Biomedical applications of nanofibers

Received: 23 April 2010, Revised: 22 July 2010, Accepted: 22 July 2010, Published online in Wiley Online Library: 29 December 2010

Biomedical applications of nanobers

Victor Leunga and Frank Koa*

Nanober technology is an exciting area attracting the attention of many researchers as a potential solution to thecurrent challenges in the biomedical eld such as burn and wound care, organ repair, and treatment for osteoporosisand various diseases. Nanobers are attractive in this eld for several reasons. First, surface area on nanobers ismuch higher compared to bulk materials, which allows for enhanced adhesion of cells, proteins, and drugs. Second,nanobers can be fabricated into sophisticated macro-scale structures. The ability to fabricate nanobers allowsrenewed efforts in developing hierarchical structures that mimic those in animals and human. On top of that, a widerange of polymers can be fabricated into nanobers to suit different applications. Nanobers are most commonlyfabricated through electrospinning, which is a low cost method that allows control over ber morphology and iscapable of being scaled-up for mass production. This review explored two popular areas of biomedical nanoberdevelopment: tissue regeneration and drug delivery, and included discussions on the basic principles for hownanobers promote tissue regeneration and drug delivery, the parameters that affect nanober performance and therecent progress in these areas. The recent work on biomedical nanobers showed that the large surface area onnanobers could be translated into enhanced cell activities, drug encapsulation, and drug release rate control.Furthermore, by optimizing the electrospinning process via adjusting the material choices and ber orientation, forexample, further enhancement in cell differentiation and drug release control could be achieved. Copyright 2010John Wiley & Sons, Ltd.

Keywords: nanobers; biomedical; tissue regeneration; drug delivery

INTRODUCTION

In the recent decade, the medical industry has experienced anunprecedented rate of advancement, with innovations and newdiscoveries made each year. Despite the fast growth in researchand development in the eld, patients and medical practitionersstill face many challenges that current technology is eitherineffective or inadequate in addressing. In the wound care sector,500,000 patients receivemedical treatment on burn wounds eachyear in the US.[1] Among the burn patients, those with moresevere injuries require more effective alternatives to autograftingand traditional wound dressings such that larger burn areas canbe covered and healing can take place in a timelier manner. Asanother example, osteoporosis threatens over 44 millionAmericans,[2] and improved bone healing and bone tissuemaintenance technology is required. On top of that, millions ofpatients worldwide suffer from failures of various organs such asliver and heart. These patients will greatly benet from advancedtissue regeneration technologies, which can promote restorationof the failing organ functions, or facilitate improved healing afterorgan transplantation. These examples have shown that themarket for wound care and tissue regeneration is great and hasopportunities for further innovations. Another sector that hasexperienced even more rapid growth is the pharmaceuticalsector, with its 2009 annual worldwide sales estimated at US$750billion.[3] The pharmaceutical market also actively seeks solutionsto its challenges, an example of which being the search for moreeffective and controlled carrier for various drugs for curing someof the worlds most threatening diseases, such as cancer.

An exciting area that many researchers are investigating in as apotential solution to the challenges is nanotechnology. In fact, in2004, the industry and governments worldwide have investedmore than US$10 billion in research. In the United States, aprogram known as the National Nanotechnology Initiative (NNI)

was initiated to encourage research in nanotechnology, andmorethan US$5 billion has been spent since 2001.[4] Nanotechnologyhas attracted much attention because in the nano-scale,materials can exhibit properties that are greatly different thanin their bulk forms, and in some cases, these unique propertiescan outperform any existing materials. These size-dependentproperties include biological, optical, chemical, mechanical,thermal, electrical, and magnetic. Perhaps the most famousexample is the great enhancement in mechanical and electricalproperties of materials using carbon nanotubes.[5,6] Also, as thesize-scale of a material decreases, the amount of surface area aswell as surface energy increase dramatically and may becomeuseful in many biomedical applications.

Among various nanomaterials, nanobers are a unique classbecause, similar to natural biological tissues, they can beorganized into porous ber architectures useful in manyapplications. Nanobers attract attention in the biomedical eldfor several reasons. First, as mentioned, surface area and surfaceenergy are much higher for nanobers compared to bulkmaterials, which allows for enhanced adhesion with cells,proteins, and drugs. Indeed, many in vitro studies on nanobrouswound dressings, tissue engineering scaffolds, and drug carriershave shown that they can outperform their micro or macro-metric scale counterparts, even when they are composed of thesame material, and examples of these will be discussed in latersections. Second, properties of nanober assemblies, such as

(wileyonlinelibrary.com) DOI: 10.1002/pat.1813

Special Issue: Review

* Correspondence to: F. Ko, Department of Materials Engineering, University ofBritish Columbia, Vancouver, British Columbia, Canada.E-mail: [email protected]

a V. Leung, F. Ko

Department of Materials Engineering, University of British Columbia,

Vancouver, British Columbia, Canada350

Polym. Adv. Technol. 2011, 22 350365 Copyright 2010 John Wiley & Sons, Ltd.

exibility, can be customized to great extent. A wide range ofpolymers can be electrospun to suit different applications. Also,compared to bulk materials, nanober assemblies can have veryhigh porosity while surface area remains high, which is useful forcell activities.

Although different geometries have their uniqueness, nano-materials in brous form are considered in this review, based ontheir high aspect ratio, connectivity, exibility, and the possibilityto fabricate structures with high strength or directional proper-ties. For example, compared to other geometries such as foamand gel lms, which many tissue scaffolds today adopt, bers canbe fabricated such that the surface area is higher on an equalvolume basis, by reducing the ber diameter. Furthermore, berscan contain pores, enabling each ber to behave as foam butwith more exposed surfaces. The effect of ber size on cellproliferation and differentiation have been detailed in the workby Ayutsede et al.[7] and Ko and Gandhi,[8] and will be furtherdiscussed later in this review. On the other hand, although somegeometries such as nanoparticles may have a higher surface areathan nanobers, nanobers have the advantage that they can befabricated into more sophisticated macroscopic structures suchas sutures and scaffolds. In addition, nanobers are a closerstructural mimic of the structure in the native extracellular matrix(ECM) which is mainly composed of nanobrous collagen, andthus are more desirable for biomedical applications such as tissueengineering.[9] The unique properties of brous structures can beuseful for many biomedical purposes, and several application-specic examples will be explored in more detail in the followingsections.

Besides the nanometric size scale, the ability to fabricatehierarchical structures with component sizes ranging frommacroscopic to nanoscopic is also of great interest to themedical industry. The importance of the ability to fabricatehierarchical structures is apparent from the fact that humantissues, such as skin and bone, are hierarchical, with size scalesranging from nanometer to centimeter. An example that outlinesthe importance of hierarchical structures is a cancer therapeuticutilizing the enhanced permeability and retention (EPR) effect.The EPR effect is a phenomenon in which macromolecules areretained in tumors for extended time, thereby enhancing thetherapeutic effect. The ability of nanoparticles to utilize the EPReffect has been shown. Moreover, the nanoparticle can beincorporated into a macroscopic drug carrying system. Melankoet al.[10] have shown an example of a potential therapeutic usingcarbon nanotubes clustered into a carbon nanohorn structurewith an anticancer agent incorporated. Considering the examplepresented by Melanko et al., it is also possible to hostnanoparticles in a nanobrous matrix incorporated with ananticancer agent, as a nanobrous drug system that utilizes theEPR effect.

Among the various processing methods, nanobers are mostcommonly fabricated by electrospinning. Compared to the otherfabrication techniques, such as template synthesis, drawing, andphase separation, electrospinning is a simple method thatenables control over the ber diameter. It has been demonstratedthat electrospinning is capable of being scaled up for massproduction.[11] The concept of electrospinning originates fromFormhals whose patent led in 1934 described a process thatproduces polymeric bers through the electrostatic repulsionbetween the molecules in the polymer chains. However, notmuch focus was being placed on using this technique to formpolymeric nanobers until the early 1990s when several research

groups, such as the Reneker group, showed that ultrane berscan be electrospun from various organic polymers.[12] Since thenresearch interest in electrospinning has greatly strengthened. Infact, the number of publications in this area has beenexponentially increasing since the late 1990s.[13] In terms ofnanobers for biomedical applications, Ko et al.[14] presented oneof the rst studies on nanobercell interactions for tissueengineering.

The overview above shows the possibility of combiningnanober and medical technology. However, we must alsoscrutinize the implications of integrating nanober technologyinto biomedical applications. More importantly, the interest lies inwhether the use of brous structures, especially in thenanometric scale, can more adequately address the challengesin the medical industry today, compared to existing, non-nanometric or non-brous structures. Partial answer to thisquestion can be found in the work that has been done recently.Before discussing the recent work in more details, this review willbegin with an introduction to the electrospinning process,outlining the various electrospinning techniques that have beendeveloped for producing a wider variety of nanobers withenhanced functions, as well as some of the materials that arecommonly used for biomedical nanobers. Examples of nanoberuse and their uniqueness compared to non-nanobrous structurescan be explored in more in-depth discussions on the two mainapplications of biomedical nanobers, including tissue regener-ation and drug delivery. For each of the applications, the basicprinciples of how the nanobers can perform their intendedpurposes, and examples of the recent work will be explored. At theend of this review, readers will be able to develop an understand ofwhat can be demonstrated by nanobers and how these propertiescan be useful for biomedical applications, and the new challengesthat have been unveiled by this new understanding.

