Fabrication of functional PLGA-based electrospun scaffolds and their applications in biomedical engineering

Materials Science and Engineering C 59 (2016) 1181–1194

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Fabrication of functional PLGA-based electrospun scaffolds and their applications in biomedical engineering Wen Zhao a,⁎, Jiaojiao Li a, Kaixiang Jin a, Wenlong Liu a, Xuefeng Qiu b, Chenrui Li a a b

Key Laboratory for Space Biosciences and Biotechnology, School of Life Sciences, Northwestern Polytechnical University, Xi'an, Shaanxi, China Department of Cardiovascular Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China

a r t i c l e

i n f o

Article history: Received 1 July 2015 Received in revised form 22 October 2015 Accepted 9 November 2015 Available online 11 November 2015 Keywords: Electrospun PLGA Biomedical engineering Tissue engineering Drug delivery Applications

a b s t r a c t Electrospun PLGA-based scaffolds have been applied extensively in biomedical engineering, such as tissue engineering and drug delivery system. Due to lack of the recognition sites on cells, hydropholicity and single-function, the applications of PLGA fibrous scaffolds are limited. In order to tackle these issues, many works have been done to obtain functional PLGA-based scaffolds, including surface modifications, the fabrication of PLGA-based composite scaffolds and drug-loaded scaffolds. The functional PLGA-based scaffolds have significantly improved cell adhesion, attachment and proliferation. Moreover, the current study has summarized the applications of functional PLGA-based scaffolds in wound dressing, vascular and bone tissue engineering area as well as drug delivery system. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . Functionalization methods for electrospun PLGA fibers . . . . . 2.1. Surface modification techniques of electrospun PLGA matrix 2.1.1. Plasma treatment . . . . . . . . . . . . . . . 2.1.2. Physical absorption and coating . . . . . . . . 2.1.3. Chemical methods . . . . . . . . . . . . . . 2.1.4. Surface graft polymerization . . . . . . . . . . 2.2. Composite fibers . . . . . . . . . . . . . . . . . . . 2.2.1. Blend electrospinning . . . . . . . . . . . . . 2.2.2. Co-axial electrospinning . . . . . . . . . . . . 2.2.3. Hybrid electrospinning . . . . . . . . . . . . 2.3. Drug-loaded fibers . . . . . . . . . . . . . . . . . . 2.3.1. Surface immobilization . . . . . . . . . . . . 2.3.2. Blending electrospinning . . . . . . . . . . . 2.3.3. Co-axial or emulsion electrospinning . . . . . . Application of PLGA-based scaffolds in biomedical engineering . . 3.1. Tissue engineering . . . . . . . . . . . . . . . . . .

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Abbreviations: PLGA, poly(lactide-co-glycolide); ECM, extracellular matrix; PLA, polylactide; PCL, poly(ε-caprolactone); CS, chitosan; GE, gelatin; FDA, Food and Drug Administration; PGA, polyglycolic; LA, lactide; GA, glycolide; RGD, Arg-Gly-Asp; EGF, epidermal growth factor; BMP-2, bone morphogenic protein-2; VEGF, vascular endothelial growth factor; NaOH, sodium hydroxide; SBF, simulated body fluid; ATCP, amorphous tricalcium phosphate; HA, hydroxyapatite; AA, acrylic acid; DTPA, diethylene-triaminepentaacetic acid dianhydride; PPy, polypyrrole; BMSCs, bone marrow stromal cells; CNTs, carbon nanotubes; SWNTs, single-walled carbon nanotubes; MWNTs, multi-walled carbon nanotubes; GBR, guide bone regeneration; NGF, nerve growth factor; NGCs, nerve guidance conduits; PVA, polyvinyl alcohol; PBS, phosphate buffer saline; DNA, deoxyribonucleic acid; hMSC, human mesenchymal stem cells; HNTs, halloysite nanotubes; DOX, doxorubicin hydrochloride; TCH, tetracycline hydrochloride; FA, flurbiprofen axetil; PVP, poly(viny pyrrolidone); BSA, bovine albumin; PDP, pressuredriven permeation; MSM, dimethyl sulfoxide; Lys, Lysine; SMCs, smooth muscle cells; PTX, paclitaxel; BFA, brefeldin A; RCT, rotator cuff tear; bFGF, fibroblast growth factor; Nrg, neuregulin-1; RFGD, radio-frequency glow discharge. ⁎ Corresponding author. E-mail address: [email protected] (W. Zhao).

http://dx.doi.org/10.1016/j.msec.2015.11.026 0928-4931/© 2015 Elsevier B.V. All rights reserved.

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3.1.1. Wound dressing . . . . . . . . . . . . . . 3.1.2. Vascular tissue engineering . . . . . . . . . 3.1.3. Bone tissue engineering . . . . . . . . . . 3.1.4. Applications in other tissue engineering areas 3.2. Drug delivery system . . . . . . . . . . . . . . . . 3.3. Issues remained to be solved . . . . . . . . . . . . 4. Conclusions and remarks on future challenges . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Electrospinning is a versatile fiber fabrication technique as demonstrated [1]. It has become the most reliable way to fabricate continuous fibers scaled from nanometer to micrometer, which is much smaller than the fibers obtained by the conventional techniques, such as phase separation and self-assembly. The electrospun fibers have high porosities and high surface-to-volume ratio [2], and they can perfectly mimic the native extracellular matrix(ECM). Electrospun fibrous scaffolds have been investigated as potential scaffolds for biomedical applications [3,4]. A lot of works have shown that the micro/nano-fiber structure was capable of supporting cell attachment and enhance cell proliferation. There are a wide range of materials which can be utilized in this technique, such as polymers [5] (natural and synthetic), ceramics as well as composites. Usually the natural polymers for electrospinning include collagen [6,7], gelatin(GE) [8,9], chitosan(CS) [10,11], and silk fibroin [12,13], while the synthetic polymers include polylactide(PLA) [14,15], poly(lactide-co-glycolide)(PLGA) [14,16,17], and poly(εcaprolactone)(PCL) [18,19]. Synthetic biodegradable polymers, such as PLA, PLGA and PCL, are more prevailing materials for the construction of nanofibrous scaffolds because of their good processability. PLGA, one of the most commonly used synthetic materials for preparing fibrous scaffolds in tissue engineering, has been approved for clinical application by the US Food and Drug Administration (FDA). It is a copolymer of PLA and polyglycolide (PGA) with faster biodegradation rate than PLA due to the presence of PGA. With a higher ratio of lactide (LA) to glycolide (GA) in the copolymer composition, PLGA has longer degradation time, better mechanical strength and increased hydrophobicity [20,21]. It is a promising material for tissue engineering because of its good mechanical properties, nontoxic biodegradation products and tunable biodegradation time. Electrospun PLGA nanofibrous scaffolds have been widely investigated in tissue engineering as described in many works [22–24], such as bone, wound dressing and drug delivery system. However, similar to other synthetic biopolymers, PLGA has poor hydrophilicity. It has poor absorbance of proteins, and only solves in nonpolar halogenated hydrocarbons include chloroform and methylene chloride. Furthermore, PLGA has no natural cell recognition sites on the surface of the polymers [25] leading to the poor cell affinity. In order to improve its surface performance, many surface modification techniques have been used. Although the scaffolds composed of single component of PLGA have been widely used in tissue engineering, sometimes it could be a limitation that the electrospun nanofibrous matrix has only one component or simple structure because of its low suitability to the complicated environments. Therefore, PLGA-based composites have been investigated [26–29]. Simple hybrids including synthetic and bioactive components (natural/synthetic polymer or bioactive factors) were electrospun into nanofibers with improved properties [30]. Using composite materials made from the combination of two or more components can complement the individual deficiencies of each component by the presence of the other one. Such composite matrix can be designed by the versatile electrospinning methods, such as simply blending electrospinning and co-axial electrospinning. The additional components might improve

