4C)
[26]. In the first stage, the polymer jet is stretched and accelerated smoothly under the action of electrostatic force and gravity, and only does uniaxial stretching motion, and the shape of the jet does not change with time, which is usually called the "stable motion" stage. In the second stage, the jet undergoes a period of "unstable motion" before reaching the collection device, which is usually called the "whipping motion" stage, that is, under the combined action of surface charge repulsion and strong electric field, the diameter of the jet becomes smaller and smaller until it bends and whips. As the jet accelerates, it oscillates like a whip, where the jet is thinned and a large amount of the ejected solvent is volatilized.
Figure 4D shows a schematic representation of the forces acting on the charged jet
[26]. By placing one or more charged electrodes asymmetrically, introducing azimuthal asymmetry in the radial component of the electric field, multiple ejections of a drop can be stabilized for arbitrarily long times. Fig. 4E shows an example of four jets from the same droplet recorded with a digital video camera
[26]. The straight segments of the jet are about the same diameter, but of different intensity, because some of the segments are not oriented to reflect the flash light into the digital video camera used. Finally, the nanoscale fibers formed by jet solidification are randomly deposited on the conductive collection device to form the electrospun nanofibers we need (Fig. 4F). Fig. 4G shows a wide-angle X-ray diffraction pattern formed by loosely winding electrospun nanofibers. The very dispersed equatorial diffraction spots shown in red indicate that the polymer crystallites are small and imperfect, but are generally aligned in the axial direction of the yarn
[26].
3.2 Lectrostatic spin device and method
A wide variety of nanofibers have been prepared, including organic, organic/inorganic composite and inorganic nanofibers. In addition to traditional electrospinning devices, researchers have developed new devices to control co-spinning/mixing, fiber orientation and directional collection, and have obtained nanofibers with various structures and morphologies. Fig. 5A shows a detailed schematic of each electrospinning method. According to the fiber composition, the electrostatic spinning method is divided into coaxial electrostatic spinning, fiber blending electrostatic spinning, emulsion electrostatic spinning and multi-needle electrostatic spinning; The orientation of the fiber can be controlled by an orientation collecting device, comprising the steps of rotating mandrel alignment, gap electrostatic spinning, magnetic electrostatic spinning, pattern electrostatic spinning and moving a spinneret plate; Through co-spinning, layer-by-layer water spinning and 3D electrostatic spinning, nanofibers with different network structures and components can be obtained.
According to the preparation process of spinning solution, the electrospinning process can be divided into solution electrospinning and melt electrospinning. In solution electrospinning, the polymer is dissolved in a suitable solvent, and then the nanofibers are obtained by electrospinning, while melt electrospinning requires additional heating equipment to melt the polymer. Melt electrospinning does not require a solvent to dissolve the polymer, does not have to worry about solvent recovery or removal, and has no mass loss due to solvent evaporation, so productivity is higher, but the fibers produced by this technology are coarser than those produced by solution electrospinning. Solution electrostatic spinning includes coaxial and multiaxial electrostatic spinning. The basic principle of coaxial electrospinning is that the core and shell solution are injected into two coaxial capillaries with different inner diameters respectively, and the core and shell solution converge at the end of the nozzle and solidify into core-shell composite nanofibers under the action of electric field force
[27,28]. Coaxial electrostatic spinning solves the defect that the spinning solution must be a uniform system during spinning, and uniform and continuous hollow fibers or composite nanofibers with core-shell structures are obtained. Kuang et al. prepared core (heparin) -shell (PC/SAB-MSN) structural fibers as shown in fig. 5B
[29]. Heparin was dissolved in reverse osmosis (RO) water as the core solution, and PLA-PLC (PLCL) and collagen were dissolved in a hexafluoroisopropanol (HFIP) solution containing salvianolic acid B-mesoporous nano-silicon (SAB-MSN) as the shell solution. Heparin and SAB-MSN were encapsulated in the core and shell of the nanofibers, respectively, by coaxial electrospinning to construct the inner layer of small-diameter vascular grafts. Moreover, strategies have been developed to fabricate nanofibers with complex network structures by taking advantage of the versatility of electrospinning devices. Kidoaki et al. proposed a multi-layer and hybrid electrospinning technology based on multiple nozzles. In the multi-layer electrospinning method, two or more polymer solutions are sequentially sprayed on the same static collection plate through independent nozzles to form a reticular layered structure
[30]; In the hybrid electrospinning method, different spinning solutions are simultaneously spun on the same moving collection plate to obtain a homogeneous mixture of multiple fibers (fig. 5C).