ELECTROSPUN STRUCTURES

Electrospun nanobers are highly adaptable to many differentbiomedical applications, since their properties are highlycontrollable. For example, nanober structures can be tailoredto suit different types of tissues or to load various drugs. Besidesthe wide range of materials that can be selected for electro-spinning, the control over nanober diameter, and more so thediameter, is extremely important for nanobers in biomedicalapplications. Moreover, as mentioned in the Introduction Section,bers having nanometric diameters have more available surfacearea for cell activities and drug loading. This section will begin byfocusing on the electrospinning parameters that can affect thenanober morphology, and how some of these parameters canbe t into mathematical relationships such that ber diameterscan be predicted. Besides ber morphology, the ber architectureis also a signicant variable contributing to the exibility ofnanober for specic biomedical applications. This section willalso explore several types of electrospinning set-up typically usedfor nanobers in biomedical applications, including single nozzle,multi-nozzle, and co-axial electrospinning.

Fiber diameter control

The diameter of electrospun bers can be inuenced byparameters related to both the electrospinning solution andthe equipment set-up. All the parameters are often interrelated to 3

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BIOMEDICAL APPLICATIONS OF NANOFIBERS

one another, and in this section we will discuss each of themseparately. In terms of the solution parameters, the polymersolution concentration is the most important parameter indetermining the ber diameter. The polymer solution concen-tration dictates three important solution properties: solutionviscosity, surface tension, and charge density,[12] and theseproperties will be discussed in more details.

The viscosity of a polymer solution originates from theentanglement of polymer chains. If the polymer chains are lessentangled, the solution will have a low viscosity, and vice versa.Fiber diameter tends to increase with viscosity. However, if thesolution viscosity is too low, ber jet may break into droplets dueto the lack of entanglement.[11] On the other hand, if the viscosityis too high, solution ow through the needle may becomedifcult and ultimately ber formation will be hindered.

The surface tension is the cohesive forces between liquidmolecules, and in electrospinning, the solution must be able toovercome the surface tension. To minimize surface area, surfacetension tends to convert the solution jet into spherical droplet ordroplets.[12] A polymer solution with high viscosity will have alower surface tension due to the increased interaction betweensolvent and polymer molecules, reducing the tendency for thesolvent molecules to come together,[12] whereas the opposite istrue for a low viscosity polymer. Surface tension also preventssudden changes in the shape of the ber jet.[12]

The electric eld strength and charge density in the polymerrelate to the amount of charge carried by the polymer chains inthe solution and determine the extent of electrostatic repulsionexperienced by the molecules. Electrostatic repulsion betweenpolymer molecules tends to increase the polymer surface area,meaning that the charge density in the electrospinning solutionmust be sufciently high to yield small diameter bers, yet it cannotbe too high such that the ber jet will break into droplets.[15]

Besides solution parameters, several process parameters canalso affect the nanober diameter, including voltage, solutionfeed rate, spinning distance, temperature, and humidity. Voltageis an important parameter in electrospinning because thecharges on the polymer molecules that form the ber jetoriginate from the applied voltage. At high voltage, the solutionjet will be drawn at a faster rate and will experience a greateracceleration. Moreover, the greater acceleration causes thesolvent in the solution to evaporate faster, reducing the ber jetvolume. These factors are in favor of reducing ber diameter.However, increased solution jet acceleration also reduces theight time, or in other words, the jet will reach the collectorquicker. A reduced ight time reduces the time available for thebers to stretch, leading to a greater ber diameter.[11] Thisultimately implies that as the voltage increases, ber diameterwill decrease, but when the voltage increases past an optimum,ber diameter may increase. The balance in voltage is alsoimportant for the crystallinity of the polymer. In general, since ahigher voltage causes a stronger stretch in the bers, greatercrystallinity can be achieved. However, a voltage that is too highreduces ight time, which gives the ber less time to orient in anorderly fashion, reducing crystallinity.

The electrospinning solution feed rate is the rate at which thesolution is supplied and determines the amount of solutionavailable for Taylor cone formation at a given moment. Since theTaylor cone must be kept at a certain size to be stable, a specicfeed rate is required. Increasing the feed rate past this level willcause increase in ber diameter since there is more solutionsupplied than that being withdrew from the needle tip.[11]

The spinning distance affects the ight time of the solution jet.When the distance is short, there is less time for the solvent in thesolution to dry and for the jet to stretch, which can lead to anincrease in ber diameter. Also, as the distance between theneedle tip and grounded target reduces, the electric eldstrength increases.[11] The increase in electric eld strengthaccelerates the jet, thus giving even less time for the jet to stretchand for solvent to evaporate. The effect of spinning distance onber morphology depends on the volatility of the solvent in thesolution as well as the polymer concentration.

As there are many factors that can affect the electrospinningprocess, it can be imagined that the ber diameter will be afunction of at least some of the independent parameters. Indeed,Fridrikh et al.[16] presented a model that can predict the ber jetdiameter as a function of ow rate, surface tension, and electriccurrent. Although the model bases only on three parameters,each of those in turn is related to at least one of the parametersmentioned in this section. The model presented by Fridrikhet al.[16] is shown below. In the equation, h is the radius of the berjet, g is the surface tension, e is the dielectric constant, Q is theow rate, I is the electric current, and x is the ratio of jet lengthover nozzle diameter

h g"Q2

I22

p2 lnx3 1=3

The Fritdrikh model, however, does not take solution proper-ties into consideration. In order to guide the preparation of theelectrospinning solution a dimensionless index, the Berrysnumber, has been proposed by the Ko group.[17] The Berrysnumber is dened as the product of intrinsic viscosity and thepolymer concentration of the solution. It was found thatelectrospinnability and ber diameter can be correlated to theBerry number of the polymers.[18] For example, the range ofBerrys number for PAN polymer can be divided into four regions,with the rst region corresponding to a Berrys number lowerthan 1, region 2 corresponding to a Berrys number between 1and 2.7, and region 3 corresponding to 2.73.6, whereas region 4corresponds to a Berrys number of above 3.6. When the Berrysnumber of a polymer solution is in region 1, polymer dropletrather than ber forms. The smallest diameter ber can beproduced in region 2, and then as Berry number increases, theaverage ber diameter tends to increase. The ber diameter, d,can then can be estimated using the equation below, where B isthe Berrys number, c is an experimentally determined value thatis related to but not entirely dependent on the polymercrystallinity, and a is the Mark-Houwink constant which dependson the polymer. It is important to note that since c is not entirelydependent on the polymer crystallinity, an amorphous polymerwill still have a c value that is dened

d aBc

Single nozzle electrospinning

Often viewed as the traditional approach to electrospinning,single nozzle electrospinning involves applying a high voltage toa single orice through which a polymer solution or melt ow. Aschematic of the single nozzle electrospinning set-up is shown inFig. 1. In the recent decades, nanobers of many polymer/solventsystems have been fabricated through single nozzle electrospin-ning, which has become a common method for preparing3

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V. LEUNG AND F. KO

nanobers for biomedical applications. On top of the ability toelectrospin many polymer/solvent systems, composite nano-bers can also be fabricated from a blend of polymers. Thiscapability is signicant because desirable properties of multiplepolymers can be combined. We have previously electrospunblends of sodium alginate and polyethylene oxide (PEO), as wellas chitosan and PEO. In both cases, the biomedical capabilities ofalginate and chitosan are combined with the chain entanglementcapability of PEO, leading to an electrospinnable nanober fortissue regeneration applications. Electrospinning of polymerblends is possible if the polymer solutions are compatible witheach other. Besides polymer blends, second-phase particles canalso be electrospun with a polymer matrix into nanobers. Forexample, silver nanoparticles, with its antimicrobial propertiesreported by many, can be dispersed in solutions containing abiocompatible polymer for making antimicrobial nano-bers.[1921] Electrospinning with second-phase particles is notlimited to solid. In the case of emulsion electrospinning, theparticle can be a liquid that is not compatible with the polymersolution. Xu et al.[22] presented the potential of a poly(ethyleneglycol)poly(L-lactic acid) (PEGPLLA) nanober containing ananticancer agent. In this case, the PEG-PLLA is dissolved inchloroform, which is not compatible with the water-solubleanticancer agent, doxorubicin hydrochloride (Dox). To incorpor-ate the Dox into the polymer solution, a water-in-oil emulsion isprepared by adding the Dox to the polymer solution containingthe surfactant sodium dodecyl sulfate (SDS). By vigorouslyagitating the solution, the immiscible Dox will break into smalldroplets that will be dispersed in the solution with the aid of theSDS. If the droplets become sufciently small, they can besuspended in the polymer solution, and electrospinning thissolution can yield bers containing the anticancer agent.Nanobers from emulsion electrospinning have shown potentialin drug delivery and protein release applications.