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the hydrophilicity and mechanical properties of the PLGA-based scaffolds to make the matrices more promising in biomedical application. Although PLGA is non-toxic, the accumulation of degradation products and swelling of polymer may change the environment around the scaffolds, such as pH value [31] and mechanical irritation of the surrounding tissue and sometimes result in inflammatory response [32–34]. To overcome this problem, strategies have been studied with focus on altering the polymer components or loading of anti-inflammatory drugs. Recently, PLGA electrospun fibrous scaffolds have drawn remarkable attention in the area of tissue engineering and drug delivery system due to its well-known good processability and good mechanical performance. But some essential problems still remain to be addressed, like its inherent hydropholilic and inflammatory rsponse induced by degradation products. Using ʻElectrospinningʼ and ʻPLGAʼ as the keywords for literature searching through the Web of Science, we found that, most applications of electrospun PLGA not only rely on the single PLGA material, but also account on the post-treatment after electrospinning or improved electrospinning processes. In this review, methods to obtain functional PLGA-based electrospun scaffolds and their potential biomedical applications will be discussed. 2. Functionalization methods for electrospun PLGA fibers 2.1. Surface modification techniques of electrospun PLGA matrix Although PLGA is expected to mimic natural ECM environment, its poor surface characters make the cells hard to attach to the surface of the polymers. The characteristic of the interfacial layer between cells and materials is pivotal for cell adhesion, proliferation, migration and differentiation [3]. Many works have been done to make the hydrophobic surface into a hydrophilic and affinitive one, such as plasma treatment, surface graft polymerization, surface entrapment and so on [21, 35,36]. Some modifications aim to immobilize some biomolecules on the surface of the scaffolds. Such biomolecules on the surface can be recognized by cells specifically. These biomolecules include some adhesive proteins such as collagen, fibronectin, Arg-Gly-Asp (RGD) peptides, and growth factors such as epidermal growth factor (EGF). Incorporation of these bioactive agents on the surface of electrospun fibers could make the scaffolds more biofunctional. Surface modification has advantage of maintaining the good mechanical properties of the electrospun fibrous scaffolds while giving the scaffolds improved cell adhesion property. 2.1.1. Plasma treatment Gas plasma treatment is a common post-treatment modification of electrospun fibers as demonstrated [37–39] that can generate desired functional groups, such as hydroxyl and amino groups on the surface of the polymers [40,41], to influence the cell-scaffold interactions. Plasma treatment can only affect a limited depth from the surface, so the properties of the base materials are rarely changed. Different plasma sources can create different functional groups on the surface of polymers. For instance, carboxyl or amino groups can be created under the condition of oxygen or ammonia gas. It has been reported that the electrospun PLGA nanofibers could be treated with plasma in the

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presence of either oxygen or ammonia gas to modify the surface of the nanofibrous scaffolds [41–44]. The modified electrospun PLGA nanofibrous scaffolds had increased surface hydrophilicity and improved cellular affinity. Kim and Jeong obtained PLGA fibers by meltelectrospinning method and then modified the surface of the fibers with either oxygen or ammonia gas [40]. The results showed that different plasma treatment made different influence on the fibers. The presence of amino gas made the surface more hydrophilicity than oxygen treatment. X-ray photoelectron spectroscopy analysis indicated that the number of polar groups on the surface of PLGA fibers increased after plasma treatment, such as hydroxyl and amino groups. In another research [43], ammonia gas plasma treatment was used to control the surface hydrophilicity and composition of electrospun PLGA nanofiber matrices. The stress response of fibroblasts was studied. The result showed that the nitrogen-containing functional groups on the surface had significant influence on cell adhesion and migration. Hydrophilicity and composition of the surface strongly influenced the cell response. Except the polar groups, plasma treatment can also be used to immobilize various bioactive molecules covalently on the polymer surface, such as GE, collagen, laminin and so on, to enhance cellular adhesion and proliferation [35,45]. Electrospun PLGA fibrous scaffold was modified by collagen coating after air plasma treatment to better mimic the composition of the natural bone ECM [46]. Plasma treatment facilitated the collagen immobilization on the scaffold, which could better support cell adhesion. Plasma treatment of electrospun PLGA can alter the cell-material interactions. It's a beneficial method to improve biocompatibility of the PLGA scaffolds. This approach to modify the surface properties of the tissue engineering scaffolds might be of great use in designing and tailoring of novel synthetic biomaterials in the area of tissue engineering. But according to some reviews [47,48], overtreatment might make etching and ablation damages on the surface of PLGA scaffolds. Plasma treatment mainly has four effects: surface cleaning, ablation or etching, crossing and modification of surface-chemical structure. Ablation, or plasma etching, could remove a weak boundary layer and increase surface area. But when PLGA is exposed to plasma for a long enough time, the exposed layer will etch off, etching reaction happens, and the polymer scaffolds will degrade [25]. So controlling the time of plasma treatment is important in surface modification. Remote plasma treatment was used to modified PCL nanofibers [49], which could minimize unwanted etching and ablation damages. In my opinion, despite of the etching and ablation damages, plasma treatment is an effective method to make the surface more hydrophilicity and affinitive to cells. We can easily control the accurate time and the distance from the plasma source to make it a better method in surface modification methods. Undoubtedly, more papers will be followed on the plasma modification of PLGA electrospun scaffolds. 2.1.2. Physical absorption and coating The physical method like absorbing or coating is an easy way to produce an interface more affinitive to the cells. It's the simplest but efficient way to load biomolecules (drugs or proteins, such as growth factors) on the fibrous meshes making the scaffolds more promising in biomedical applications. Electropun PLGA scaffolds modified by HFBI (a kind of hydrophobins) or HFBI/collagen were obtained by immersing in the prepared solutions [50]. The scaffolds modified by HFBI demonstrated improved hydrophilicity. Furthermore, the introduction of HFBI could promote collagen immobilization on the surface of PLGA. And both modified electrospun PLGA scaffolds showed higher efficiency in promoting cell adhesion than the native PLGA scaffolds. Bone morphogenic protein-2 (BMP-2) has been loaded on PLGA scaffolds, which had sustained release within 20 days in a research [51]. It can be concluded that BMP-2 encapsulated fibrous scaffolds were promising delivery devices. In some cases, the interaction between heparin and growth factors was used to modify the surface of fibrous electrospun fibers as well [52,53]. Small diameter vascular grafts composed of elastin,

