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Pharmaceutical Nanotechnology, 2014, 2, 23-34 23

The Multifaceted Potential of Electro-spinning in Regenerative Medicine

Kieran Fuller, Abhay Pandit and Dimitrios I. Zeugolis*

Network of Excellence for Functional Biomaterials (NFB), National University of Ireland Galway (NUI Galway), Galway, Ireland

Abstract: The increased interest in nanotechnology has resulted in an intense investigation and development of nano-fabrication methods. Among the bottom-up approaches, electro-spinning has attracted great interest in recent years. The popularity of electro-spinning lays on the fact that it is a relatively simple and economic technique that enables production of large quantities of nano- to micro-meter range fibrous materials with various morphologies and architectural features. The versatility of electro-spinning is evidenced by the range of applications being utilised, including filtration, textiles, batteries, and tissue engineering and regenerative medicine. Specifically to biomedical applications, advancements in the electro-spinning setup have allowed the development of electro-spun mats that closely imitate native extracellular matrix assemblies and provide opportunities for localised and sustained delivery of therapeutics. Herein, we are discussing dif-ferent electro-spinning setups and their distinct benefits for regenerative medicine applications.

Keywords: Architectural features, delivery of therapeutics/biologics, electro-spinning, nano- and micro-fibres, scaffold archi-tecture, tissue engineering.

1. INTRODUCTION

The nanomaterial’s sector global market was worth $15.9 billion in 2012 and is expected to rise to $37.3 billion by 2017 with a compound annual growth rate of 19.1% [1]. Top-down nano-processes, such as lithography, deposition and etching, reduce large pieces of materials to nano-scale components. However, such technologies require large amounts of starting materials and are often associated with excessive waste generation. On the other hand, bottom-up nano-fabrication approaches, such as electro-spinning and self-assembly, are utilised to build three-dimensional hierar-chical constructs by putting together atomic and molecular components in a hierarchical fashion. Such technologies have minimal processing waste and are particularly favoured for biomedical applications. Among the various bottom-up processes, electro-spinning is favoured a means to fabricate sub-micron devices, largely attributed to its inherent versatil-ity and controllability [2, 3]. Indeed, to-date, numerous natu-ral and synthetic polymers [4, 5]; ceramics [6, 7]; and metal-lic materials [8, 9] have been electro-spun that have found applications in textile [10], filtration [11] and biomedicine [12] sector/industries. The rationale of using electro-spinning in biomedicine lays on the fact that this technology can create three dimen-sional fibrous scaffolds that closely imitate the nano- to mi-cro-scale intertwined fibrillar meshwork of the extracellular matrix [12-15]. The first paper using electro-spinning a means to develop an implantable device was published in 2002 [16]. Since then, a staggering number of peer-reviewed publications (12,087 papers; Source: Scopus; Term searched: electro-spinning); patents (75,300 patents; Source: Google

*Address correspondence to this author at the Network of Excellence for Functional Biomaterials (NFB), National University of Ireland Galway (NUI Galway), Galway, Ireland; Tel: +353-(0)-9149-3166; Fax: +353-(0)-9156-3991; E-mail: dimitrios.zeugolis@nuigalway.ie

patents; Term searched: electro-spinning); clinical trials (2 trials; Source: ClinicalTRials.gov; Term searched: electro-spinning); and products (e.g. NovaMesh™, Nicast Ltd; HealSmart™, PolyRemedy® Inc.; Bioweb™, Zeus® Inc) have become available, clearly demonstrating the multifac-eted potential of this technology. In this review, we will dis-cuss how modifications in the electro-spinning setup allow (a) development of implantable devices with different archi-tectures for specific clinical targets (Fig. 1) and (b) function-alisation through the ability to deliver therapeutic molecules (Fig. 2).