Multi-nozzle electrospinning

In single nozzle electrospinning, composite nanobers can befabricated by blending polymer solutions that are compatible.However, in some cases it is desirable to fabricate compositenanobers from polymer solutions that are not miscible with oneanother. In this case, the two immiscible solutions can beelectrospun from separate orices, resulting in a compositenanober assembly rather than composite nanobers. The twonozzles can be placed side by side, or on opposite sides of arotating collector. When the nozzles are placed on opposite sides

of a rotating collector, two power supplies are required, one foreach nozzle. In the case of a side-by-side set-up, connecting eachnozzle to an individual power supply may cause interferencebetween the two nozzles due to the variations in charge for eachnozzle. The important consideration in a side-by-side set-up is theuniform charge distribution. A metallic bar can be attached to allthe nozzles in the system to distribute the charge, and the nozzlescan be charged by a common power supply by connecting it tothe metallic bar. In this set-up, rather than having a point chargeson each nozzle, a uniform, linear application of charge willconnect all nozzles simultaneously, resulting in effectiveelectrospinning in each nozzle.[23]

Co-axial electrospinning

Recent progresses in co-axial electrospinning have furtherexhibited the potential of nanobers in drug and proteindelivery applications. In short, co-axial electrospinning involves aco-axial spinneret with one nozzle inside a larger nozzle, whichcan result in a core-shell nanober structure. A co-axialelectrospinning nozzle set-up is shown in Fig. 2. It can be seenfrom the gure that two separate channels can deliver onepolymer solution in each channel to either the outside or theinside capillary. Using the set-up shown in Fig. 2, core-shellnanobers suitable for biomedical applications can be fabricated,such as one that contains poly(lactic-glycolic acid) (PLGA) as theshell and chitosan as the core in a drug delivery system.[24] In acore-shell nanober, drugs or proteins can be incorporated intothe core ber protected by the shell that can be composed of amore mechanically stable or less degradable polymer. In effect,the core-shell nanober structure adds an extra layer of controlover the release rate of drugs or proteins, allowing for a moresustained release prole. Another advantage of co-axial electro-spinning is that the core solution does not need to beelectrospinnable. The most important factor is that a shell bercan be electrospun to contain a uniform channel within to carrythe core solution. For example, Jiang et al.[25] developed acore-shell nanober containing the protein dextran in a bovineserum albumin (BSA) solution in the core, and poly(caprolactone)(PCL) as the shell polymer. PCL is generally considered to have

Figure 2. Example of a co-axial electrospinning set-up, two solutions

are fed into different capillaries such that a core-shell structure can becreated at the capillary tip. This gure is available in colour online at

wileyonlinelibrary.com/journal/pat.

Figure 1. Single-nozzle electrospinning process. This gure is available

in colour online at wileyonlinelibrary.com/journal/pat.

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lower biodegradability among polymers used for biomedicalapplications, and the use of PCL as the ber shell with an optimalporosity can slow the release rate of the protein from the core.Indeed, Jiang et al. reported that reduction in burst release can beobserved from the core-shell bers, showing potential of such astructure in release rate control.

The methods to fabricate different kinds of nanobers discussedabove expand the variety of applications in which nanobers canbe used. Among the applications, the area of biomedical nanobershas found extensive use of single nozzle and co-axial electrospin-ning. The following sections will explore the use of nanobers invarious biomedical applications.

MATERIALS

While the electrospinning process is important for controlling thenanober diameter and architecture of the resulting structure,thematerial choice is also an important consideration in controllingthe function of the nanobers for biomedical applications. Somematerials, especially natural polymers, may have properties that arespecic to certain cells or proteins, such as adhesion, or promotionof growth factors. Several polymers have structural or chemicalresemblance to materials in the human body, which maytranslate into favorable biomedical properties when nanobersof these polymers are used. Gelatin, for example, is derived fromcollagen, the main protein in connective tissues. In addition, thematerial choice also plays a signicant role in determining thebiodegradability, which is important in many biomedicalapplications. Furthermore, certain properties of the polymers,such as surface charge, determine the mechanisms of drug orprotein binding for release applications. For this review, biomedicalpolymers can be grouped into two main categories, natural andsynthetic polymers. Only linear polymers will be considered in thisreview due to their electrospinnability.

Natural polymers can be plant or animal-based and aregenerally considered a renewable resource, with many beingbiocompatible, especially those derived from materials found inanimal or human bodies. Many natural polymers have beenextensively explored for biomedical nanobers applications, andthis review will focus on several examples, including alginate,chitosan, and hyaluronic acid. Alginate, derived from brownseaweed, is a polysaccharide that is biodegradable andbiocompatible. Crosslinked alginate, such as calcium alginate,has been investigated as a potential material in tissueregeneration scaffold and drug carriers.[2630] Chitosan, likealginate, is a biodegradable and biocompatible polysaccharide.The use of chitosan for applications such as drug delivery andwound dressing has also been extensively studied.[31,32] Chitosanis produced by deacetylation of chitin. The degree of deacetyla-tion and the pH governs the charge density of chitosan insolution. This property of chitosan attracts attention becausesince the polymer is positively charged, it can bind withnegatively charged drugs or proteins. Hyaluronic acid, anothercommonly investigated polymer, is an anionic linear polysac-charide that can be found in human tissues as a non-sulfatedglycosaminoglycan (GAG). Due to its ability to bind with cellsurfaces and proteins in the ECM, hyaluronic acid has beeninvestigated for tissue regeneration and drug delivery appli-cations.[33] Other natural polymers that are often exploredinclude collagen, which is the main protein in human connectivetissues, spider silk,[7,8,34] and Bombyx mori silk,[3537] which has

superior mechanical properties, and gelatin, which is a hydrolysisproduct of collagen.

Nanobers of many synthetic polymers have also beenfabricated for biomedical applications, especially in tissue repairand drug delivery. Some examples include PCL, PLGA, andpoly(vinyl alcohol) (PVA). PCL is semi-crystalline polyester that isbiodegradable. It is widely used in drug delivery systems. It is alsoknown that compared to other biodegradable polymers, PCL hasa slow rate of degradation,[38] and thus is desirable when thenanobers are required to remain in the body for an extendedtime. PLGA is a copolymer of poly(lactic acid) (PLA) andpoly(glycolic acid) (PGA). Several properties of PLGA, such asbiodegradability, can be varied through adjusting the PLA to PGAratio in the copolymer,[39] providing a control mechanism for invivo degradation or drug release to a certain extent. Similar toPCL, PLGA shows superior mechanical integrity in aqueousenvironment and does not swell as readily as chitosan or alginate.PVA, on the other hand, is a water-soluble polymer with arelatively fast degradation in aqueous environments, especially atelevated temperatures. As a result, PVA is desirable in applicationsin which quick release of drugs is required or as a temporarytissue scaffold.

Besides natural or synthetic polymers, another relevant way togroup biocompatible polymers is by their biodegradability.Polymers with relatively fast biodegradability include PVA,alginate, chitosan, and hyaluronic acid, whereas those withslower biodegradability include PCL and PLGA. Some polymers,such as poly(ethylene vinyl acetate) (PEVA), are non-degradable.[40]

Use of biodegradable polymers can eliminate the need for surgicalremoval after the nanobers have performed its function, providedthat the body can tolerate the degradation products. Non-degradable polymers, on the other hand, require surgical removalbut can be desirable when maintenance of structural integrity ornanober morphology is important for the biomedical function.Table 1 summarizes some of the common natural and syntheticpolymers used in electrospinning for biomedical applications andtheir biodegradability.

The variety of polymers available allows selection based ondifferent criteria specic to each application. This versatility,combined with the control of properties from electrospinningparameters, such as ber diameter and porosity, enablesnanober technology to become a highly adaptive method forsolving biomedical challenges. The following sections will look atseveral areas in which nanober technology is applied.

TISSUE REGENERATION

The previous sections have provided an understanding on thecontrol over physical and functional properties of electrospunnanobers via solution and electrospinning parameters, andmaterial selection. With this understanding, we can beginexamining the implication of nanober technology integrationto the medical industry. Translation from the unique structure ofthe nanobers to practical use can be appreciated throughexploring two of the main applications of biomedical nanobers:tissue regeneration and drug delivery. This section will focus ontissue regeneration, which has already been recognized 20 yearsago by the US National Science Foundation as an emerging areaof national importance.[41] The examples given in the Introduc-tion Section outlined the importance of improvements in tissueregeneration technology. Current means of tissue regeneration3

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commonly involves autografts and allografts. Autografts are thetreatment of choice due to their identical genetic origin, but thistechnique depends greatly on the availability of donor sites andcan become a great challenge in the case of large injured areas.Furthermore, the donor site also suffers from damage due toremoval of tissues. Allografts, on the other hand, can be rejectedby the recipient body due to immune responses against theintroduction of an object of different genetic origin. The goal indeveloping new tissue regeneration technology is therefore toobtain a biocompatible platform that can act as a temporary hostfor tissue cells, which must be able to attach, proliferate, anddifferentiate into the specic tissue that needs to be repaired.Attachment, proliferation, and differentiation are thus the threemain requirements for a nanobrous scaffold, and they havebeen further detailed by Ko and Gandhi.[8] As mentioned at thebeginning of this review, nanobers are suitable for tissueregeneration due to the large surface area available for tissue cellactivities, and the structural resemblance to our ECMs.[9] Inaddition, the large number of biocompatible polymers availablefor nanober fabrication is also a driving force behind thiscombination of nanotechnology and biomedical technology. Thissection will focus on nanobers for tissue regeneration scaffolds,with a brief overview exploring the basic components, berarchitectures, scaffold performance. Also, some examples ofcurrent work on regeneration of several types of tissues will beoutlined.