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PLGA and PCL were fabricated by electrospinning, and heparin and vascular endothelial growth factor (VEGF) were added to prevent thrombosis and promote endothelial cell growth [54]. Sometimes, perhaps the ways like absorbing or coating are simple to fabricate modified surface, but the biomolecules on the surface might have a weak interaction with the biomaterials [35]. Accordingly, surface entrapment is used as an easy physical way to produce strong interaction between the biomolecules and the surface of the scaffolds, which was firstly proposed by Desai and Hubbell [55]. The polymer matrix can swell in the excellent solvent and deswell in the poor solvent which could immobilize the natural macromolecules on the surface of scaffolds. PLGA nanofibrous scaffolds modified by GE and sodium alginate/GE were fabricated by surface entrapment and entrapment-graft treatment [56]. Both nanofibrous membranes exhibited high hydrophilicity, improved biocompatibility and enhanced tensile strength compared to pure PLGA membranes. The scaffolds modified by physical methods may have a weak bond, but this character can be used in drug delivery system to obtain a high drug concentration during the burst-release stage. 2.1.3. Chemical methods Although plasma treatment is popular, it fails to modify the deeply located fibers in the scaffolds. Chemical methods are better choices to modify the thick fibrous meshes [21]. Surface hydrolysis or aminolysis is a simple and effective way to increase the surface hydrophilicity and wettability of the electrospun scaffolds or to create new functional groups for immobilizing biomolecules on the surface, such as collagen, CS and peptides [57,58]. The most common reagent is sodium hydroxide (NaOH). NaOH treatment can produce carboxyl groups on the fiber surface to improve the wettability of the materials [57]. But papers about this method applied on modifying electrospun PLGA fibers are few. Immersion in simulated body fluid(SBF) is another common wet chemical method that can deposit hydroxyapatite(HA) to mineralize the electrospun fiber surface [59]. Mineralized PLGA electrospun nanofibers with calcium phosphate apatite on the surface have been fabricated by immersing in SBF in a recent study [60]. This method created an osteophilic environment similar to the natural ECM for bone cells. Amorphous tricalcium phosphate (ATCP) is a precursor of HAp formation. PLGA/ATCP fibers were fabricated by an electrospinning process and then soaked in SBF for the deposition of HAp [61]. The HAp on the surface of the nanocomposite revealed a highly increased bioactivity compared with pure PLGA. In an another research, electrospun PLGA nanofibrous mesh was pretreated with CS and heparin after plasma treatment, then immersed in 10SBF for mineralization [62]. These treatments could facilitate the formation of thick, uniform HAp coating on the fibrous mesh. The HAp coating improved the mechanical properties of the scaffolds. However, the wet chemical methods, like immersing in SBF, need one or more weeks to prepare biomimetic deposition. The time is too long for practical applications. Fibers with larger specific surface area will shorten the time needed in wet chemical method but not short enough. Chemical method is a flexible and functional way to modify thick fibrous meshes. It can improve the interactions between cells and materials. But this method hasn't appeal enough attention on the modification of electrospun PLGA fibers. 2.1.4. Surface graft polymerization Surface graft polymerization is also an efficient way to modify the surface of electrospun fibers. It is usually used in combination with plasma treatment or wet chemical method. Bioactive molecules can be covalently immobilized on the surface of fibers after the treatment with plasma or UV radiation or chemical reagents to enhance cell adhesion, proliferation and migration [63–66]. Particular functional groups or macromolecules grafted on the surface of the electrospun PLGA scaffolds could improve the performance and make them more promising in biomedical applications. Electrospun fibrous scaffolds from PLLA,

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PGA and PLGA have been modified by the combined method of oxygen plasma treatment and situ-grafting of hydrophilic acrylic acid (AA) [67]. Carboxylic functional groups were introduced on the surface of the scaffolds and made them more hydrophilic and cell-compatible. Their interactions with fibroblasts had been studied as well in this study. The in vitro experiment showed that the surface modified scaffolds made significant improvement on cell attachment and proliferation. Arg-Gly-Asp (RGD) was modified on the surface of PLGA nanofiber mesh to better mimick ECM [29]. In this study, PLGA and PLGA-b-PEGNH2 di-block copolymer were electrospun to fabricate PLGA nanofibers with amino groups on the surface. Then RGDDY was conjugated on the surface of the electrospun nanofibers. NIH3T3 fibroblast cell adhesion and proliferation were studied. The results exhibited enhanced cell adhesion, proliferation and migration. In another research, radionuclide was conjugated on the modified surface of electrospun PLGA nanofibrous membrane [68]. Electropun PLGA membranes were immersed in NaOH solution firstly, then immersed in DMTMM aqueous solutions to activated carboxyl groups on the surface of membranes and incubated in GE aqueous solution secondly, and then the PLGA-gGE membranes were incubated in diethylene-triaminepentaacetic acid dianhydride (DTPA) solution thirdly to obtain PLGA-g-GE-DTPA nanofibrous membranes. The obtained membranes were then conjugated with radioactive yttrium 90Y for tumor internal radiotherapy. The modified electrospun PLGA membrane showed good stability in saline, and has good hydrophilic and mechanical properties. In some special area like neural tissue engineering, electroconducting polymers polypyrrole(PPy) have been deposited on the electrospun PLGA fiber templates to produce electroconducting nanofibers by the method of polymerization in a solution containing porrole, pTS, and FeCl3 [69]. The PPy-coating meshes displayed good electrical activity and performed better in modulating cellular interactions compared to PLGA control nanofibers (Fig. 1). All these cases suggested that functional groups on the surface might serve as surface-modification agents of many bioactive molecules or drugs. Surface graft polymerization might have a strong connection between the matrices and bioactive molecules, the modified PLGA fibers might be more stable to be applied in more areas. Surface modification can make the surface more hydrophilic and affinitive to cells without changing the bulk properties of the nanofibrous scaffolds. With well-defined surface modifications, the modified PLGA with good mechanical properties become more hydrophilic and biocompatible. The present surface modification methods might make the surface-functional PLGA fibers more promising in biomedical engineering. But considering the modification conditions, such as plasma, acid, and high temperature, the future work will pay special attention to find moderate conditions to protect the fibers from destruction and degradation. 2.2. Composite fibers The fabrication of composites will be a more facile and cost-effective way to modify the material properties. Composite fibers may possess better hydrophilicity and provide cell recognition sites on the surface of the scaffolds to promote cell adhesion, proliferation, migration and differentiation. Composite fibers can be divided into three categories based on their structure: randomly blended structure, core-shell structure, and mingle fibers, that can be fabricated by blend electrospinning, co-axial electrospinning and hybrid electrospinning respectively. 2.2.1. Blend electrospinning The electrospun blended composite nanofibrous scaffolds not only overcome the limitations associated with a single polymer, but also create a new component without the need for synthesizing a new copolymer, which is difficult and complex. Electrospinning multicomponent scaffolds has attracted much attention recently.

Fig. 1. PPy-coated PLGA meshes. (a) Uncoated PLGA meshes (white, left) and PPy–PLGA meshes (black, right). (b) SEM micrograph of single strands of PPy–PLGA fibers. (c) SEM image of section of the PPy–PLGAmeshes [69].

The components blended composite nanofibers can be divided into two categories: organic–organic blends and organic–inorganic blends as described [70]. The organic–organic blends are made from synthetic and natural polymers. The synthetic biomaterial PLGA with good mechanical properties is poor in hydrophilicity and lacking in cell-recognition signals. In contrast, the natural polymers, such as CS, collagen, and so on, have the potential advantages of specific cell interactions and a hydrophilic nature but possess poor mechanical properties. Many researchers have explored an amount of organic–organic blends to improve the bioactivity and functions of the electrospun scaffolds. The proposed tubular longitudinally aligned and random PLGA/PCL scaffolds were fabricated by electrosponning the blends of PLGA and PCL [71]. PCL is flexible so that it can overcome the brittle and low elongating nature of PLGA. The result showed that the PLGA/PCL composite scaffold could offer good flexibility, desired porosity, slow degradability and topographical cue with better biological properties. Furthermore, cell proliferation on the aligned nanofibers was significant higher than on random ones. The hybrid PLGA/PCL electrospun scaffolds have been studied in many works [70,72,73], all the scaffolds showed better biocompatibility than pure PLGA scaffolds. In an early study [26], composite scaffolds were fabricated by co-electrospinning from a blend of PLGA (10% solution) and two natural proteins, GE (8% solution) and α-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The obtained fibers have smaller