2. ELECTRO-SPINNING SETUP AND PARAMETERS AFFECTING ELECTRO-SPINABILITY OF MATE-RIALS

A typical electro-spinning setup is made up of a syringe containing the material to be extruded suspended in a highly volatile solvent; a syringe pump that controls the flow rate of the extrudate; a power supply that controls the voltage; a metallic spinneret; and a metallic collector. During extrusion, the electrostatic force, applied by the high voltage power supply, overcomes the surface tension and forms a Taylor cone. A jet ejection is formed at the tip of the metallic spin-neret and is subsequently attracted towards the grounded metallic collector. The initial ejection is in a uniaxial direc-tion, which subsequently develops into a conical envelope through electrostatic and fluid dynamic instabilities [17]. As the solvent evaporates, there is a significant reduction in the ejected material size, which results in fibre formation that is collected on the metallic collector. An alternative to the tra-ditional setups is needless electro-spinning. This is a tech-nique which is gaining increasing interest and there are vari-ous methods of electro-spinning without a needle such as bubble electro-spinning whereby a gas is passed through a solution and the bubbles disruption of the surface creates a Taylor’s cone [18], and another method uses a variety of

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24 Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 Fuller et al.

spinnerets which rotate through a solution and when in close proximity to the collector where the electrostatic force is sufficient for Taylor cone formation, ejection of the solution occurs [19]. Operating parameters are crucial for the production of reproducible electro-spun products. Indeed, selection of an appropriate volatile solvent, compatible with the material to be electro-spun is crucial for production of consistent fibres [20]. Temperature [21, 22] and humidity [21, 23] have been reported to significantly alter material properties and solvent evaporation rate. Material and solution properties, such as concentration and molecular weight [24]; viscosity [25]; conductivity [24]; and surface tension [24] are also crucial for homogeneous fibre production. Fibre diameter can be controlled by varying the extrusion rate [26]; the voltage / charge of the material [27]; the spinneret/needle gauge [25]; and the distance between the spinneret and the collector [28].

Alterations in the collector setup offer opportunities for the development of custom-made materials that closely imi-tate native extracellular matrix assemblies. Indeed, the first generation of electro-spun setups utilised a flat and static collector that gave rise to random fibrous mats suitable for

wound healing applications [29]. Subsequent use of rotating collectors gave rise to aligned mats, suitable for tendon [30, 31], cornea [32, 33] and neural [34, 35] applications. There are numerous modifications for the fabrication of aligned electro-spun materials, such as parallel electrodes, rotating wire drum collectors and disc collectors [36]. Additionally, function specific collectors can be utilised for the develop-ment of more complex architectures. Indeed, a cylindrical tube can be used for the production of cylindrical, three-dimensional electro-spun constructs suitable for vascular prostheses [37, 38] or peripheral nerve repair [39, 40] and electrodes with a peripheral ring and central point have been used for dura mater formation [41]. The utilisation of porous collectors enabled production of macro-porous electro-spun products that offer better cell infiltration capacity [42-44] and are bypassing multistep processes, such as physical re-moval of material [45]; salt leeching [46]; gas foaming [46]; and dissolution of one of the co-spun polymers [47]. Near field electro-spinning allows for the fabrication of scaffolds, where finely controlled topographies are required [48]; how-ever this technique is mostly employed for electronics appli-cations.

Fig. (1). Variations in the electro-spinning setup allow development of three-dimensional implantable devices that closely imitate architec-tural features of the tissue to be replaced.

The Multifaceted Potential of Electro-spinning in Regenerative Medicine Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 25

Fig. (2). The superiority of electro-spinning in regenerative medicine lies on the fact that offers several opportunities for sustained and local-ised delivery of therapeutics. 3. ELECTRO-SPUN SCAFFOLDS AS DELIVERY VE-HICLES

Biomaterials design has evolved from basic constructs that imitate native tissue architecture and material properties to more sophisticated products that aim to interact with the host, promoting repair and regeneration. It has been postu-lated that sustained and localised delivery of therapeutics will alleviate issues associated with conventional drug deliv-ery platforms, such as high dosage regime and drug toxicity. It soon became apparent that electro-spun devices can act as ideal delivery vehicles, as appropriate selection of polymer will enable sustained delivery of the cargo through its con-trollable degradation rate, whilst the high surface area of the scaffold will allow delivery of much larger cargos or cargos

with short half-life [49, 50]. To-date, electro-spun scaffolds have been used to deliver numerous therapeutics / bioactive molecules, utilising a range of different delivery methods.