Components of a nanobrous scaffold

As the main purpose of the nanobers is to encourage cellattachment, proliferation, and adhesion, there are threecomponents to a nanobrous scaffold system. First, there isthe nanober, which can be incorporated with bioactivemolecules, such as growth factors or ligands, to help cells attach,proliferate, or differentiate. Finally, the system includes the tissuecell that is to be grown on the scaffold, which may be pre-seededor can be attached after the scaffold is applied. Figure 3 outlinesthe various aspects of a tissue regeneration scaffold system. It isimportant to note that the three components mentioned closelyinteract with one another. The nanober scaffold can bind cellsand act as the platform for their activity, which is very similar to

the ECMs in native tissue, which can provide cell anchoragethrough ligands, and can inuence cell activity. On top of that, thescaffold can also protect the bioactivemolecules. Lim andMao[42]

mentioned that growth factors can degrade in the bulk liquid, butwhen they can concentrate on the, cell signaling can be achieved.

Fiber architectures

Nanober scaffolds are most commonly applied as a non-wovenfabric, and individual bers can be randomly oriented or highlyaligned, depending on the specic application. For example,aligned nanobers are benecial when directional growth of cellsor anisotropic mechanical properties are required. Furthermore,nanobers can also be woven or braided into various structuressuitable for biomedical applications.[8] Different woven or braidedstructures that can be useful for surgical implants are shown inFig. 4 and are also detailed by Ko.[43] It can be observed thattextile technology, such as weaving and braiding, can be used tofabricate hierarchical brous structures with nanobers being atthe smallest level, which can be a closer structural mimic of thehierarchical brous structures in biological tissues. Such hierarchicalstructures can result in a range of size scales and porosities whichmay be advantageous in certain surgical applications and inobtaining desired mechanical properties. Examples of yarns andfabrics suitable for various surgical applications have also beenoutlined in by Ko.[44]

Scaffold performance

As nanober scaffolds act as the host for cell activities, variousber properties can signicantly affect scaffold performance. Inthis section, a more detailed overview of the scaffoldtissueinteraction will rst be explored. In addition, the impact of berproperties such as size, porosity, mechanical properties, andgeometry will be discussed.

When tissues are damaged due to injuries or diseases, thestructural support for tissue cells is compromised due to the lossof ECMs. Since tissue cells and cell signaling molecules can betransferred by the ow of blood, new tissue can be generatedthrough ECM synthesis by tissue cells that have repopulated thedefect site. However, the lack of connecting tissue at the defect

Table 1. Summary of natural and synthetic polymers for biomedical nanobers

Material Typical solvent Biodegradability

NaturalAlginate Water FastChitosan Water/acetic acid FastCollagen 1,1,1,3,3,3 Hexauoro-2-propanol SlowGelatin 1,1,1,3,3,3 Hexauoro-2-propanol 2,2,2-triuorethanol SlowSilk Formic acid SlowCellulose Acetic acid SlowHyaluronic acid Water Fast

SyntheticPoly(vinyl alcohol) Water FastPoly(caprolactone) Chloroform methylene chloride SlowPoly(lactic-glycolic acid) Tetrahydrofuran/dimethylformamide SlowPoly(urethane) Tetrahydrofuran/dimethylformamide NoPoly(ethylene vinyl acetate) Chloroform No

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site can greatly compromise the healing process due to the lowintegrity of the tissue cells, and thus the primary function of ananobrous scaffold is to serve as a temporary ECM to providestructural support until the natural ECM is regenerated. There aretwo requirements for the scaffold: surface and structuralcompatibility.[45] For surface compatibility, the surface chemistryscaffoldmust be suitable for adhesion of tissue cells and signalingmolecules that regulate cell activities. For structural compatibility,on the other hand, the architecture and scaffold morphologymust conform to the defect site and be able to support cellmigration and differentiation. Tissue cells are able to senserestraining forces in its surroundings and respond accordingly byadjusting their linkages in each other.[46] As a result, having low

structural compatibility may negatively affect the properties ofregenerated tissues. In making nanobrous scaffolds, there aredifferent strategies to satisfy both requirements. For surface,material choice plays an important role in deciding the surfacechemistry of the nanobers. In addition, various polymers andproteins can be added to enhance cell interaction, as will bediscussed later. For structural compatibility, the electrospinningprocess allows optimization ber morphology, size, and porosity.Furthermore, since the natural ECM is composed of proteogly-cans and network of nanobrous collagen, electrospun nano-bers are a closer structural mimic to the ECM. Anotherconsideration for structural compatibility is the biodegradabilityof the scaffold, which can be customized through the material

Figure 4. Textile structures for surgical implants. There are many possible ways to assemble brous structures into macrometric forms (from[8]).

Figure 3. Main aspects of a tissue regeneration scaffold system, with the base scaffold containing tissue cells cultured in vitro. The scaffold may also

contain signaling molecules to regulate cell functions (from[8]). This gure is available in colour online at wileyonlinelibrary.com/journal/pat.

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choice. The biodegradability is important as the healing processcan be hindered by intact nanobers in the scaffold that restrainthe proliferating cells and thus the synthesis of new ECM.

Having explored the requirements on the scaffold as a whole, itis now important to discuss the signicance of the bers in thescaffold. In studying nanobrous scaffolds, ber size is perhapsthe most studied parameter. In general, the smaller the berdiameter, the larger the surface area to volume ratio will be. Alarge surface area is generally considered benecial for cellattachment, provided that the cells can access the surfaces.Porosity plays a signicant role in cell access to ber surfaces andwill be discussed shortly. The effects on nanober size fromdifferent voltage, concentration, and drug loading on PLGA bershave been extensively studied by Katti et al.[47] However, forperformance in terms of cell activity, comparisons betweennanobers and microbers are most commonly made. In ourwork on calcium alginate nanobers, we have compared theattachment and proliferation of lung broblast cells between ourelectrospun nanobers, with an average diameter of 200 nm, andKaltostat, a commercially available alginate microber wounddressing, with an observed ber diameter of approximately30mm. Cell proliferation tests using Alamar blue assay showedthat broblast proliferation on the nanober scaffold is superiorto the Kaltostat. Also, Fig. 5 shows a comparison betweenbroblast cell morphology on natural silk microbers and onregenerated silk nanobers.[8] From Fig. 5 it can clearly be seenthat cells adopt different morphologies when cultured on bersof different sizes.

Porosity is another signicant factor in nanober scaffoldsbecause without porosity, cells cannot access the availablesurfaces on the bers. On top of that, pores through the thicknessof the nanober scaffold must be interconnected to promote cellmigration, otherwise proliferation can only occur on the nanobermat surface, limiting the effective surface area and thus the cellproliferation on the scaffold. Porosity in a nanober scaffold iscommonly measured via mercury porosimetry, which forcesmercury through the scaffold at high pressures and providesinformation about the pore size and the total pore volume.

Mechanical properties that are relevant for tissue regenerationscaffolds typically include the strength of the nanober assembly,the failure strain of the scaffold, and stiffness. These propertiescan be measured via a tensile or compression test, depending onthe application. For skin applications it is more relevant to pursuetensile testing, whereas for bone implant applications it is usefulto obtain compression test data. Specic mechanical propertyrequirements are different for each application and should besimilar to the specic tissue considered. For example, a skinscaffold should be stretchable, with a failure strain on the order of20%. Figure 6 shows the tensile testing results for a spider silkprotein nanober sample, and the same sample but with theaddition of 1% carbon nanotubes.[8] As seen in the gure,addition of carbon nanotubes greatly strengthened the nano-bers, at the expense of reduced failure strain. Indeed, the use ofcarbon nanotubes in biomedical applications has sparked muchinterest due to their superior mechanical properties. However,information on the biocompatibility of carbon nanotubes havebeen contradictory, with some suggesting that exposure to the

Figure 5. Chondrosarcoma cell morphology on natural silkworm silkbers (left) and nanobers (right). The images show that cell differen-

tiation can be different depending on the surface morphology and the

size scale of the scaffold.

Figure 6. Tensile strength of spider silk nanobers, upon adding CNT, tensile strength of nanobers can signicantly increase, but with reduced tensile

strain (from[8]). This gure is available in colour online at wileyonlinelibrary.com/journal/pat. 357



nanotubes can cause toxic effects, while others reported nosignicant effects from exposure. Smart et al.[48] reviewed severalstudies on carbon nanotube toxicity and concluded that whiletoxic effects can result from exposure to unrened carbonnanotubes, pristine nanotubes cause minimal toxic effects due tothe lack of transition metal catalysts. Furthermore, studies haveshown that carbon nanotubes can be functionalized, withcarboxyl or ammonia groups, for example to enhance biocom-patibility for biomedical applications.[49] Mechanical properties ofnanobers can also be greatly affected by post-electrospinningtreatments. In our work on alginate nanobers, we observed asharp reduction in failure strain from 15% to 2% after thenanobers are crosslinked with calcium ions.