3.3 The Influence of Electrospinning Process Parameters
The nanofiber diameter, surface morphology, mechanical properties, porosity, and pore size distribution are highly dependent on the parameters used in the electrospinning process. Since the formation, properties, and structure of electrospun nanofibers are affected by the close interaction of multiple controllable process parameters, it is challenging to optimize the electrospinning manufacturing process to obtain the desired nanofiber properties
[32].
As shown in Table 1, any parameter can significantly affect the final fiber morphology. These parameters include polymer molecular weight, polymer solution properties (viscosity and concentration), applied voltage, working distance (distance between spinneret and collecting device), polymer solution flow rate, solvent volatility, type of collecting device, tip tip design, and environmental parameters. The molecular weight of polymer directly affects the viscosity, surface tension and conductivity of electrospun nanofibers. The molecular weight of the polymer used for electrospinning is generally between 30 kD and 400 kD, and the polymer with sufficient molecular weight and viscosity can provide good mechanical properties to prevent the fracture of the formed electrospun nanofibers
[36,59]. The concentration and viscosity of the spinning solution have a significant effect on the formation of continuous and uniform electrospun fibers
[60]. If the concentration of the spinning solution is too low, it is easy to form a beaded structure due to the lack of viscosity and sufficient chain entanglement of the solution. An increase in polymer concentration correlates with an increase in fiber diameter and a decrease in bead formation. Too high a polymer concentration will result in too high a viscosity of the solution that is resistant to the applied electric field, thereby preventing the formation of the necessary polymer jet
[61].
Fiber diameter and morphology also depend on processing parameters such as working voltage, working distance (distance between spinneret and collection device), and spinning solution flow rate. With the increase of the voltage applied to the polymer solution, the electrostatic force of the system increases, the splitting ability of droplets is enhanced, and the diameter of the resulting fibers tends to decrease. However, if the electric field intensity is too high, the jet speed of the spinning solution will be accelerated, which is not conducive to the stretching and splitting of the spinning solution, thus deteriorating the uniformity of the fibers and forming beaded or beaded nanofibers. The normal working distance is 8 to 15 cm to allow sufficient time for the fibers to dry before they reach the collection device and to avoid beading at too large or too small a distance
[50,62]. Likewise, since the solvent does not have sufficient time to evaporate before reaching the collection device, too high a dope flow rate can produce fibers with a beaded structure, while a lower dope flow rate can obtain fibers with a smaller diameter
[62⇓~64]. The highly volatile solvent will cause the spinning solution to dry at the nozzle, thus blocking the needle tip and hindering the electrospinning process. Also, a less volatile solvent may result in insufficient time for the solvent to evaporate before reaching the collection device, and the deposition of solvent-containing nanofibers on the collection plate may result in the formation of nanofibers having a beaded structure.
The choice of collection device can change the distribution and arrangement of fibers, and directly affect the final mechanical properties of electrospun nanofibers. Aluminum foil is the most commonly used collection device because it is inexpensive, electrically conductive, and when grounded will maintain the proper voltage between the tip of the needle and the receiving device. Other electrostatic spinning receiving devices include a metal mesh, a tip, a rotating disk frame, a cylindrical rotating shaft, a coagulation cell, and a parallel electrode receiving device
[65][55,66][67,68][69,70][71⇓~73][74⇓~76]. And these substrates are widely used in various tissue engineering scaffolds. Rotating cylindrical collection devices can produce tubular structures commonly used for engineered vascular grafts or to make channels to guide directional tissue formation. Porous collection devices, such as copper mesh, form less of a mesh structure than aluminum foil
[77]. Needle design is another important parameter affecting fiber morphology. Spinnerets with multiple tips have been developed. Coaxial or dual capillary spinnerets can produce hybrid nanofiber mats of hollow fibers and immiscible solutions. Core-shell and multilayer nanofibers can be obtained by triaxial electrospinning
[28][78].