3.1. Blending

Blending the therapeutic into the extrudate solution was the first, and remains the simplest, technology having been assessed [50]. The elution rate of the therapeutic is dependent on the degradation rate of the carrier material. This delivery system has been extensively assessed in various clinical targets and bioactive molecules, including vascular tissue repair with heparin [51]; neural regeneration with growth factors/genes [52]; dermal [53], brain [54] and bone [55] tissue infection with various antibiotics (Table 1) [51, 53-63]. Despite the

26 Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 Fuller et al.

Table 1. Examples of blending electro-spinning technologies as means to deliver therapeutics and biologics in a range of clinical targets.

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

Hyper branched polyglyc-erol and C. officinalis extracts.

Random. C. officinalis extracts enhanced the bioadhesive properties, tensile prop-erties and swelling.

No difference between sili-con in an irritation test. Full re-epithelialisation after 6 days.

[56]

PVA and quaternised chitosan.

Random. All growth of S. aureus and E. coli was prevented within 2 hours.

- [57] Wound healing

PEO:PDLLA 50:50 and ßnisin.

Random. Sustained release of nisin for 4 days. Enhanced wound closer and significantly reduced the CFU.

[53]

PCL with heparin. Random.

Sustained heparin release for 14 days, without a pro inflammatory response or alteration in biological functionality.

- [51]

POC and PLCL. Random The POC at 25 and 40% signifi-cantly increased cell proliferation.

- [58] Cardiac

PDO and Elastin. Random. Varying the component ratios affect the tensile properties.

- [59]

PLGA with Vancomycin. Random. Sustained release for 6 weeks. No infection or inflammation for up to 8 weeks.

[54]

PCL with BDNF. Partially aligned. -

BDNF improved microglial integration to a untreated injury levels but also en-hanced neurite sprouting.

[60] Neural

PCL and Gelatin. Random and aligned. Gelatin significantly reduced tensile properties but increased cell viability.

- [61]

Bone PLDL with HAP. Random. The presence of HAP significantly unregulated mineralisation and os-teoblastic differentiation.

- [62]

Antibacterial PLGA, Rifampicin and Fusidic acid.

Random.

Initial burst release followed by a sustained release for 35 days. Co-loaded agents were fully bactericidal in 48hrs.

Co-loading of the drugs significantly reduced CFU numbers.

[55]

Not specified PDO and PMeDO. Random.

PMeDO addition did not increase the crystallisation rate for e-spun meshes unlike the properties in films, which correlated with mechanical proper-ties and sustained degradation up to 5 weeks. The increased levels of PMeDO correlated with increased cell proliferation.

- [63]

efficacious pre-clinical data obtained to-date, the main limi-tation of this method stems from the uncontrolled release profile in heterogeneous solutions, which also results in pockets of decreased mechanical properties.

3.2. Dual Electro-spinning

Alternatively, pre-fabrication methods are based on modification of the electro-spinning setup. For example, dual

electro-spinning utilises two separate spinnerets that produce fibres on the same collector simultaneously. The spinnerets can be loaded with different therapeutics to create an inte-grated fibrous hybrid structure. To avoid electrostatic inter-action of the spinnerets, they are kept apart by 90° or more. This technology has tremendous potential in biomedicine (Table 2), [64-73] especially in clinical indications, where a delivery platform is required in conjunction with appropriate

The Multifaceted Potential of Electro-spinning in Regenerative Medicine Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 27

mechanical properties [64]. This method has been utilised for antibacterial delivery to promote dermal [65] and abdominal wall regeneration [66] and enhanced bone regeneration, through protein delivery [67]. Although dual electro-spinning offers the possibility of eluting a molecule, whilst retaining some of the physical characteristics of the scaffold, reduction of mechanical properties, delamination and separa-tion may occur, once the eluting material has degraded; these issues stemmed research for alternative strategies.