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average diameter (380 ± 80 nm) compared with pure PLGA, and were homogeneous in appearance. The scaffolds displayed suitable mechanical properties and an enhance capacity to support 3D tissue-like assembly when compared with electrospun nanofibrous scaffolds made from single component PLGA. The cultured H9C2 rat cardiac myoblast and rat bone marrow stromal cells (BMSCs) were found to grow well. This kind of scaffolds was proposed for engineering soft tissues, such as heart, lung and blood vessels. This novel composite scaffold exhibited advantages beyond those of the individual component, providing the proof-of-concept for tailoring scaffolds for specific tissue engineering purposes. Randomly-oriented and the aligned PLGA and PLGA/GE scaffolds were fabricated through electrospinning [74]. As the GE content increased, the nanofiber diameter decreased and the hydrophilicity increased, but the mechanical properties of the scaffold decreased. The result showed that the addition of GE enhanced the adhesion and proliferation of the cells. In a word, the introduction of structural proteins such as collagen and elastin can improve the physicochemical and biological properties of the electrospun PLGA fibrous scaffolds [75,76]. Recently, the organic–inorganic hybrid scaffolds have gained more and more attention in biomaterials science. Inorganic nanoparticles have often been incorporated to improve mechanical properties of the polymer scaffolds for bone tissue engineering [30]. As reported, inorganic nanoparticles such as Ag [77], nano-hydroxyapatite (nHA) [78–80], carbon nanotubes (CNT) [81] have been used in fabricating nanofibrous composite scaffolds for tissue engineering. HA is among the family of calcium phosphate-based bioceramics [82,83]. The incorporation of nHA into the electrospun scaffolds can not only mimic the natural bone structure but also can enhance the mechanical properties of the scaffolds because it is nontoxic, bioactive and osteoconductive, and its chemical and crystalline structure are similar with the nature bone minerals [84]. Moreover, incorporation of nHA particles into the polymer matrix is considered to slow down the degradation process by neutralizing of buffering the pH changes caused by the typical acidic degradation products of polyesters [85,86]. Considering these advantages listed up, compounding of the biopolymers and HA is one of the effective ways to fabricate bone tissue engineering scaffolds. Aligned nanofibrous scaffolds based on PLGA and nHA particles were synthesized by electrospinning for bone tissue engineering [79]. Results showed that the inorganic nHA particles acted as reinforcements at lower concentrations (1% and 5%) but acted as defects at higher concentrations (10% and 20%). The composite scaffolds had improved mechanical properties and protein adsorption, while maintaining high porosity and suitable microarchitecture. CNTs, which exist as either single-walled carbon nanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs), are allotropes of carbon. They have caught widely interest in biomedical scaffolds since the discovery in 1991. CNTs have been reported to promote human osteoblasts attachment, proliferation, and differentiation [87, 88]. CNTs exhibit the potential to mimic the role of collagen and serve as scaffolds for the growth of hydroxyapatite [89]. All these unique advantages make CNTs a potential material in the area of tissue engineering. It is also reported that the incorporation of even small quantities of CNTs into polymeric materials significantly improves their mechanical, optical, electrical, and thermal properties, and their biocompatibilities [90,91]. In a work [92], PLGA, MWNTs, and wool keratin were successfully electrospun into nanofibrous membranes. The mechanical properties of the composites were significantly enhanced by the incorporation of the MWNTs, and it showed great bioactivity. This study showed great potential in the guide bone regeneration (GBR). PLGA/MWNTs nanofibrous scaffolds have been fabricated by electrospinning in many present works [90,93,94], the scaffolds were composed of continuous and uniform fibers, their 3D structure mimicked the ECM structure well, and possessed improved mechanical properties as well as significantly increased the attachment and proliferation of cells compared with the pure PLGA scaffolds.

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2.2.2. Co-axial electrospinning The second category of composite nanofibers is in the form of coreshell or core-sheath structure. Just as its name, it consists of a core of one polymer and a shell of another polymer. This kind of fibers can be produced by electrospinning as well. Co-axial electrospinning is a modification technique of ordinary electrospinning process, which was first reported in 2002 to prepare nanofibers with core-shell structures [95]. Nanofibrous composite with core-shell structure can be fabricated by co-axial electrospinning well [95,96]. In co-axial electrospinning, two solutions are coaxially and simultaneously electrospun through different feeding capillary channels into one nozzle to generate composite nanofibers with a core-shell structure. It has numerous advantages comparing with ordinary electrospinning. For example, the shell outside can protect the core material from the hostile environment. Coaxial electrospinning has attracted more and more attention because it can endow biomaterials with functional property for different applications. Modified PLGA/starch fibrous scaffolds were obtained by coaxial electrospinning method as demonstrated [97]. The composite fibers had improved hydrophilicity, biocompatibility and degradation after adding starch into PLGA fibers. Core-shell PLGA/CS fibrous membranes fabricated by co-axial electrospinning were studied [27]. The scaffolds exhibited higher hydrophilicity than pure PLGA scaffolds. The evidence on cytocompatibility manifested that the membranes could facilitate cell adhesion and migration. The obtained PLGA/CS fibrous membranes with core-shell structure might have potential applications in drug release and skin restoration. Using the PLGA as core and the natural polymers as shell, fibers with good mechanical properties and affinitive surface could be fabricated by co-axial electrospinning to solve the problem of the poor biocompatibility of electrospun PLGA nanofibers. As mentioned up, bioactive additives can be incorporated into the nanofibers by simply blending in the electrospun solution. Coaxial electrospinning provide a new way to fabricate fibrous scaffolds with bioactive additives. Biomolecules, such as anti-cancers, antibiotics, enzymes and proteins, can also be introduced as the core phase by coaxial electrospinning. In a study [98], nerve growth factor (NGF) was incorporated into the aligned core-shell nanofibers by co-axial electrospinning, in which NGF was the core layer and PLGA was the shell layer (Fig. 2). The aligned PLGA/NGF nanofibers were then reeled to develop aligned nerve guidance conduits (NGCs). The result showed that NGF was successfully incorporated into the core-shell nanofibers, a sustained release was observed for 1 month. The in vivo experiment indicated that the nerve regeneration in PLGA/NGF NGC was significant better than that in PLGA NGC. It could be concluded that the aligned PLGA/NGF NGCs had a potential application in the peripheral nerve regeneration. 2.2.3. Hybrid electrospinning Mingled fibrous composite was defined as the hybridization of two or more different nanofibers. Different biomaterials are electrospun individually into fibers to obtain random or homogenous structures. With this electrospinning process, not only some advantages in physical and mechanical properties can be obtained, but also the cell penetration can be improved in the electrospun nanofibrous scaffolds. Although surface modifications can improve the wetting property of PLGA surface, the modification process may be very complicate and it may result in some toxicity problems. To solve these issues, the nanofibers mingled structures could be a choice. In a study [99], PLGA–HA nano-hybrid scaffolds were obtained by the multi-spinning process. The hybrid scaffolds showed a negligible toxic influence in cell culture tests, and the addition of HA improved the wettability of the scaffolds, enhanced the attachment and proliferation of cells. PLGA and CS/polyvinyl alcohol (PVA) were simultaneously electrospun from two syringes to fabricate PLGA–CS/PVA nanofibrous composite membrane [100]. The introduction of CS/PVA changed the hydrophilic/hydrophobic balance, influenced the mechanical properties, degradation behavior and improved cell proliferation and

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Fig. 2. TEM images of (A) PLGA and (B) PLGA/NGF nanofibers [98].