In addition to solution and processing parameters, environmental parameters (temperature, relative humidity, and air flow rate) also affect the final morphology of the fiber mat
[45,58]. Humidity has a significant effect on the electrical conductivity of the air and causes changes in the electric field, as well as affecting the evaporation rate of the solvent
[79]. An increase in humidity can lead to that formation and coalescence of surface pore
[35,44,70]. Although electrospinning is usually carried out at room temperature, increasing the temperature from 25 ° C to 60 ° C decreases the fiber diameter
[44,45]. An excessively high air flow rate around the spinneret tends to increase the evaporation rate due to convection, resulting in a larger fiber diameter
[44].
The morphology, diameter, and arrangement of the fibers can be changed by adjusting the above preparation parameters, and the preparation of electrospun nanofibers with different structures (fig. 6) provides an opportunity to create tissue-specific scaffolds with specific functions. The main disadvantage of electrospinning is the lack of independent control of all electrospinning parameters, since changing one parameter affects the others simultaneously. In view of the influence of these complex factors on fiber morphology, it is necessary to further study the influence of electrospinning parameters and environmental parameters on the biophysical properties of electrospun fibers in order to improve the production of fibers for specific applications.
4 Surface Modification of Electrospun Nanofiber
Due to the chemical flexibility and high specific surface area of electrospun nanofibers, the surface of electrospun nanofibers can be modified by a variety of bioactive molecules. Surface modification methods can be used to modify the surface chemistry of electrospun nanofibers, aiming at improving the hydrophilicity of scaffolds, enhancing their biocompatibility, or inducing specific biological responses of attached cells to provide a more ideal microenvironment for the growth of surrounding tissues and cells
[90].
4.1 Plasma modification technique
Plasma technology is used to modify the surface of nanofibers in order to produce polar groups on the surface, such as hydroxyl, carboxyl or amino
[91,92]. The presence of these functional groups can lead to changes in fiber surface structure or chemical properties, such as wettability, polarity, and bioadhesion
[92,93]. Carboxyl groups can be introduced onto the surface of electrospun PLGA nanofibers under oxygen and gas-phase acrylic acid plasma treatment, thereby enhancing the cell adhesion and proliferation ability of electrospun PLGA nanofibers
[94]. In addition, various extracellular matrix proteins such as gelatin, collagen, laminin and fibronectin are immobilized on the surface of nanofibers by plasma technology to enhance cell adhesion and proliferation
[95,96]. When PCL electrospun nanofibers were modified by argon plasma, a large number of carboxyl groups were produced on the surface. When the surface-modified fibers were immersed in simulated body fluid with a concentration of 10 times that of human body fluid, it was found that bone calcium phosphate mineralization occurred on the surface of the nanofibers. The scaffold material has great potential application value in bone transplantation
[97].
Plasma modification has significant advantages, almost all polymer fiber surfaces can be modified by plasma, and plasma technology can uniformly modify nanofibers on the whole surface.The plasma modification is limited on the surface of the nanofiber (the depth is about tens of nanometers), but the overall performance of the matrix material is not affected, the surface of the fiber can be modified in different ways by changing the type of plasma used, and the use of plasma technology can avoid the introduction of other components on the fiber surface.
4.2 Surface grafting modification technology
Graft copolymerization mainly refers to the combination of appropriate branched chains or functional side groups on the macromolecular chain through chemical bonds, which connects two polymers with different properties to form graft copolymers with special properties. The surface grafting modification reaction only occurs on the surface of the material to be modified, and a new grafted polymer layer with special functions is obtained on the surface of the matrix material through grafting macromolecular chains, thereby achieving a significant surface modification effect. The main advantage of surface grafting modification technology is that the same polymer can be modified by choosing different monomers, so that the polymer surface can obtain more properties, while the original properties of the matrix material are almost unchanged. The surface grafting modification technology can endow the nanofiber surface with a variety of active functional groups, and fix bioactive molecules and cell recognition ligands on the nanofiber surface, which can interact with biological tissues or cells to achieve the purpose of cell adhesion, proliferation and differentiation
[98⇓~100].