3.3. Co-axial Electro-spinning

Co-axial electro-spinning gives rise to a multi-compartmental fibre with a uniform [74] or interrupted [75] internal core or cores[76] and external sheath which can have a single [76] or multiple layers [77]. This set-up can be used

to deliver multiple molecules in a single material, but it is more commonly used to deliver a material with an internal core, which provides structural support and a sheath contain-ing a molecule to be released in a controlled manner. This method has been used for numerous clinical targets [74, 78] including in vivo delivery of antibacterial drugs [78]; non-steroidal anti-inflammatory drugs to prevent adhesions in tendon defect model [79]; and growth factor delivery in wound healing models (Table 3) [80-87].

3.4. Surface Treatment

Immersion of the electro-spun mats into a solution con-taining the molecule of interest has also been extensively studied for numerous clinical targets (Table 4). Immersion has been used for a variety of pre-clinical applications, including

Table 2. Examples of dual electro-spinning technologies as means to deliver therapeutics and biologics in a range of clinical targets.

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

CA and with PHMB. Random. CA enhanced hydrophobicity and 50% release of PHMB within 24h resulting in reduced microbial activity by 96% +.

In a burn model the co electro-spun 4:1 PEU: CA with PHMB resulted in the most significant healing after 16 days.

[65]

PEO with HA-DTPH and PEGDA

Random. Dual spinning of the crosslinked al-lowed in situ crosslinking.

- [68] Wound healing

PLLA with Lidocaine and mupirocin.

Random. The dual spinning allowed for an initial burst of lidocaine and a sustained re-lease of mupirocin up to 72 hours.

- [69]

Cardiac PCL: Gelatin/ PLGA: Gelatin and PLGA: Chitosan.

Random. The scaffold is cytocompatible for fi-broblasts and endothelial cells.

- [64]

PEO and PCL. Random. Tensile properties were a function of the polymer ratios. High PEO content in-crease cell integration once removed.

- [70]

PLLA and PEO alone and blended.

Random. The removal of PEO drastically en-hanced cell integration due to higher porosities.

- [71] Bone

PLLA with rhBMP-2 and PLGA with Ca-P.

Random. Fabricated evenly distributed bicompo-nent scaffolds.

- [67]

PLLA: Col and PCL: Col 1:1.

Random.

Distinct mechanical properties scaffold within the same scaffold for muscle and tendon. The scaffolds allowed cell adhe-sion and differentiation of myoblasts into myotubes.

- [72]

Interfaces

PCL with HAP and PUR.

Random.

Bone and ligament scaffold. Gene ex-pression of BMP-2 and osteopontin were up regulated on the mineral con-taining region and the opposite was seen for ALP mRNA.

- [73]

Anti-bacterial PEUU/ PLGA with tet.

Random.

Antibiotic activity retained up to 7 days. Addition of PEUU to PLGA reduced tensile properties but prevented shrink-age after 24hrs.

The presents of tet significantly reduced skin dehiscence and abscess formation.

[66]

28 Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 Fuller et al.

Table 3. Examples of co-axial electro-spinning technologies as means to deliver therapeutics and biologics in a range of clinical targets.

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

PELA with bFGF core. Random. After an initial burst release, sus-tained release of bFGF occurred until 25 days.

Meshes significantly enhanced re-epithelialisation and mature capil-lary vessel formation.

[80]

Wound healing

PLA with a PEG and salicylic acid core.

Random.

Porous sheaths were created by increasing the DMAC solvent concentration which facilitated faster drug release.

- [81]

Neural PLCL with a BSA and NGF core.

Random. -

The fibres with a BSA/ NGF core nerve reconstruction were similar to an autograft and significantly better to hollow PLLA fibres.

[82]

PLLA with a CFX-Na core.

Aligned and subse-quently braided.

Co-axially spun fibres had a more gradual release profile up to 250hrs and had better tensile properties than blended fibres.