attachment on the membranes. Furthermore, the composite scaffold showed higher phosphate buffer saline (PBS) absorption than electrospun pure PLGA membrane. This scaffold had a little shrinkage after incubation in PBS for 24 h and possessed moderate tensile strength and tensile modulus and larger elongation. The result showed that the scaffold combined with the advantages of both PLGA and CS would have a potential use in skin tissue engineering. In another research [76], the blended nanofibers of PLGA/GE (50:50) was applied to treat myocardial infarction. The addition of GE increased the hydrophilicity and biocompatibility of the scaffolds. But the tensile and elastic strength of PLGA was reduced. It was concluded that the blended nanofibers were more acceptable than PLGA alone in the treatment of myocardial infarction. This result indicated this scaffold could be novel soft tissue materials in the tissue engineering area. 2.3. Drug-loaded fibers Electrospun nanofibers loaded with drugs will be more promising in tissue engineering. PLGA fibers have been considered as ideal drug carriers as demonstrated [101]. Drugs (antibiotics, anticancer drugs), proteins, and genes can be incorporated into the fibrous membranes by many methods, for example, surface modifications, blend electrospinning and co-axial electrospinning [102]. 2.3.1. Surface immobilization Drugs can be immobilized physically or chemically on the surface of the fibrous membranes [37,103]. Physical absorption is the simplest way to load drugs on the fibrous meshes. Drugs can be immobilized on the surface by direct deposition and other indirect methods [37]. Naked deoxyribonucleic acid (DNA) was coated onto the electrospun PLGA/HAp composite scaffolds, which could be used to deliver gene into human mesenchymal stem cells (hMSCs) in vitro and in bone regeneration [104]. BMP-2 loaded PLGA/HAp composite scaffolds have been studied in a series works [51,105]. The drug delivery systems had potential application in bone regeneration. But surface modifications could hardly regulate the drugs' location and distribution on the fibers surface, along with burst release. Usually this method is seldom

used to load proteins or genes on the surface of electrospun fibrous scaffolds due to the uncontrolled release profiles. 2.3.2. Blending electrospinning Drugs can be mixed into the polymer solution and then electrospun to obtain drug-loaded fibers [106,107]. A novel electrospun composite nanofiber-based drug delivery system was obtained as described in a study [108]. The model drug-loaded halloysite nanotubes (HNTs) were mixed with PLGA polymer for electrospinning to obtain drugloaded nanofibrous mats. The drug-loaded HNTs had good encapsulation efficiency, and HNTs improved the tensile strength of the nanofibrous mats. As both PLGA and HNTs were drug carriers, the burst release of the drug reduced. This double-container drug delivery system might have potential applications in tissue engineering and pharmaceutical sciences. In another study, CNTs can also be used as drug containers [109]. Doxorubicin hydrochloride (DOX) loaded-CNTs were mixed with PLGA solution, and then a drug delivery system was prepared by electrospinning. This composite delivery system prolonged the release rate of the drug and eliminated the initial burst release. The developed system would have widely applications in tissue engineering and pharmaceutical sciences. PLGA-based electrospun fibrous scaffolds could also be used as protein release systems by electrospinning the blends of PLGA and protein [110]. Sometimes, growth factors could be mixed in PLGA electrospun scaffolds to release by a sustained rate [111–114]. However, there still remain many challenges, for example denaturing due to touching with organic solvent and burst release from the scaffolds. 2.3.3. Co-axial or emulsion electrospinning Co-axial or emulsion electrospinning is a potential approach to overcome these drawbacks of blending electrospinning and surface modifications. In this process, drugs or proteins can be incorporated into the core polymer to reach the purpose of sustained release with the polymer shell as barrier [115–118]. Emulsion electrospinning is a promising method in the delivery of hydrophilic drugs via W/O emulsion to reduce the initial burst release. The core-shell structure has a controlled release profile, potentially yields a zero-order profile. The drug release kinetics can be controlled by the thickness of the polymer shell. The core-shell

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structure nanofibers with bioactive additives as core are potential in solving the denaturing and burst release problems. Drug loaded coreshell nanofibrous scaffolds with tetracycline hydrochloride(TCH) as core and PLGA as shell were prepared by co-axial electrospinning [119]. The core of TCH endowed the scaffolds of antibiotic. It has been verified that the nanofibrous PLGA scaffolds had the potential use in wound healing area. In another research [120], flurbiprofen axetil(FA)-loaded poly(viny pyrrolidone) (PVP)-nanopoly(lactic-coglycolic) core-shell composite nanofibers were successfully fabricated by a facile co-axial electrospinning. FA was first loaded in PVP via physical blending. Then FA-loaded PVP and PLGA solutions were co-axial electrospun to obtain drug-loaded PLGA/PVP nanofibers. The drugloaded PLGA/PVP nanofibrous membranes had improved adhesion prevention activity and significantly reduced the initial burst release of FA. Both of the core and shell materials acted as barriers to slow down the release rate of FA. This kind of drug delivery system might have various applications in tissue engineering and pharmaceutical science. Growth factors can be encapsulated in the core material by co-axial electrospinning [121]. Growth factors are more fragile than drugs or bovine albumin (BSA). The shell polymer can avoid the factors attaching organic solvent. This core-shell structure can deliver growth factors in a sustained way with maintained activity. In future work, the study may focus on the research of the delivery of plasmid DNA, large protein drugs and genes to the target sites. Electrospun PLGA fibers are promising drug carriers for various applications in biomedical engineering. But the incompatibility between PLGA and the loaded drugs must draw more attention to control the release of the drugs. On the other hand, the burst release of the drugs in the electrospun PLGA-based fibrous scaffolds is another difficult problem. 3. Application of PLGA-based scaffolds in biomedical engineering Electrospun nanofibers have been verified to be of great use in tissue engineering and drug delivery system [122]. PLGA has been electrospun for various applications in biomedical engineering because of its biocompatibility, biodegradability, nontoxicity and tunable mechanical properties. In this paper, we will summarize some applications of electrospun PLGA nanofibers in biomedical engineering and some issues remained to be solved. 3.1. Tissue engineering 3.1.1. Wound dressing Skin is the largest body organ, functioning as a barrier to harmful mediums, preventing pathogens from entering into the body. A wound is result from physical, chemical, mechanical and/or thermal damages. The natural healing process of skin is complex and continuous. The treatment of skin lesions is a critical issue in healthcare. Usually, the treatment for skin loss is traditional autografts and allografts. As the tissue engineering is rising, it has emerged as an alternative treatment for