4.3 Surface chemical modification technology
Surface chemical modification technology is a method that uses some functional groups (carboxyl, hydroxyl, amino, ester, etc.) Of the polymer substrate itself to react with other functional compounds to change the surface chemical and physical properties of the substrate material, including surface chemical immobilization reaction method, wet chemical treatment method, etc.
When the polymer molecule contains benzene ring, hydroxyl, double bond, halogen, ester and other functional groups, it is easy to form an electrophilic or nucleophilic environment, which can promote the chemical immobilization reaction. Biomacromolecules can be immobilized on the surface of nanofibers by surface chemical immobilization
[101]. Amino and carboxyl functional groups have been widely used in surface chemical immobilization modification of electrospun nanofibers. Collagen molecules can be conjugated and immobilized on the surface of electrospun polyacrylic acid nanofibers containing carboxyl groups by chemical immobilization modification technology, so as to achieve the purpose of changing the surface function of the fibers
[102]. In addition, polyacrylic acid nanofibers can be conjugated with amino functional groups by surface curing modification technology to prepare electrospun nanofibers with aminated surfaces
[103,104].
The surface of thick nanofiber membranes can be modified by wet chemical treatment
[105]. During the surface modification of polymers by wet chemical methods, oxygen-containing functional groups such as carbonyl, hydroxyl and carboxyl groups on the surface of polymers are usually used to effectively transport gas-phase reagents or oxidant solutions for oxidation or hydrolysis reactions. The increase in the number of oxygen-containing functional groups can enhance the ability to form hydrogen bonds, thereby improving the wettability and adhesion of the material. The poly (L-lactic acid) nanofiber hydrolyzed by the sodium hydroxide solution can be used for the mineralization of hydroxyapatite because carboxyl on the surface of the poly (L-lactic acid) nanofiber subjected to the hydrolysis treatment can be chelated with calcium ions,O as to accelerate the formation and growth of crystal nuclei on the surface of the levorotatory polylactic acid nanofibers and promote the mineralization of hydroxyapatite
[106].
5 Application of Electrospinning in Bone Tissue Engineering
Considering the highly complex and hierarchical structure of human bone, fibers are mainly used as reinforcing phases of composite or hydrogel biomaterials or as temporary ECM-mimicking fiber scaffolds for bone in bone tissue engineering
[18]. These fiber-based composites have been widely used in the fields of bone, cartilage and osteochondral regeneration due to their superior properties over single-phase materials.
5.1 Bone defect regeneration
Whang et al. developed an electrospun PCL/Gel nanofiber scaffold based on mesoporous silicate nanoparticles (MSN) to achieve dual delivery of alendronate (ALN) and silicate.Thus having a synergistic effect in regulating bone remodeling, where ALN inhibits the bone resorption process by preventing the expression of guanine triphosphate and guanine-related proteins, and silicate promotes the bone formation process by improving angiogenesis and bone calcification
[107]. The results of bone repair in a rat critical-size skull defect model suggest that the electrospun PCL/Gel nanofiber scaffold based on dual delivery of ALN and silicate is promising for clinical bone repair.
Ye et al. developed a 3D nanofiber scaffold with a porous structure for bone tissue engineering (fig. 7)
[108]. The n-HA/PLA/Gel three-dimensional nanofiber scaffolds were prepared from electrospun nanofibers by homogenization, freeze-drying and heat treatment. A polydopamine-assisted coating method was used to immobilize BMP-2-derived polypeptide (PEP) on 3D scaffolds to obtain n-HA/PLA/Gel-PEP scaffolds capable of sustained release of BMP-2 polypeptide. The in vitro study of n-HA/PLA/GEL-PEP showed that the scaffold could promote the alkaline phosphatase activity of BMSCs and the expression of osteogenic differentiation related genes. In addition, an in vivo evaluation was performed using a rat skull defect model, and the results of radiological and histological analyses indicated that the scaffold promoted bone formation in the defect area
[108].