The PLLA with CFX-Na showed less inflammation and infection than commercial silk or PLLA alone.

[78] Sutures

PLLA with a TCH core. Aligned. The encapsulated core prevented an initial burst release profile.

- [83]

Gelatin with a PCL core.

Random.

The PCL core significantly in-creased tensile properties and the sheath provided a porous structure for cell proliferation and adhesion.

- [84]

PCL and PEG with a BSA core.

Random.

Heterogeneous release profile from blended meshes and homo-geneous release profile from coax-ial meshes.

- [74]

PCL/PEG with a BSA and PDGF core.

Aligned.

Rapid initial release occurred which plateaued after 15 days. The PEG levels allowed for con-trollable release.

- [85]

PCL with a PEG, pDNA and PEI-HA core.

Random. The release profile was found to be most dependant concentration and MW of the core polymer.

- [86]

Not specified

Various combinations of PHB and PDLLA were tested as either the core or sheath polymer with the location of DMOG also mixed.

Random.

Varying the thickness of the PHB sheath allowed for sustained re-lease, while all other parameters resulted in a burst release.

- [87]

delivery of BMP-1 [88], simvastatin [89] and collagen/nano hydroxyapatite [90] for guided bone repair and delivery of antibiotics to abdominal wall [91]. Although plasma treat-ment has been shown to increase cell proliferation, this treatment has been shown to have detrimental effect on the mechanical properties of meshes [92]. To this end, bi- and multi-functional conjugation strategies based on transglu-taminase [93] and PEG-based systems [94, 95] respectively have been introduced (Table 4) [44, 88-91, 94-97].

3.5. Multi-layering

Multi-layering of electro-spun meshes can be beneficial for designing a material which will traverse multiple cell types for interfacial tissue engineering application and com-

plex organs, such as cartilage-bone interface [98] and arterial wall reconstruction [99] respectively. This technique has the potential to utilise materials with different mechanical / deg-radation properties and biomimetic characteristics to form a functional composite that would closely imitate complex native extracellular matrix assemblies. Electro-spun multi-layers with different materials/cargos have been used in a variety of complex pre-clinical studies, including osteogenic regeneration in cranial defects [100]; cell delivery for sus-tained insulin release [101]; and subcutaneous inflammation [102]. Despite the wide potential of multi-layered electro-spinning in biomedicine (Table 5) [99-101, 103-106], it is worth pointing out that potential delamination issues should be addressed to avoid implant failure in vivo.

The Multifaceted Potential of Electro-spinning in Regenerative Medicine Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 29

Table 4. Examples of surface treatment of electro-spun fibres as a means to deliver therapeutics and biologics in a range of clinical targets.

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

PCL and PEG with a LPEI /hEGF coating.

Random. -

Burns in diabetic animals coated meshes had a rapid initial closure rate but were equivalent to controls at day 14.

[95]

PCL and PEG with a LPEI /DNA coating.

Random. DNA release was significantly higher in response to MMP-2.

Dorsal wounds in diabetic and normal animals showed highest DNA incorpo-ration for coated samples.

[94] Wound healing

PCL immersed in Bit-eral®.

Random. 100% release achieved after 24 hours.

The PCL with Biteral showed adhe-sion levels similar to a surgical sham.

[91]

Cardio

Plasma treated PU films were grafted onto elec-trospun PLGA, which were again plasma treated with microwave induced argon.

Random.

Plasma treatment increased hydrophilicity and surface roughness correlating with and cell proliferation.

- [96]

Neural PCL and immersed in fibronectin.

Aligned.

Radial alignment enhanced cell migration over random align-ment. Coating enhanced cell elongation and cell migration speeds.

- [41]

PLGA with a BFP-1 coating.

Random.

ALP activity and calcium deposition were significantly unregulated after 14 days with the mesh.

Cranial wounds after 2 months were 57.29±15.24% healed compared to 23.02±6.54% with fibres alone.

[88]

PCL with simvastatin dropped onto it.