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excessive skin loss. Wound dressing has attracted great interest in this area. Wound dressing materials have changed continuously and significantly during the past years. In present time, nanopatterned electrospun fiber meshes are shown to be helpful in treating a variety of wound conditions [123]. Due to their high surface area to volume ratio and high porosity, the nanofibers can cover and protect the wound, protect the wound area from the loss of fluid and proteins, drain the wound exudates, isolate the wound from bacterial infection and their 3D structure allow gas and nutrients transportation. They could enhance the healing process of wounds or skin regeneration significantly. In the electrospinning process, nanopatterned fiber meshes are collected on a plat grounded collector. Nowadays many polymers, including natural materials, synthetic materials and combinations of both types, have been electrospun to obtain nanofibers meshes for the application of skin regeneration. PLGA have been widely electrospun into nanofibrous membranes to be used as wound dressing as described [124,125]. It has been found that the PLGA-based hybrid nanofibrous scaffolds exhibited better performance in wound healing process [125,126]. In a work [127], porous PLGA and PLGA/MWCNTs membranes were prepared by electrospinning process. Cell viability studies on these two types of scaffolds showed that cell attachment and proliferation on PLGA/MWCNTs membranes were better than pure PLGA scaffolds. The results showed a rational design for artificial skins. To inhibit hypertrophic scars of the skin, a novel PLGA-Rg3/CS electrospun nanofibrous membranes were studied [128]. Ginsenoside-Rg3 (Rg3) has poor solubility, and PLGA is hydrophobic. Due to these disadvantages, PLGA-Rg3 mixed fibers tend to be relatively hydropholic which is not conducive for cell adhesion and growth on the surface of the scaffolds. CS is a natural polysaccharide which is not only non-toxic and degradable, but also has been verified to show hemostasis, odynolysis, bacteriostasis and promotes cell growth and accelerated wound healing [129]. Coating the surface of PLGA scaffolds with CS can improve the surface hydrophilicity. PLGARg3 electrospun fibrous membranes coated with CS were fabricated by combining electrospinning and pressure-driven permeation (PDP) technology. The obtained scaffolds showed better effect of inhibiting hypertrophic scar formation than PLGA-Rg3 or PLGA/CS membranes alone (Fig. 3). PLGA-based multicomponent nanofibrous membranes fabricated by electrospinning method have been studied in many researches to examine their potential applications in wound healing [130]. Antibacterial property is a critical function of wound dressing. Sometimes, the incorporation of antibacterial agents into the scaffolds will decrease infections and significantly speed up the wound healing process. Antibacterial dressings containing nano-silver powders (nAg)/dimethyl sulfoxide (MSM)/PLGA were fabricated by electrospinning [131]. The results showed that with an increase of n-Ag content, the strength of the fibers and water absorption ability enhanced. Due to the n-Ag, the scaffolds showed a good antibacterial ability against the Gram-positive staphylococci and Gram-negative Escherichia

Fig. 3. Electrospun membranes in wound healing time. (A) Photographic imaging of the status of wound healing. (B) The wound healing time of different groups [129].

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coli. It can be concluded that this kind of wound dressing had a biocompatibility and antibacterial properties, and would have a broad application in the wound dressing area. Electrospinning PLGA with 3D nanofibrous structure as drug carriers, could be used as excellent skin repair scaffolds to help heal wound [126,132,133]. To mimic the real structure of skin, bilayer scaffolds have been developed [134,135]. The complex structure of two layers can provide several advantages. The dense upper layer is used to prevent further infection and decrease the loss of fluid. The porous lower layer is thicker and it can absorb the wound exudates well. In a recent study [135], a novel bilayer scaffold consisted of electrospun PCL and PLGA membrane and glutaraldehyde (3.5% v/v) cross-linked CS/GE hydrogel was fabricated using electrospinning and casting. This structure mimicked the twolayer structure of the native skin. This scaffold could exhibit advantages not found in single-layered scaffold. In this bilayer scaffold, the synthetic polymers provided improved physical properties and mechanical strength; the hydrogel layer was observed to be very biocompatible, evidenced by high proliferation and favorable adhesion of human dermal fibroblast cells but without eliciting immune response. This bilayer structure scaffold must have great potential in wound dressing area. Nanofibrous PLGA-base scaffolds made by electrospinning have played a vital role in the wound dressing area. Hybrid scaffolds of natural and synthetic polymers can result in excellent scaffolds with desired physic-chemical properties with biocompatibility. Growth factors and drugs can be incorporated into the scaffolds to accelerate wound healing process. But it's still a challenge to fabricate nanofibrous scaffolds to intimate the real structure of the skin, especially the regeneration of new capillary and nerve. 3.1.2. Vascular tissue engineering Nowadays the need of vascular grafts, especially small diameter (b6 mm) grafts, is becoming more and more serious year by year. The available substitutes of vascular grafts have been limited by the high cost in clinic application, and the hierarchical structure is another challenge for vascular graft development. Instinctively, these problems require unique strategies for fabricating suitable synthetic biomaterials for vascular application. The engineered vascular grafts should be compatible with the host vessels nearby [136]. Electrospinning provides an efficient but low-cost method to obtain vascular grafts with different diameters to fit the specifications of any vascular conduit. It has shown potential use in producing tubular scaffolds which could satisfy the need of vascular grafts. The electrospun nanofibrous scaffolds have already been studied in vascular tissue engineering application [137–139]. And vascular graft scaffolds, fabricated by electrospinning PLGA, have been studied in a series of works [16, 140,141]. These studies indicated that the electrospun PLGA scaffolds possessed ultrafine fibrous and porous structure, had adequate mechanical properties, easy controllability of diameter, and alignment, which is suitable for blood vessel substitutes. After implantation, the vascular scaffolds, with large surface area, high porosity and good flexibility, perform well in transporting nutrients and oxygen for the native living cells. In general, synthetic polymers PLGA are easily electrospun into desired structures with good mechanical properties and controllable degradation rate. Despite these advantages, the PLGA vascular scaffolds are insufficient for cell-recognition signals, and their hydrophobic property will obstruct the cells seeding. Researchers have explored many methods to improve the current application situation of the electrospun PLGA scaffolds, such as blending PLGA with other natural polymers, introducing bio-additives into PLGA scaffolds, and so on. Vascular scaffolds electrospun from synthetic and natural materials have been successfully obtained with smaller inner diameter. In a recent work [54], vascular grafts composed of PLGA, PCL and elastin were fabricated by electrospinning technique with smaller diameter (3 mm). In particular, heparin and VEGF were introduced to the scaffolds. They could prevent the thrombosis formation and promote endothelia cells growth on the scaffolds surface. In vivo experiments found that the

scaffolds could promote the recovery process of broken vessels, enhance cells attachment, migration and proliferation on the scaffolds. The electrospun nanocomposite will be of great potential in the vascular tissue engineering with smaller diameter. In a modified electrospinning technique, VEGF was successfully incorporated into core-shell ultrafine fibers by coaxial electrospinning, using PLGA as the shell polymer [142]. The data demonstrated that the VEGF-incorporating PLGA core-shell nanofibrous membrane could have potential application in vascular tissue engineering. In general, the acidic degraded products of PLGA will hindered the cells proliferation. To tackle this problem, the basic amino acid Lysine (Lys) was introduced into the PLGA nanofibers to alleviate the acidity of PLGA nanofiber degradation in vitro [143]. Pure PLGA nanofibers were electrospun as a control. Vascular smooth muscle cells (SMCs) were cultured with the fluid containing degraded product to assess their cytotoxicity. The result showed that the incorporation of Lys enhanced the degradation process of PLGA and mitigated the decrease of PBS solution pH (acidity). It was also find that SMCs grew and proliferated well in the solution containing PLGA degraded products and Lys. This study provided an improvement of PLGA application in vascular tissue engineering. Tubular type PLGA scaffold fabricated by electrospinning have been widely used in the vascular tissue engineering area. The functional tubular PLGA scaffold can be obtained by the modified technique coaxial electrospinning, or incorporating the bio-additives into the matrix. The future work may be concentrated on the construction of hierarchical vascular scaffolds with smaller diameters (b6 mm) but without thrombosis and subsequent occlusion. 3.1.3. Bone tissue engineering The bone damage is becoming an increasing clinical problem. Nowadays, bone tissue engineering is the most promising field in tissue engineering area. It has become a rapidly expanding research area since it offers a new and promising approach for bone repair and regeneration. In present time, the electrospun nanofibrous scaffolds are of great potential use in bone repairation and regeneration. The scaffolds serve to support the cell growth and maintain phenotype. The electrospun fibers have high surface-to-volume ratio and high porosity, which provides a microenvironment for cell growth and facilitates nutrition transportation. In particular, the nanofibrous structure is similar to the natural bone extracellular matrix, and bring stimulus to the cultured cells toward bone formation [144]. PLGA is a prospective material in bone tissue engineering area since it could manipulate the mechanical properties and biodegradation. PLGA-based random and aligned fibers fabricated by electrospinning seeded with BMSCs were studied [145]. The results showed that PLGA-based scaffolds could provide a promising platform to guide bone repair and reconstruction. However, PLGA will hinder cellular affinity and bioresponsivity because of its hydrophobility. Many measures have been taken to improve this phenomenon. For instance, PLGA can be used to blend with different types of bio-additives to improve its initial properties or endow the scaffolds with more biocompatibility or improved functions. Many bioactive macromolecules, such as collagen, GE, angiogenic growth factors, MWNTs, nHA and amorphous calcium phosphate and so on, can be incorporated in the engineered scaffolds. Collagen is one of the major components of bone ECM and can enhance cell adhesion and proliferation [147]. Aligned PLGA/collagen blended nanofibrous scaffolds were obtained by electrospinning, and characterized for bone tissue engineering application [148]. The scaffolds behaved higher biocompatibility after collagen incorporation. Mineralized PLGA/GE electrospun nanofibers have been fabricated by surface modification and its potential application in bone tissue engineering have been studied in a research [60]. The CaP coating had positive effect on cell adhesion and migration, which showed good biocompatibility. This study indicated that the mineralized PLGA/GE scaffold would be a suitable choice for bone tissue engineering. Although these electrospun matrices