The importance of ber geometry, whether randomly orientedor aligned, depends greatly on the specic applications. The bergeometry can be modied by changing the movement rate ofthe collector, or by using a magnetic eld. To obtain directionallyaligned nanobers, one can electrospun onto a collector spinningat a high rate. Alternatively, one can apply two parallel-positionedmagnets on the collector to inuence the electric eld near thecollector, much like the approach adopted in the work of Wanget al.[50] on iron oxide nanobers.

Hydrophilicity of the nanobers is also important for tissueregeneration applications, since the scaffold must interact withsurrounding tissues in an aqueous environment. Hydrophobicnanobers may prevent cell attachment due to their repulsionfrom aqueous phases. Hydrophilic nanobers are therefore desiredfor cellscaffold interaction. However, the use of hydrophobicnanobers in tissue regeneration is possible, provided that thenanobers contain second-phase materials that are hydrophilic. Insuch systems the hydrophilicity of hydrophobic nanobers can beregarded as being modied by the second-phase material. Oneexample of such a system is a PLGAhydroxyapatite compositenanober, with hydroxyapatite being a nanosized ceramic particle. Inthis case, the hydrophilic hydroxyapatite can modify the hydro-phobic PLGA nanober such that it can be used as a tissueregeneration scaffold.[51]

Tissue applications

Nanobers for tissue regeneration are highly adaptive because ofthe wide range of biocompatible polymers available, and theexibility in process optimization. Several polymers have been

shown to have desirable biomedical properties, such as chitosanbeing able to aid blood clotting, and thus can be considered inapplications to specic tissues. Moreover, the electrospinningprocesses, such as the techniques mentioned at the beginning,can be used to further enhance nanober scaffold performance.Also, bioactive agents such as proteins can be added to thenanober scaffold as well,[52] further enhancing the performance.Some of the recent work in scaffold development for severaldifferent types of tissue is explored in this section.

For skin tissue regeneration, it is desirable to nd betterperforming, efcient alternatives to existing solutions such asbandages and skin grafts. Examples of materials that have shownpotential as skin tissue scaffold include collagen, silk, chitosan,and alginate. The SEM images in Fig. 7 show examples ofelectrospun chitosan and alginate fabricated by our group at theUniversity of British Columbia.[53] We have electrospun sodiumalginate nanobers with poly(ethylene oxide) which aids berformation. The resultant nanobers were crosslinked withcalcium nitrate to enhance structural integrity in aqueousenvironments. Also, since it has been demonstrated in the pastthat poly(L-lysine) can enhance cell adhesion, we added a smallamount of poly(L-lysine) into the crosslinking bath, entrappingthe protein in the crosslinked alginate matrix. To observe theeffect on cell attachment, we cultured broblast cells on alginatenanober scaffolds with and without poly(L-lysine). With thepoly(L-lysine), we noticed a signicant increase in cell attachmentcompared to the alginate nanober scaffold without the protein.

Whilst nanober scaffolds such as the alginate one mentionedcan be used to treat supercial wounds and minor burns, moresophisticated solutions are required for treating more severeinjuries. Herndon et al.[54] mentioned that if the loss of fullthickness skin reaches a diameter of 4 cm, grafting proceduresare required. It can be expected that grafting is required becausethe tissue cells required for regeneration can only be sufcientlysupplied by the graft. Indeed, the epidermis layer will onlyregenerate if the amount of epidermal cells in the uninjureddermis layer underneath is sufcient.[55] In order to promotetissue regeneration without the use of grafts in the case of severeinjuries, several groups have attempted to electrospin nanoberswith keratin.[5658] Keratin is the major protein found inkeratinocytes that constitute 95% of the cells in the epidermis.Both Yuan et al.[58] and Li et al.[57] have electrospun PLAnanobers with keratin derived from wool. Yuan et al. reported

Figure 7. Electrospun nanobers of alginate (left) and chitosan (right). These images show that a range of materials can be electrospun into nanobers

with similar morphology.358


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superior viability and proliferation of NIH-3T3 broblast cells onPLA-keratin nanobers compared to plain PLA nanobers,whereas Li et al. seeded MC3T3 osteoblast cells and reportedan greater number of cells observed on the PLA-keratin scaffoldthan on the plain nanober control. These studies have demon-strated that nanobers can be modied to enhance interaction withtissue cells. On top of encouraging tissue cells to attach andproliferate in three dimensions to ll the defect the injury site, it isalso possible to pre-seed keratinocytes or broblasts onto thescaffolds and then applying to the injured site, which can be greatlybenecial for more severe injuries. Also, by optimizing theelectrospinning process, one can mimic the layered structure ofhuman skin, and, through pre-seeding keratinocytes in theepidermal mimic layer, and broblasts in the hypodermal mimiclayer, one can synthesize an articial skin that is a close mimic of thenative structure.

Besides skins, potential in treating many other types of tissueshave been demonstrated using nanobrous scaffolds. Lim andMaos[42] review on electrospun scaffolds for stem cells provided adetailed overview of this signicant technology. The reviewbrings out the message that through optimizing the material andelectrospinning process, and the appropriate choice of growthfactors, it is possible to treat a wide range of tissues with stemcells due to their ability to differentiate specically into a varietyof tissue cells.

In bone regeneration, the incorporation of calcium phosphateinto polymer nanober matrix has been widely studied. Variousforms of calcium phosphate, such as hydroxyapatite, tricalciumphosphate (TCP), and dicalcium phosphate anhydrate (DCPA) areattractive in bone tissue regeneration studies due to theirosteoconductivity and osteointegration, as well as their similarityto the apatite found in our bodies.[59] In addition, since the mainmechanical function of bones is load bearing, incorporation ofcalcium phosphate into nanobers has an added purpose ofenhancing scaffold properties such as compressive strength. Theability of the scaffold to sustain physiological loads has a largeimpact on the overall bone regeneration process. Jose et al.[51]

have demonstrated the fabrication of PLGAhydroxyapatitenanobers and reported that porosity of the scaffold dependson ber alignment and the incorporation of hydroxyapatite. Nieet al.[24] also demonstrated the use of PLGAhydroxyapatitenanobers, but containing bone morphogenetic protein-2(BMP-2) plasmid encapsulated in chitosan. BMP-2 is known tobe able to induce bone healing, whereas hydroxyapatite canenhance attachment to bone cells. Furthermore, the presence ofhydroxyapatite enhanced the release of the BMP-2 due to theirhydrophilicity in a hydrophobic matrix. The authors alsoimplanted the PLGA scaffolds into mice and reported that thePLGAhydroxyapatite composite nanober with BMP-2 inchitosan can enhance the healing of bone segmental defects.Furthermore, transfection efciency of the BMP-2 wasmaintainedat a high level.[24] Another method for incorporating hydro-xyapatite into nanober scaffold is to synthesize them on thenanobers in situ. Chae et al.[60] demonstrated electrospinningalginate nanobers containing phosphate, from which hydro-xyapatite nanocrystals were synthesized in a crosslinking bathcontaining calcium ions. In an example that does not involvehydroxyapatite, Yoshimoto et al.[61] demonstrated bone cellregeneration by a PCL electrospun scaffold via culturingmesenchymal stem cells (MSC) on the 3D scaffold with anosteogenic supplement to promote stem cell differentiation intoosteoblasts.

BMP-2 can also be useful for orthopedic implants, due to theirbone healing effects. A nanober scaffold that can release BMP-2in a controlled manner can be processed into an injectableformulation for delivery to an implant site,[62] or used to coat anorthopedic implant. The interest in using nanobers inorthopedic implants arises from several challenges with existingimplant systems, one of which being the osteodegradation of thebones surrounding the implant.[63] Coating the implants withhydroxyapatite can induce osteoconductivity and osteointegra-tion, but since hydroxyapatite is soluble in water to an extent, thelifetime of the coating is limited. Upon degradation of thehydroxyapatite coating, the boneimplant interface may weaken,leading to loosening of the implant. Besides the PLGAhydroxyapatite composite nanober with BMP-2, Price et al.[64]

have examined the use of carbon nanobers as a possiblescaffold for osteoblast attachment. In this case, the carbonnanober is synthesized through chemical vapor deposition(CVD). Price et al. reported that osteoblast cell adhesion on thecarbon nanober increases with decreasing carbon ber size andincreasing ber surface energy, and that adhesion of competingcells such as broblasts can be reduced through adjustment ofthe ber size and surface energy. More importantly, theosteoblast adhesion is observed to be higher than the metalsused in existing implants.[64]

Connecting between bones is the ligament. Ligament injuriesoccur more commonly in some ligaments, such as the anteriorcruciate ligament (ACL), than the others. Unfortunately, surgeriesare often required for ACL injuries due to poor self-healing. Toenhancing ligament healing, electrospun nanobers have beensuggested by various groups, due to the large surface areaprovided by nanobrous scaffolds, and the customizableporosity.[65,66] Lee et al. cultured human ligament broblastson polyurethane nanobers and cast polyurethane and reportedsuperior cell attachment and proliferation on the nanobercompared to the cast lm. Moreover, aligned polyurethanenanober exhibited enhanced collagen production in 7 dayscompared to the randomly oriented polyurethane nanober. Inaddition, the natural ACL has a hierarchical structure with berbundles arranged into different sections for accommodatingmotions and the resulting friction and deformation, leading tointerests in studying 3D arrangements of brous structures forACL scaffolds.[66] For example, Cooper et al.[66] examined abraided structure containing PLGA bers. Culture of ACL cellsshowed that the PLGA braided scaffold is biocompatible and cellscan attach and proliferate. Although the work by Cooper et al. isnot done on nanobers, it nonetheless showed the potential thatnanobers can be braided into structures useful for tissueregeneration if a hierarchical structure or if a certain geometry isrequired.