5.2 Cartilage defect repair
Articular cartilage plays an important role in joint movement, and its structure is fine. Cartilage tissue is composed of chondrocytes, matrix and fibers. Various joint diseases caused by cartilage wear, injury and deformation are a serious threat to human health. At present, the application of electrospinning technology, which can simulate the characteristics of extracellular matrix, in cartilage regeneration has attracted great attention of researchers. Li et al. designed some PCL nanofiber scaffolds and studied their ability to promote chondrogenesis of MSCs in vitro to promote cartilage regeneration
[109]. The results show that the nanofibers are uniformly oriented and have a diameter of 700 nm. MSCs cultured in scaffolds differentiated into cells with a chondrocyte phenotype in the presence of transforming growth factor-β (TGF-β) -1.
For the first time, Chen et al. processed cartilage acellular matrix (CDM) into powder, dispersed and processed electrospun Gel/PLGA fibers into dry short fibers as a reinforcing material to overcome the softness and mechanical instability of 3D-printed CDM scaffolds
[110]. By combining a series of processing techniques with chemical treatment, 3D printed fiber-reinforced CDM (CDM-fiber) scaffolds with adjustable shape and controllable internal structure were successfully prepared by adding dispersed electrospun Gel/PLGA fibers in CDM ink (Figure 8 a). The feasibility of cartilage regeneration in vivo was explored by implanting the cell-scaffold construct into the subcutaneous tissue of nude mice. After 8 weeks of implantation, white cartilage-like tissue was formed in both the fiber-3DP group and the CDM-fiber 50% group, but the cell-scaffold structure of the CDM-fiber 50% group remained its original square shape and formed a more dense and uniform tissue. Histological examination further found that cartilage-like tissue appeared in both fiber-3DP and CDM-fiber 50% groups, and typical cartilage lacunae gradually formed with the increase of culture time in vivo. In the CDM-fiber 50% specimen, cartilage lacunae with denser connections were observed. Pure scaffolds were implanted into articular cartilage defects in rabbits to evaluate the feasibility of cartilage repair in situ (Fig. 8B). After 12 weeks of implantation, from the large in vitro view of the cartilage tissue, there was a significant unrepaired defect in the untreated group, while there was a partial filling of the defect in the fiber-3DP group, and complete filling of the defect was observed in the CDM-fiber 50% group. These results indicate that 50% CDM fibers can promote the regeneration and reconstruction of cartilage defects in rabbits. This study shows that CDM-fiber 50% scaffold has potential application value in the repair of cartilage defects.
5.3 Osteochondral tissue engineering
Osteochondral tissue is a combination of two different tissues with a gradient in composition and complex physiological characteristics. Osteochondral defects (defects affecting articular cartilage and underlying subchondral bone) usually originate from trauma or chondritis of bone injury, which is a common disease in orthopaedics, and its clinical treatment effect has been unsatisfactory
[111]. At present, the construction of composite structures simulating natural osteochondral tissue based on electrospun scaffolds provides a new hope for its repair. In order to repair osteochondral tissue with multiple cell types formed in a specific way, it is necessary to design special functional gradient osteochondral scaffolds with physical and chemical properties.
A large number of studies have shown that electrospun nanofiber scaffolds combined with other substances/structures in a specific way can form three-dimensional scaffolds with gradient properties to reproduce the ribbon-like matrix at the bone-cartilage interface.
Liu et al. prepared a novel osteochondral tissue engineering scaffold by combining 3D bioprinting and multi-nozzle electrospinning
[112]. To achieve sustained release of gentamicin sulfate (GS) and deferoxamine (DFO), co-electrospun GS/PVA fibers and co-electrospun PVA-DFO/PCL (core-shell) composite fibers were deposited in scaffolds. The composite scaffold has the potential to release multiple biomolecules in different spaces and times to achieve the special characteristics of osteochondral scaffolds.