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act as barriers for controlling the intrusion of soft tissues, they have no bioactivity or enhanced bone-formation ability. HA, another major component of bone, can promote the formation of bone-like apatite on the surface of the scaffolds [149]. It is widely used in the bone tissue engineering. The nano-hydroxyapatite particles (nHAp) can improve the mechanical properties and the mineralization ability of the scaffolds [150,151,78]. In a recent research, primary human osteblasts cell behaviors on the electrospun PLGA and PLGA/nHA scaffolds have been compared [146]. Fig. 4 showed the confocal fluorescent microscopic images of the cells on the PLGA-alone scaffolds and PLGA/nHA composite fibers. The results showed that the cells on composite fibers had more pseudopodia and spread better than PLGA-alone scaffolds, which meant the embedded nHA could significantly enhance the formation of the bone-like apatites on the surface of the scaffolds. This research showed a promising way for human bone repair. The PLGA/HA nanofibrous nanocomposite scaffolds for bone tissue engineering have been researched in many reports. All the results showed that the incorporation of nHA could improve the process of bone formation, and the nHA/PLGA composite nanofibrous scaffolds showed higher cellular adhesion, proliferation, and enhanced osteogenesis performance [78,79, 93,152]. However, the adhesion force between PLGA matrix and HA particles is so weak that it will result in deteriorating the mechanics properties. HA grafted PLLA/PLGA nanofibers were fabricated by electrospinning for bone regeneration membrane [153]. In this study, L-lactide on HA surfaces (HA-g-PLLA) was synthesized by the method of ring-opening polymerization. A certain amount of HA-g-PLLA powders were uniformly suspended in chloroform, which was mixed with 8 wt.% PLGA/chloroform solution. Then blending solution was electrospinning under optimize conditions to get nanofibrous membranes. At 5 wt.% HA-g-PLLA content, the composite membrane reached the best tensile strength compared with pristine PLGA. The content of HA-g-PLLA would significantly influence their wettability, degradation, and bioactivity. This study made a tight combination between PLGA and nanoparticles. It also made PLGA more popular in the bone tissue engineering. In a work, a series of PLGA/MWNTs/wool keratin membranes were successfully electrospun to meet therapeutic application for different bone defects [92]. The composites possessed high

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bioactivity, presented good values of ultimate strength, elongation at break, and Young's modulus, high thermal and thermooxidative stabilities. An electrospun bio-hybrid scaffold consisting of silk fibroin, calcium phosphate and PLGA, which was considered as an effective scaffold for bone tissue engineering application, was studied [112]. The composite nanofibrous scaffolds were used as VEGF delivery system. The release profile of VEGF during 28 days verified the efficacy of the scaffold as a sustained delivery system. The scaffolds could induce angiogenesis process during bone regeneration. Moreover, osteoblast cells next to the scaffold exhibited good adhesion, proliferation and alkaline phosphatase production. In summary, these functional nanocomposites were promising in bone tissue engineering applications. From all these examples above, we can conclude that PLGA-based nanofibrous scaffolds incorporated with other natural polymers or inorganic nanoparticles are of great potential in the bone tissue engineering area. From my opinion, although the inorganic nanoparticles might improve the mechanical properties of PLGA-based electrospun scaffolds, the strength would not be enough. Bone defect has been an inevitable problem, ideal bone substitutes with appropriate mechanical strength and bioactivity are still in great need, and PLGA-based scaffolds might be of great applications in soft tissues. 3.1.4. Applications in other tissue engineering areas PLGA-based fibrous scaffolds have also been widely used in other tissue engineering areas, such as nerve [69,73], cornea [154,155], cartilage [156], ligament [157] and ligament/tendon [111,158] and so on. Panseri [73] and Subramanian [71] obtained electrospun scaffolds from a blend of PLGA/PCL to be used in nerve tissue engineering. The former one concentrated on the in vivo experiments and the latter one concentrated on in vitro experiments. Both works showed positive performance in the process of nerve regeneration. In another research [159], a range of PLGA membranes with different proportions of collagen and hydroxyapatite nanoparticles were fabricated by electrospinning. These scaffolds had a potential use in bone/cartilage tissue engineering. A hybrid nano-microfibrous PLGA scaffold was demonstrated in 2006 [158]. The scaffold processed superior mechanical strength, integrity, and large surface area and better hydrophilicity. And this scaffold seeded with BMSCs could improve tendon/ligament healing and be used in

Fig. 4. Confocal fluorescent microscopic images of human osteoblasts (HOB) cells cultured on 20% PLGA mat, 20% PLGA/nano-HA mat, and glass after 14 days. The three bottom images share the same scale bar. HOB cells were stained with blue and green [146].

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tendon/ligament tissue engineering. In a recent work [160], electrospun PLGA membrane with microfabricated pockets was used to deliver cultured cells limbal explants to wounded corneas. In conclusion, PLGAbased scaffolds are promising in various tissue engineering areas. PLGA is a biomaterial approved by FDA for clinical use. Electrospun PLGA-based fibrous scaffolds satisfy the requirements for cell adhesion and proliferation with the potential applications in tissue engineering. The further work will be focus on in vivo experiments to demonstrate the effectiveness of these scaffolds. 3.2. Drug delivery system In recent years, drug delivery has attracted more and more attention in tissue regeneration and the treatment of various diseases. Electrospun fibers can act as drug carriers in the field of drug delivery [125,161,162]. Various biomolecules have been successfully incorporated into/onto electrospun fibers for controlled release [104,163–165]. Drug-loaded PLGA fibrous scaffolds have widely application in wound dressing [132] and cancer treatment [166,167]. Antibioticloaded wound dressing based on PLGA has been studied to reduce infections caused by bacteria [168–170]. In a recent work, paclitaxel(PTX) and polymeric micelles contained brefeldin A(BFA) were loaded into the eletrospun PLGA composite nanofibers [171] (Fig. 5). In vitro cytotoxicity studies revealed that the nanofibers with two hydrophobic drugs restrained HepG-2 cells more efficiently. The composite nanofibers with polymeric micelles contained hydrophobic drugs might be used as an effective controlled dual release system for postoperative chemotherapy of cancers. Curcumin loaded PLGA nanofibers were obtained by electrospinning for the treatment of carcinoma in a recent research [172]. The scaffolds exhibits high drug efficiency and sustained release but with no burst release. The drug-loaded PLGA scaffolds do also have applications in other clinical areas, such as shoulder pathologies, cardiovascular diseases and inflammation inhibition. A core-shell structure of PLGA fibrous scaffold loaded with bFGF for repairing rotator cuff tear (RCT) was fabricated by emulsion electrospinning [121]. The scaffold showed a sustained release of fibroblast growth factor (bFGF) for 3 weeks. Compared with PLGA-alone scaffold, the bFGF–PLGA scaffold had better biocompatibility and biodegradability, a higher load-to-failure and stiffness. The bFGF–PLGA membranes had a more pronounced effect in the treatment of RCT. A hydrophilic antibiotic drug (Mefoxin, cefoxitin sodium) was successfully incorporated in electrospun PLGA-based nanofibrous scaffolds [163]. In vitro release studies demonstrated that the PLGAbased scaffolds provided a sustained drug release over 1 week.