Chung et al.[27] explored the feasibility of an alginate-basedscaffold for growing hepatocytes. Hepatocytes are liver cells, anda scaffold for growing hepatocytes will be helpful for regenerat-ing damaged or diseased parts of the liver, which may become apromising alternative to liver transplantation. Despite being aneffective treatment to liver failure, transplantation is plagued byproblems such as donor shortage and hepatic failure.[27] Thealginate scaffold by Chung et al. was modied by addinggalactosylated chitosan, an adhesive ligand, to the alginate, andthey reported that hepatocyte attachment onto the scaffold wasimproved. Although the work by Chung et al. was on alginatefoam, the same technique of adding anchoring groups can beapplied on electrospun nanobers, and the larger surface area 3

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available in a nanobrous scaffold may translate into furtherimprovement in hepatocyte attachment and proliferation. Also,Chu et al.[67] have developed a 3D chitosan nanober scaffold forhepatocyte culturing. Compared to a chitosan cast-lm,hepatocyte adhesion on the nanober scaffold was superior,and cells remained viable. To assess cell activity, Chu et al.compared hepatocyte functions through the synthesis ofproteins such as albumin, glycogen, and urea, and reported ahigher extent of protein synthesis on the nanober scaffold,implying superior cell proliferation. Chua et al.[68] have alsoreported a hepatocyte nanobrous scaffold composed of PCLEEPcontaining galactose ligand, which can aid cell attachment,spheroid formation, and functional maintenance of hepatocytes.

Cartilage tissue repair using nanobrous scaffold has also beendemonstrated. The aim for creating cartilage scaffolds is to repairdefects or lesions in cartilages resulting from injuries and diseasessuch as osteoarthritis.[69] Defects in cartilage tissue generally donot heal and thus surgical techniques have traditionally beenemployed for repair, such as inserting osteochondral plugs from aless crucial area into the defects, or facilitating bleeding from themedullary cavity in order to ll the defect with MSC that candifferentiate into chondrocytes.[70] With cartilage scaffolds,however, MSC and chondrocytes can be pre-seeded on thescaffold and then placed into the defects. In such applications, itis important that the scaffold be biodegradable and bioresorb-able such that it will not interfere with the repopulation of thedefect by regenerated cartilage tissues. For nanobers, thebiodegradability and bioresorbability can be customized throughthe material choice. Li et al. and Janjanin et al. have presented thefeasibility of using PCL nanobers as a scaffold for bone-marrow-derived MSC.[71,72] To promote stem cell differentiationinto chondrocytes, transforming growth factor b (TGF-b) wasincorporated. Li reported that the level of chondrogenesis ishigher with the TGF-b incorporated nanobers, compared to thecell pellet culture control. Besides PCL, the use of PLGA andchitosan as cartilage scaffolds has also been demonstrated byShin et al.[73] and Subramanian et al.[74]

Another signicant area of research is vascular tissue scaffolds,due to the widespread of vascular diseases like atherosclerosis.Traditionally, vascular repair is achieved through grafts. However,supplies are often limited for autografts whereas immunogenicresponses have been associated with allografts.[75] Moreover,requirements for dimensions and mechanical properties onvascular grafts also vary depending on the patients age andgrafting location.[76] Nanobers are attractive for this applicationdue to the exibility in dimension and property control. Anotherrequirement for vascular grafts is that thrombosis and hyper-plasia must be prevented, which remained a challenge for manysmall diameter blood vessel grafts. A proven solution to suchissues as thrombosis and hyperplasia is to pre-seed a monolayerof endothelial cells (EC) onto the graft prior to application on thedamaged vascular tissue, due to the signalingmolecules secretedby these cells.[77] In addition, pre-seeding EC can preventincomplete cell coverage in vivo, which can cause hyperplasia.[77]

Nanobers are benecial due to its high surface area and porosityfor complete cell coverage. The monolayer cell pre-seedingrequirement is an example in which a scaffold with a 2Dnanobrous structure may be of interest over 3D, since uniformityof a monolayer of seeded cell is important for ensuring thepatency of the graft. Recently, several materials have beenelectrospun into nanobers for vascular graft, with varyingdegrees of success. He et al.[78] presented an electrospun scaffold

from a co-polymer of PLA and PCL coated with collagen, whereasMa et al.[77] electrospun a polymer obtained from graftingpoly(methacrylic acid) (PMMA) onto poly(ethylene terephthalate)(PET), and then grafting again with gelatin. In both studies, theviability of EC on the scaffold is shown. The natural artery iscomposed of three layers: the intima, media, and adventitia.[79] ECare present as a lining in the intima which is the innermost layerof a artery. SMC, on the other hand, appear in sheets in themedia.The adventitia contains mainly bers of collagen and elastin.Stitzel et al.[76] mimicked the arterial wall by electrospinning ascaffold with a blend of collagen, elastin, and PLGA, andexamined the cell viability using both EC and smoothmuscle cells(SMC). Lee et al.[75] also investigated the mechanical properties ofand SMC viability of nanobers spun from various blendedpolymers such as collagen/elastin, and collagen/elastin blendedwith PCL, PLGA, or PLA.

Scaffolds for repairing neural tissues have also been intensivelystudied as alternative treatments for spinal cord injuries. Ingeneral, the goals for neural tissue scaffolds are to support axonregrowth and grey matter repair, due to their insufcientself-regeneration.[80] Axon regeneration poses a directionalrequirement on the scaffold design because axons are protru-sions from neurons that connect to nearby cells, and the directionof axon growth is therefore an important consideration. Theelectrospinning technique can be employed in fabricating neuraltissue scaffolds because the nanober morphology can becustomized for directional growth of neural cells. Xie et al.[81]

examined the effect of embryonic stem cell differentiation onrandomly and uniaxially oriented PCL nanobers. In this case,embryonic stem cells can be induced to differentiate into neurallineage cells by treating with retinoic acid. The authors found thatneurite outgrowth is higher for the aligned nanober scaffold,since the stem cells tend to elongate parallel to the bers. Thesame observation was also made by Yang et al.,[82] whose groupreported that neurite outgrowth is higher on aligned PLA bers.On top of hat, Yang et al. reported that the differentiation rate onPLA nanobers is higher than PLAmicrobers. These studies haveshown the potential of 1D nanobrous structures as opposedto 3D, due to the directional nature required for cell growth. If a3D scaffold was used for this application, neural cells wouldhave three directions for growth, which is not desirable for axonregeneration. Schnell et al.[83] also prepared PCL nanoberscoated collagen and found that while viability of Schwann cells,olfactory ensheathing cells, and broblasts were evident,the cell migration and neurite orientation are superior onscaffolds that are coated with collagen as opposed to a PCL-onlyscaffold.

Besides the common biopolymers mentioned, the use ofconducting polymers in tissue regeneration has also beenexplored. Li et al.[84] investigated on nanober scaffolds ofelectrospun gelatin containing polyaniline (PANi) for cardiactissue repair. Gelatin is known to be suitable for tissueregeneration due to its origin as a hydrolysis product of collagen.Li et al. reported that addition of PANi to gelatin aided theelectrospinning process, since the ber diameter decreased asthe PANi concentration increased. Also, the authors showed thatthe nanober scaffold supported the attachment and prolifer-ation of H9c2 rat cardiac myoblasts, to a similar degree as thetissue culture-treated plastic (TCP) control. Whilst the tissueregeneration performance of nanober scaffold is not superior tothe control in this case, this study showed the potential ofcreating a basis for electrical stimulation studies. Since PANi is3

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conductive, it is possible to study the effect of electricalstimulation on the functionality of the H9c2 cells, or maybeeven to produce a scaffold that allows control of cell activitiesthrough electrical signals.

The examples of current work reviewed herein provide anoutline of the current ability to effectively utilize the uniqueproperties of electrospinning and nanobers to enhance tissueregeneration. In many cases the nanobrous scaffolds have beencompared to microbers and gel lms, showing that the largesurface area on nanobers can enhance cell attachment andproliferation. Moreover, the study by Xie et al.[81] and Yanget al.[82] showed that the ability to induce directional growth viaaligned nanobers could be translated to controlling thedirection of cell growth. Co-axial electrospinning and fabricationof polymer composite nanobers such as the gelatinPANiscaffold outline the possibility in making smart scaffolds in whichcell differentiation can be controlled through mechanisms suchas growth factor release and electrical signaling. On top of anappropriate nanober scaffold that can support cell activities,growth factors and other proteins can be incorporated toenhance cell activities. An important consideration in incorpor-ating these macromolecules to nanober scaffold is that theirdelivery to the target tissue must be controlled. The next sectionwill focus on the delivery of these macromolecules, as well asother therapeutics, using nanober carriers.