For the first time, Zhang et al. combined collagen and electrospun PLA nanofibers (COL-nanofibers) to prepare a double-layer microporous scaffold to synergistically promote osteochondral regeneration
[113]. Mesenchymal stem cells were cultured on double-layer scaffolds, and their adhesion, proliferation and differentiation were observed. In addition, a rabbit model of osteochondral defect was established and implanted with collagen nanofiber scaffolds, and the regeneration of cartilage and subchondral bone was evaluated at 6 and 12 weeks after operation, respectively. As shown in fig. 9, compared with the COL scaffold, the cells on the COL-nanofiber scaffold showed stronger osteogenic differentiation ability, as indicated by the high expression of OCN and Runx2 genes and the accumulation of calcium nodules. In addition, implantation of the cell-loaded COL-nanofiber scaffold induced faster growth of subchondral bone and better formation of cartilage, and histological staining, biomechanical testing, and microcomputed tomography data showed better functional repair of osteochondral defects. This study demonstrates the potential of double-layered microporous COL-nanofiber scaffolds for the treatment of deep osteochondral defects.
6 Conclusion and prospect
A fundamental goal of bone tissue engineering is to develop materials that effectively mimic the structure and function of the extracellular matrix of natural bone tissue, while allowing the replacement material to degrade continuously during the regeneration of new bone tissue. Electrospinning is considered to be one of the most efficient techniques for the synthesis of nanomaterials. Inspired by the highly directional characteristics of the natural extracellular matrix, electrospun nanofiber scaffolds with desired structure and function (fiber arrangement, patterned structure, enhanced porosity, improved mechanical properties, partial gradient of composition or function) can be designed by improving the electrospinning device or changing the electrospinning parameters. In addition, electrospinning provides many pathways to direct cellular responses and enhance tissue regeneration. More and more polymers and high molecular weight compounds with sufficient viscosity have been electrospun and widely used in bone tissue engineering.
Although electrospun nanofibers have made great progress in recent years, there are still some limitations and challenges before the clinical application of electrospun bone grafts. Improving the biomimetic function of scaffolds requires precise and repeatable control of fiber morphology, structure, composition and uniform cell distribution. Insufficient cell infiltration is still the focus of current research, and new technologies developed to solve this problem include reducing fiber packing density, multi-layer electrospinning, dynamic cell culture, and electrospraying cells. In addition, the collection speed of electrospun nanofibers is relatively slow, and further exploration is needed to realize the large-scale production of electrospun nanofibers. Finally, the functional application of electrospun composite nanostructures is still a great challenge.
In recent years, a variety of materials with good biodegradability and biocompatibility have been prepared into electrospun nanofiber scaffolds (including natural materials and synthetic polymer materials) with different structures by electrospinning technology, which have been widely used in tissue engineering and regenerative medicine. Electrospun nanofibers with nanostructure, tunable porosity and high specific surface area can produce a fibrous matrix with similar size to ECM to more closely mimic the structure and biological function of natural ECM. Electrospun oriented nanofiber scaffolds can promote cell adhesion and migration through contact guidance, and the combination with cells or growth factors can further promote cell proliferation and differentiation, and ultimately achieve tissue regeneration. With the innovation and optimization of electrospinning technology and devices, electrospun nanofibers as scaffold materials have shown great potential and broad application prospects in the fields of bone, cartilage, osteochondral regeneration and wound healing.
Current research on electrospinning is still in the early stages of addressing clinical challenges, but the versatility of electrospinning devices combined with innovative approaches to biomimicry has made a huge impact in the field. Research on electrospun nanofibers has a richer field of exploration, including deepening the understanding of electrospinning process parameters, creating improved systems for studying cell-structure interactions, and increasing the biological function of nanofibers through ligand immobilization or growth factor encapsulation.
National Natural Science Foundation of China(11632013)
National Natural Science Foundation of China(11502158)
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