Random. -

Simvastatin coating was not as effec-tive as internalisation for long term release. The cranial defect closure was enhanced by simvastatin compared to PCL control.

[89] Bone

PLGA with collagen and HAP.

Random. - After 8 weeks in a cranial model, Col and HAP combined showed significant increase in regrowth.

[90]

Ocular surface PCL plasma treated with He/O2.

Random. Plasma treatment significantly enhanced hydrophilicity.

- [97]

Table 5. Examples of multi-layered electro-spinning technologies as a means to deliver therapeutics and biologics in a range of

clinical targets.

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

PCL/Elastin/Col in dif-ferent ratios.

Random.

The triple layer scaffold com-bines to increase the overall mechanical properties where a single layer would fail.

- [99]

Cardio Internal silicon tube or nylon mesh and nylon outer layers of 15 and 40%w/v with cells in-jected 5 weeks post op.

Random. - The implanted cells remained viable and within the scaffold for 6 weeks.

[101]

30 Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 Fuller et al.

(Table 5) Contd….

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

Internal layer of chitosan hydrogel/ PELCL loaded with VEGF and an outer layer of emulsion/ PELCL electro-spun membrane-loaded with PDGF.

Random.

Proliferation rates of VECs increased and VSMC initially slowed followed by rapid in-crease after 6 days.

VECs and VSMCs developed on the lumen and exterior respectively with no appearance of thrombus or burst.

[103]

PLA and PCL. Aligned and Ran-dom.

Cells did not fully penetrate the scaffold’s surface. Low collagen and GAG content were found compared to por-cine pulmonary valve.

- [104]

Bone PCL/PEG/ PCL with HAP

Random. There was a slow initial degrada-tion profile but it accelerated rapidly after 4 weeks.

The treated group significantly in-creased bone formation up to 20 weeks in cranial bone with minimal inflam-mation.

[100]

Anti-cancer

The four layers are PLCL with ChroB, PLCL, PLCL with TPPS and PLCL.

Random.

PLCL created a barrier causing distinct pause, thickness de-pendant, in the release of TPPS.

- [105]

Not specified

Ibuprofen intercalated layered double hydroxide with either PCL or PLA and with or without Pluronic.

Random.

Increased controllability of ibuprofen release profile achieved through polymer ratios alterations.

- [106]

Table 6. Examples of electro-spinning-co-electro-spraying technologies as a means to deliver therapeutics and biologics in a range

of clinical targets.

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

Wound healing Electro-spun PEUU with electro-sprayed porcine derived ECM.

Random. Tuneable tensile properties based on component ratios.

In a full thickness abdominal wall defect, no infection or herniation oc-curred after 4 weeks.

[111]

Cardio Electro-spun PCL with electro-sprayed PLGA microspheres with bFGF.

Random. Release of bFGF created a gradient that increased in-depth cell migration.

In a subcutaneous mice model, it in-creased cell migration onto the scaf-fold and increased the density of newly formed blood vessels.

[112]

Cell delivery Electro-spun PEUU with electro-sprayed vascular smooth muscle cells.

Random. Significantly higher cell popu-lations integrated into the scaffold at 7 days.

- [113]

Increased cell infiltration

PCL/Col with PEO or Gelatin fibres or Hep nanoparticles.

Random. PCL/Col with Hep coating had significantly deeper cell infil-tration after 10 days.

- [108]

Electro-spun PLGA/ PEGDA or PUR /PEGDA and electro-sprayed C3H10T1/2.

Random.

Composite scaffold was cytocompatible. Cell distribution was uneven throughout the scaffold.

- [114]

The Multifaceted Potential of Electro-spinning in Regenerative Medicine Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 31

(Table 6) Contd….

Clinical target Polymer and molecule Scaffold orientation In vitro results In vivo results Ref.

PHB/gelatin electro-sprayed or blended with HAP.

Random.

Electro-sprayed samples were significantly higher in ALP activity and mineralisation rates.

- [115]

PCL electro-sprayed with PLGA micro-particles.