DRUG DELIVERY

Nanobers can also be used to deliver proteins to target tissuesin a controlled manner. In fact, the use of nanobers inencapsulating and delivering therapeutics is another area offocus in biomedical nanobers. Nanobers are attractive for twomain reasons. First, nanobers have a large surface area tovolume ratio, which is even higher considering the pores thatexist inside the bers. Not only can the large surface area ensure ahigh therapeutics take-up, it can also reduce the constraint todrug diffusion leading to increases in the total fraction of drugthat can be released. Secondly, relevant nanober properties,such as ber diameter, porosity, and drug binding mechanisms,are highly customizable through process parameters andmaterial choice, the rate of drug release can be tailored foreach application.

The most efcient way to cure a diseased organ is to focustherapeutics on their intended sites of action. Most drugs in thepharmaceutical market today can be classied into site-specicdelivery, or carriage through the blood stream. In site-specic drugdelivery, drugs are placed directly into or adjacent to the affectedarea. Examples of these systems include ointments and inhalers.Whilst there are research focusing on new methods of site specicdrug delivery such as antibody conjugated microcapsules,products available in the market today can only reach organsthat are supercial and easy to reach. Drug delivery by circulation isintended to reach sites that are inaccessible to direct placement,and examples include orally taken medicine and injection.However, the effect of drugs delivered by circulation tend to besystemic and thus non-diseased areas can be affected, causing sideeffects that can be severe in some cases. For example, drugs forcancer therapy can cause hair andweight loss because these drugscan shut down rapidly dividing cells but are unable to differentiatebetween cancer cells or the other rapidly dividing cells. In this

regard, the drug dosage control is also important, as it is desirableto apply theminimum amount of drug that is sufcient to treat thediseased area but not enough to cause adverse side effects. Moreideally, the release of drugs can be sustained at the required levelfor a period of time, such that the need of multiple drugapplications can be eliminated. Figure 8 shows the release offusidic acid in PMMA and is an example of signicant burst release,which should be avoided for some applications. The mainobjectives in developing new drug carriers are therefore two-fold:to allow drug carrier placement at the target site and controllingthe rate of drug release. Polymer matrices such as those fabricatedfrom electrospinning are capable of holding therapeutics andreleasing them in a controlled manner.

Drug loading

When using nanobers as drug carriers, therapeutics must rst beimmobilized in the polymer matrix in order for the release controlmechanisms to operate. The method depends on the polymerand the therapeutic, but immobilization can generally becategorized through entrapment or binding.[85] Entrapment isa physical means to contain drugs or proteins in a polymermatrix.In nanober drug carriers, examples of drug entrapment can bedone through crosslinking the polymers, or by entrapping thedrug in an intermediate carrier which in turn contained a polymermatrix that forms the nanober. Alginate, when crosslinked withdivalent ions such as calcium, is a common polymer forentrapment in its bulk form.[85] We have previously electrospunalginate nanobers and incorporated poly-L-lysine into thematrixby the addition of the protein into the crosslinking reaction. Forintermediate carriers in a polymer matrix, the most commonexample is a core-shell nanober drug carrier such as the PCLshell, BSAdextran core ber developed by Jiang et al.[25] Besidesentrapment, drugs can also be bound to polymers that formnanobers. The binding can be due to the formation of hydrogenbonds, hydrophobicity, electrostatic interaction, and more. Forexample, Patel et al.[86] showed, through molecular dynamicsimulation, that two anticancer drugs, Cucurbitacin B andCucurbitacin I, can be bound to PCL through both hydrophobicinteractions and hydrogen bonding. On the other hand, chitosan,a cationic polysaccharide, can bind with negatively chargeddrugs through electrostatic interactions.[87]

Figure 8. Release of fusidic acid in PMMA, demonstrating burst release.

This gure is available in colour online at wileyonlinelibrary.com/journal/pat.

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Drug release

Drug release from nanobers can be described through threemechanisms: desorption from ber surface, solid-state diffusionthrough bers, and in vivo ber degradation. Drug release testsfrom nanobers are commonly conducted in phosphate-bufferedsaline (PBS) solutions. When the nanober drug carrier issubjected to PBS, the bers will be surrounded by the solution.The solution will also penetrate the space in between individualnanobers. When the nanober drug carrier is swollen by theaqueous phase, drugs or proteins attached to the ber surfacescan be released. Drug release from nanober surface is a two-stepmechanism, starting from desorption of drugs from the bersurface, followed by fast diffusion into the aqueous phase. Thedesorption mechanism is not limited to the outer surface of thenanobers but also includes drugs on the surfaces of thenanopores inside the nanobers. Considering the nanometer sizescale of the inner pores, and that the nanopores are most likelyinterconnected to some degree, the surface area would be muchlarger than the ber outer surface area. This view is supported bySrikar et al.[88] whose team studied release of a rhodamine 610chloride uorescent dye from PCL and poly(methylmethacrylate)(PMMA). Srikar et al. also developed a model to predict the dyerelease through time, by considering the mass balance on thenanopores surface, desorption of the dye, diffusion of the dye inwater, and the concentration eld of the dye in the nanopores.The equation is shown below, where Gt is the mass released attime t, Md0 is the initial mass of the dye, a is the nanoporosityfactor dened by the initial mass of dye on pore surface over thetotal initial mass of dye, and tr is the characteristic time that isdependent of the nanopores length and the effective diffusivity.It can be seen from the Srikar equation that the amount of releaseincreases with time, but is controlled by the nanoporosity factorand the characteristic time, which depends on the surface area,both on the outer surface and inside the pores, and poregeometry, respectively. This equation shows that through thecontrol of porosity and surface area, the drug release rate can becontrolled. Furthermore, electrospinning allows the control ofsurface area and porosity through varying ber diameter, and thenumber of factors that can affect diameter control has beendiscussed in the electrospinning section. In addition, due to thelarge surface area and porosity in nanobrous structures, the G/Mratio can be very high compared to geometries with smallersurface area at an equal volume basis, indicating that thecumulative portion of drug release from nanobers can be higherthan in other geometries such as gel lms

GtMd0

a 1exp p2

8

t

tr

Srikar et al. were able to establish a close match betweenexperimental data and the release predicted by the model.Gandhi et al.[89] also generated predictions from the same modeland establish a close t experimental data on the release of BSAwith anti-integrin antibody from PCL nanobers. Both Srikaret al.[88] and Gandhi et al.[89] asserted that for slow degradingpolymers such as PCL and PMMA, the desorption mechanism isthe dominant release mechanism, as opposed to solid-statediffusion of drugs through solid nanobers. Consistent with theirarguments, both authors found that by reducing nanoporosityvia increasing electrospinning solution concentration, the releaserate can be reduced. The authors argued that if solid-statediffusion is the primary release mechanism, then 100% release of

drugs is expected which does not happen in almost every case.The inability for complete drug release is attributed to theentrapment of drugs inside regions of more concentrationpolymers or higher molecular weight, which is inconsistent ifsolid-state diffusion as the primary mechanism is assumed.

Among the existing work that neglected the desorptionmechanism, most asserted that drug release is mainly throughdiffusion. For example, Luong-Van et al.[90] investigated thecontrolled release of heparin, a treatment for intimal hyperplasia,from electrospun PCL bers. The authors were able to achieve asustained release of heparin. Moreover, when plotting thefractional release of heparin versus the square root of time,Luong-Van et al. were able to obtain a plot with linear correlation,implying that the heparin release followed Fickian diffusion.Chew et al.[91] studied the release of human b-nerve growthfactor (NGF) from PCL-ethyl ethylene phosphate (PCLEEP) bers,and established that diffusion is the main release mechanism dueto the low degree of ber degradation. Chew et al. also comparedexperimental data to estimates from 1D Fickian diffusion, underthe assumptions that the bers are non-swellable and that theyare a monodispersion of cylinders. However, the estimates didnot match well with the experimental data. The same trend canbe observed in the work on delivery of Paclitaxel by Xie andWang.[92] It should be noted that in all the cases discussed, as wellas other existing work on nanober drug delivery, the termdiffusion is used extensively, but investigators rarely explainedthe nature of the diffusion, i.e. whether or not it is solid state. It isalso likely that the diffusion effect observed in existing work isfrom the diffusion of desorbed drugs into the bulk liquid, whichsupport the assertion by Srikar et al. and Gandhi et al. The balancebetween desorption-based mechanism and solid-state diffusionmechanism still remains ambiguous and thus is an area ofpotential future research.

Drug release can also be achieved through degradation of thenanober. Upon degradation, drugs that are entrapped inside thepolymer matrix can be freed. This mechanism plays a moresignicant role in fast-degrading polymers such as chitosan,alginate, and PVA, and less in those that were mentionedpreviously. The work of Zeng et al.[93] on BSA release from PVAbers represents a case in which drug release rate was too fastdue to polymer degradation. Signicant burst release wasnoticed on PVA nanobers which experience fast degradation inwater. However, when the bers are coated with parylene whichis less degradable, the release rate is greatly reduced. Theconcept of surface and volume degradation is also important fornanober drug carriers as the drug release rates according to thetwo denitions of degradation can be different, especially whencomparing between homogenous nanobers and core-shellnanobers.