Neuregulin-1(Nrg) have been encapsulated in the electrospun PLGA/ NCO-sP(EO-stat-PO) fibers to treat cardiovascular disease [173]. This Nrg-containing biomaterial was experimented on a rat model of myocardial ischemia to evaluate the biocompatibility and degradation. The introduction of NCO-sP(EO-stat-PO) improved the hydrophilic of the surface. The incorporation of Nrg could facilitate heart tissue regeneration. This scaffold might represent a promising strategy for cardiovascular disease. To address the inflammatory caused by degradation, anti-inflammatory drugs can be loaded in the electrospun PLGA scaffolds. In a recent study, PLGA fibrous scaffold loaded with esterasesensitive prodrug was obtained to study the release profile and the inhibition of inflammation [174]. In this study, the drug release showed an enzyme-triggered release process. The prodrug-loaded scaffold showed drug release kinetic that matched to PLGA biodegradation rates and fullcourse inhibition of inflammation in vivo. This strategy offered a facile and general way to address inflammation response. The drug-loaded PLGA-based electrospun scaffold might have greater use in biomedical engineering. A fiber-microsphere composite scaffold with simvastin-loaded fibers and BMP-2-loaded microspheres was fabricated in our early work. This hybrid structure could load different drugs with different hydrophilic–hydrophobic property, which will be promising in tissue engineering and drug delivery system. The future work may focus on the encapsulation of genes and macromolecule proteins in biomimic scaffolds to be used in genetic therapy and other tissue regeneration process. 3.3. Issues remained to be solved PLGA is a biodegradable synthetic copolymer, the degradation and sterilization issues should be taken into consideration before applying. The scaffolds' degradation behavior depends on many parameters, such as the LA/GA ratio, molecular weight, and the shape and structure of the matrix [175]. With higher content of GA, PLGA will degrade faster; PLGA with lower molecular weight degrades faster than the higher molecular weight scaffold; thinner scaffold with larger surface area and interconnected holes degrades faster than thicker scaffold with semiconnected holes. An ideal substitute used in tissue engineering area should have an appropriate degradation rate which matches with the rate of new tissue formation [175]. In drug-delivery system ondemand release of drugs during the degradation of biomaterials is very attractive [174]. Degradation behavior of PLGA-based scaffolds could be influenced by many aspects, such as surface properties and other components. Plasma-treated PLGA degraded much faster than non-treated PLGA due to the improved hydrophilicity [41]. The addition

Fig. 5. TEM images of electrospun nanofibers: (A) PTX/PLGA and (B) PTX/PLGA@BFA-PM [171].

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of some components may accelerate or slow down the degradation rate of the scaffolds [85,132,176]. For example, CS-g-PLGA copolymers with a higher CS content had faster degradation rates due to the hydrophilicity of CS [176]. The addition of nHA could slow down the degradation process by neutralizing of buffering the pH changes caused by the typical acidic degradation products of polyesters [85,86]. Sterilization is a necessary pretreatment before the scaffolds would be transplanted into body or seeded with cells. There are many methods to sterilize the polymer scaffolds, such as sterilized by 75 ± 5% ethanol solution, UV irradiation, and 60Co γ-ray and plasma treatment [46,141,177]. However, ethanol sterilization is not a recognized sterilization method that can be taken to the clinic. In our recent work, we sterilized the PLGA scaffolds by 75 ± 5% ethanol solution. We found the morphology of the PLGA scaffolds changed a lot, the fibers swelling and the pore sizes became smaller. In our future work, we will adopt other methods to sterilize the scaffolds. Sometimes, ethylene oxide and 0.01% peracetic acid could be used to sterilize the electrospun scaffolds as well [154, 178]. Among these techniques, plasma sterilization is more effective than the other methods [179,180]. It can provide sterile surfaces during the preparation process. And this technique could be scaled up to industrial production. Irradiation, acid and ethylene oxide treatment may cause extensive deformation and accelerated polymer degradation [181,182]. In the future work, new methods like low-temperature radio-frequency glow discharge (RFGD) plasma treatment that sterilize PLGA electrospun scaffolds without any destructions would be optimal for in vivo use. Furthermore, electrospun fibers usually have dense packing and small pores, which limits the cell infiltration into the scaffolds and slows down the efficiency of recovery and reconstruction. Enlarging the pore sizes of the electrospun scaffolds might be another vital problem. Some works have been done to verified the larger pores had positive effects in tissue engineering area [183,184], such as vascular [185] and bone [186] tissue engineering. Our work is devoted to fabricate thicker electrospun PLGA scaffolds with larger pores and higher porosities, which could be combined with drugs (especially growth factors or chemokines) and be used in tissue engineering area. The hybrid fiber-microsphere scaffolds are fabricated by simultaneous electrospinning of PLGA fibers and electrospraying of PEG microspheres. Then the sacrificial PEG microspheres will be removed by immersing in water for 24 h to make larger pores. On the other hand, interfacetissue engineering plays an important role in tissue regeneration and recovery. The interface usually composed of different structures, different cells and different growth factors. Functional gradient scaffolds might be more and more attractive in this area. Based on the works we have done, the future work will focus on the fabrication of functional gradient PLGA scaffolds loaded with growth factors to be used in interface-tissue engineering.

4. Conclusions and remarks on future challenges Electrospun PLGA-based scaffolds have various applications in biomedical engineering, such as tissue engineering and drug delivery system. Nevertheless, the lack of cell recognition sites, hydropholicity and single-function limit the applications of PLGA fibrous scaffolds. In order to tackle these issues, many works have been done to obtain functional PLGA scaffolds including surface modifications, fabrication of PLGA-based composite scaffolds and drug-loaded scaffolds. PLGAbased fibrous scaffolds have great potential in biomedical engineering. However, there still remain some serious problems with regard to the application of electrospun PLGA scaffolds. For example, the mimic of the real microenvironment, the match of the material degradation rate with tissue regeneration and the inflammation caused by the accumulation of biodegradation products. More works should be done to improve the fabrication of functional PLGA-based scaffolds. Furthermore, the application of PLGA-based scaffolds has become an important technique

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in in-situ tissue engineering, which is a promising area in biomedical studies.

Acknowledgments This work was financially supported by the Fundamental Research Funds for the Central Universities (Program No. 3102015ZY085), Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2015JM5177) and National Natural Science Foundation of China (Program No. 81170110).

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