Random.

Decrease in initial burst release in electro-sprayed particles. Cells spread and remained viable.

- [116] Bone

PLCL/Gelatin electro-sprayed or blended with HAP.

Random. Electro-spraying enhanced cell proliferation, mineralisation and ALP activity.

- [117]

3.6. Electro-spinning-co-electro-spraying

Carrier systems can be added to the electro-spun solution prior to electro-spinning [107] or during the fabrication process, resulting in an integrated material [108]. Electro-spraying has been studied extensively in biomedicine as a de-livery platform in vitro [109, 110], but only few studies have been conducted in vivo (Table 6) [108, 111-117]. We believe that multilayer electro-spinning bypasses the need for crea-tion of complex structures (particles and fibres), with poten-tially difficult or even impossible regulatory clearance.

4. FUTURE PERSPECTIVES

Electro-spinning has revolutionised the field of regenera-tive medicine due to its innate ability to create implantable devices that closely imitate native extracellular matrix archi-tectures and have controlled and sustained delivery capacity of therapeutics. We believe that problems associated with electro-spinning of biopolymers [118, 119] soon would be addressed and will open new avenues for clinical translation and commercialisation of such technologies.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flicts of interest.

ACKNOWLEDGMENTS

We thank Mr. Maciek Doczyk (http://doczykdesign.com) for his support in the preparation of the Figures for this manuscript. This work is supported by the: Irish Research Council, under the Enterprise Partnership Scheme, in collaboration with Vornia Biomaterials Ltd to D.Z. and K.F.; European Union Seventh Framework Programme (FP7/2007-2013) under agreement number 263289 (Green Nano Mesh) to D.Z.; European Union Seventh Framework Programme (FP7/2007-2013), under grant agreement num-ber 251385 (Tendon Regeneration) to D.Z.; Science Founda-tion Ireland, Research Frontiers Programme, under the grant agreement number 09/RFP/ENM2483 to D.Z.; Health Re-search Board, Health Research Awards Programme, under the grant agreement number HRA_POR/2011/84 to D.Z.

LIST OF ABBREVIATIONS

ALP = Alkaline phosphatase BDNF = Brain-derived neurotrophic factor bFGF = Basic fibroblast growth factor BFP-1 = Bone-forming peptide-1 BMP-2 = Bone morphogenetic protein 2 BSA = Bovine serum albumin CA = Cellulose acetate Ca-P = Calcium phosphate CFU = Colony forming unit CFX-Na = Cefotamime sodium Col = Collagen DMAC = Dimethylacetamide DMOG = Dimethyloxallyl glycine ECM = Extra cellular matrix GAG = Glycosaminoglycan HA-DTPH = 3,3 -dithiobis (propanoic dihydrazide)-

modified HA HAP = Hydroxyapatite Hep = Heprasal™ LPEI = Linear polyethyleneimine mRNA = Messenger ribonucleic acid NGF = Nerve growth factor PCL = Polycaprolactone PDGF = Platelet derived growth factor-bb PDLLA = Poly-DL-lactic acid PDO = Polydioxanone PEG = Polyethylene Glycol PEGDA = Poly(ethylene glycol) diacrylate PEI-HA = Poly(ethyelenimine)-hyaluronic acid

32 Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 Fuller et al.

PELA = Poly(ethylene glycol)-poly(DL-lactide) PELCL = Poly(ethylene glycol)-b-poly(l-lactide-co-

caprolactone) PEO = Polyethylene Oxide PEU = Polyester urethane PEUU = Polyester urethane urea PHB = Poly(3-hydroxybutyrate) PHMB = Polyhexamethylene biguanide PLCL = Poly (lactic acid-caprolactone) PLLA = Poly-L-lactic acid PMeDO = PolyDL-3-methyl-1,4-dioxan-2-one POC = Poly(1,8-octanediol-co-citrate) pRNA = Plasma ribonucleic acid PU = Poly urethane PUR = Poly(ester urethane urea) PVA = Polyvinyl alcohol rhBMP-2 = Recombinant human bone morphogenetic

protein 2 TCH = Tetracycline hydrochloride Tet = Tetracycline VEC = Vascular endothelial cells VSMC = Vascular smooth muscle cells