Among the mechanisms described, the desorption releasemechanism is a relatively fast process, due to the close proximitybetween drugs on the nanober surface and the surroundingliquid, and thus can play a signicant contribution to burstrelease. Different strategies exist to further control the releaserate, such as blending with other polymers to reduce the amountof drug-bearing surface. Another technique that has recentlygained popularity is co-axial electrospinning, as mentioned in theprevious section. By placing the drug-bearing polymer in thecore, encapsulated in the shell ber, the distances that bulk liquidmust travel to penetrate the available surfaces of the corepolymer, and that the drug in the polymer must diffuse through isincreased. Also, the shell polymer presents as an extra layer of3

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material that must be degraded before full drug release can beachieved. Yang et al., who investigated the release of lysozyme ina methyl cellulose core, poly(DL-lactic acid) (PDLLA), demon-strated that the extent of burst release can be reduced throughadjusting the thickness and permeability of the shell.[94]

Applications

Whilst there is an immense effort on understanding the drugrelease mechanism in nanobers, much research has also beenconducted on specic applications of nanobrous drug carriers.As mentioned in the previous section, the work of Luong-Vanet al.[90] focuses on the heparin-releasing PCL electrospun bersas a treatment for intimal hyperplasia. The hyperplasia causesthickening of the inner layer of the blood vessel, tunica intima,following a surgery or injury of the tissue, due to the proliferationof vascular soft muscle cells (VSMC). Heparin is chosen in this casebecause it resembles heparan sulfate GAGs in uninjured bloodvessels, which help maintain VSMC at a non-proliferative state.[90]

The other main objective in this study is to control the rate ofheparin release because a burst release may lead to bleedingcomplications. The release study is done in PBS solution, and theauthors reported a total heparin release of around 5060% inthe 14-day experiment, with approximately 2025% released inthe rst 24 hr. More importantly, Luong-Van et al. reported a 40%reduction in the net growth of VSMC in the heparin-loadednanober, compared to a heparin-free control.

Another area in which the use of nanober drug carrier isinvestigated is prevention of abdominal adhesion, which is aninammatory response commonly originating from abdominalsurgical procedures. Abdominal adhesion may cause organsaround the abdomen to attach to the surgical site or othersurrounding organs. To prevent abdominal adhesion, antibioticsare usually applied. Zong et al.[95] examined the release of anantibiotic, cefoxitin sodium, on PLGA scaffolds as well as a PLGAblend with a poly(ethylene glycol)poly(lactic acid) (PEGPLA)co-polymer. The performance of the scaffolds was assessed byevaluating the extent of adhesion with a numeric score andsumming up the scores from multiple tests for each scaffold.Zong et al. reported that evenwithout antibiotics, by applying thePLGA scaffold, adhesion is reduced from 78% to 40%, and to 22%when the PLGA/PEGPLA scaffold is applied. When the cefoxitinsodium is added to the scaffolds, adhesion is reduced to 0%.Reduction in abdominal adhesion successful on the nanoberscaffolds without antibiotics is likely due to the increasedresistance to tissue cell migration compared to the case when thecells are free to move. This study nonetheless showed thepotential of nanobrous antibiotic carrier on eliminatingabdominal adhesion. Bolgen et al.[96] also reported the feasibilityof electrospun PCL bers carrying a commercial antibiotic, Biteral,in signicantly reducing abdominal adhesion.

Kenawy et al.[40] examined electrospun bers of PEVA, PLA, andblends of the two as carriers of tetracycline hydrochloride (TCH)for treating periodontal diseases. The authors compared therelease on nanobers to that on cast lms and reported that thetotal fraction of drug released is higher on nanobers, which islikely due to the increased surface area of nanobers.

Besides drugs, DNA can also be released from nanobrouscarriers for therapeutic uses. Saraf et al.[97] presented their workon plasmid DNA delivery to cells for facilitating production ofgrowth factors or signaling bioactive molecules, which may beuseful for tissue regeneration. In their work, Saraf et al. adopted a

core-shell ber design, with the core being the plasmid DNAcontained in a PEG matrix, and the shell being PCL containingpoly(ethylenimine-hyaluronic acid), which is a non-viral genedelivery vector. Since the plasmid DNA is negatively charged, andthe gene delivery vector is positively charged, upon release fromthe core, the plasmid DNA will rst bind with the gene deliveryvector, which will in turn be released from the ber. The genedelivery vector will then aid the transfection of cells with plasmidDNA. Although it is difcult to measure the level of DNA releasedue to its binding with the delivery vector, Saraf et al. reportedthat transfection efciency is maintained for 60 days, showing thepotential of such a system in gene therapy.

The potential for nanobrous drug carriers in cancer therapyhas also been shown in recent work. Xie and Wang[92] looked attreating C6 glioma using electrospun PLGA ber scaffolds withaverage diameters ranging from 30 nm to 10mm. Paclitaxel, anantineoplastic agent that can be used for various types of cancer,is loaded into the PLGA bers. Being an antineoplastic agent,Paclitaxel can combat the abnormal proliferation of cells, as is thecase in cancer.[92] However, Paclitaxel can also act on healthy cellsthat are constantly dividing, and therefore the release rate of thedrug must be controlled to prevent adverse side effects. Xie et al.reported among the total mass of drugs loaded, 22% wasreleased in the rst 24 hr, while a sustained release wasmaintained for the next 2 months with 80% of the drugsreleased at the end. Additionally, total drug release from berswith average diameter in the nanometer scale was around 20%higher than those in the micrometer scale. Cell viability test alsoshowed approximately 80% reduction in C6 cell viability with thePaclitaxel-loaded nanobers.

Up until this point, only polymer drug carrier systems havebeen discussed, but the work by Wang et al. outlines a case inwhich metallic particles can be used. In their work, Wang et al.[50]

electrospun poly(vinyl pyrrolidone) (PVP) with a high concen-tration of the magnetic iron oxide nanoparticles. By placing twoparallel positioned bar magnets on the ber collector, themagnetic eld generated in between the magnets allowedalignment of the electrospun bers, which formed a parallel array.The nanobers were then treated with oxygen plasma to removeall organic constituents in the bers, leaving a brous structurethat contains only the iron oxide nanoparticle. In this case beralignment during electrospinning with PVP is important to obtainaligned, cohesive strings of iron oxide nanoparticles. Moreimportantly, Wang et al. reported that magnetic property couldbe retained in this structure. Although no drugs were loaded intothe iron oxide bers, the system shows potential as a magnetictargeting device that can be especially useful for minimallyinvasive cancer treatment. By using nano-sized iron oxideparticles, cancer therapeutics can be focused to a very smallarea, reducing side effects often associated with chemotherapy.The iron oxide nanober can be carried to a diseased area usingmagnets. Once attached to the cancer area, the nanober may killcancer cells either by hyperthermia or by drug release. Inhyperthermia, the nanoparticles can be heated up by applying analternating magnetic eld (AMF),[98] under which polarity of theparticles change tens of thousands of times in a minute, toachieve the temperature of 40508C.[99] For drug release, it ispossible to rst coat the nanoparticle surface with PEG, and thenhave drugs bond to the PEG coating.[100] The ability to incorporatemagnetic elements into nanober membranes also bringspossibilities for nanober-based magnetic actuators, which canbe useful for drug delivery and medical imaging devices.[101] 3

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The adaptability of nanobrous drug carriers allows thistechnology to show potential in treating many types of diseases.Like the examples in tissue regeneration studies, the perform-ance of nanober drug carriers, in terms of total amount of drugreleased, is shown to be greater than microbers and gel lms.This observation is in accordance to the equation formulated bySrikar et al. and shows the potential advantage of using ananometric and brous structure in drug delivery. Furthermore,the example of a drug delivery system utilizing the EPR effectdiscussed in the Introduction Section outlines the potential inadopting a hierarchical structure using a nanober as a matrix forfunctionalized nanoparticles and drug molecules. Despite havinggained signicant understanding in nanober drug delivery,there are still several aspects that are not well understood. Thefuture work must focus on better understanding the drug releasemechanism, and drug carrier response in biological systemsrather than PBS test solutions.

CONCLUSION

In the recent decade, tremendous efforts have been put oninvestigating nanobers for biomedical applications. Theseefforts have led to a signicant increase in the number ofbiocompatible polymers that can be electrospun into nanobers.The availability of such variety of biocompatible polymers, eachwith their distinct properties, also prompts researchers to seekways to combine desirable properties of multiple polymers, aswell as bioactive materials that cannot otherwise be electrospundirectly. This in turn became the driving force behind improvingelectrospinning techniques and led to useful variations such asemulsion electrospinning and co-axial electrospinning. Suchrapid developments in electrospinning technology propellednanobers to become a promising class of novel materials aspotential solutions to the challenges faced by the medicalindustry today.

This review outlined some of the recent work in nanober drugcarriers and nanober tissue regeneration scaffolds, such as therelease of antibiotics, anti-cancer therapeutics, proteins, DNA,and growth factors from nanobers, and tissue scaffoldapplications on skin, bone, ligament, cartilage, vascular, andneural tissues. In each application, some of the backgroundprinciples on the role of nanober in performing the intendedfunction have been discussed, such as drug loading and releasemechanisms, and parameters that affect tissue regenerationscaffold performance. In tissue regeneration, the importance ofber architecture, diameter, porosity, mechanical propertie

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