REFERENCES [1] McWilliams, A. Nanotechnology: A realistic market assessment

BCC research, 2012. NAN031E 2012. [2] Vasita R, Katti DS. Nanofibers and their applications in tissue

engineering. Int J Nanomed 2006; 1(1): 15-30. [3] Persano L, Camposeo A, Tekmen C, Pisignano D. Industrial

upscaling of electrospinning and applications of polymer nanofibers: A rview. Macromol Mater Eng 2013; 298(5): 504-20.

[4] Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Compos Sci Technol 2003; 63(15): 2223-53.

[5] Pham QP, Sharma U, Mikos AG. Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Eng 2006; 12(5): 1197-211.

[6] Dai Y, Liu W, Formo E, Sun Y, Xia Y. Ceramic nanofibers fabricated by electrospinning and their applications in catalysis, environmental science, and energy technology. Polym Adv Technol 2011; 22(3): 326-38.

[7] Chronakis IS. Novel nanocomposites and nanoceramics based on polymer nanofibers using electrospinning process-a review. J Mater Process Technol 2005; 167(2-3): 283-93.

[8] Wu H, Hu L, Rowell MW, et al. Electrospun metal nanofiber webs as high-performance transparent electrode. Nano Lett 2010; 10(10): 4242-8.

[9] Gardella L, Basso A, Prato M, Monticelli O. PLA/POSS nanofibers: A novel system for the immobilization of metal nanoparticles. ACS Appl Mater Interfaces 2013; 5(16): 7688-92.

[10] Deitzel JM, Kleinmeyer J, Harris D, Beck Tan NC. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 2001; 42(1): 261-72.

[11] Gopal R, Kaur S, Ma Z, Chan C, Ramakrishna S, Matsuura T. Electrospun nanofibrous filtration membrane. J Membr Sci 2006; 28 (1-2): 581-6.

[12] Agarwal S, Wendorff JH, Greiner A. Use of electrospinning technique for biomedical applications. Polymer 2008; 49(26): 5603-21.

[13] Ridley AJ, Schwartz MA, Burridge K, et al. Cell migration: Integrating signals from front to back. Science 2003; 302(5651): 1704-9.

[14] Saino E, Focarete ML, Gualandi C, et al. Effect of electrospun fiber diameter and alignment on macrophage activation and secretion of proinflammatory cytokines and chemokines. Biomacromolecules 2011; 12(5): 1900-11.

[15] Xia Y. Nanomaterials at work in biomedical research. Nat Mater 2008; 7(10): 758-60.

[16] Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: A novel scaffold for tissue engineering. J Biomed Mater Res 2002; 60(4): 613-21.

[17] Frenot A, Chronakis IS. Polymer nanofibers assembled by electrospinning. Curr Opin Colloid Interface Sci 2003; 8(1): 64-75.

[18] Liu Y, Dong L, Fan J, Wang R, Yu JY. Effect of applied voltage on diameter and morphology of ultrafine fibers in bubble electrospinning. J Appl Polym Sci 2011; 120(1): 592-8.

[19] Niu H, Lin T. Fiber generators in needleless electrospinning. J Nanomater 2012; (725950): 1-13.

[20] Luo CJ, Nangrejo M, Edirisinghe M. A novel method of selecting solvents for polymer electrospinning. Polymer 2010; 51(7): 1654-62.

[21] Vrieze S, Camp T, Nelvig A, Hagström B, Westbroek P, Clerck K. The effect of temperature and humidity on electrospinning. J Mater Sci 2009; 44(5): 1357-62.

[22] Pakravan M, Heuzey MC, Ajji A. A fundamental study of chitosan/PEO electrospinning. Polymer 2011; 52(21): 4813-24.

[23] Van der Schueren L, De Schoenmaker B, Kalaoglu ÖI, De Clerck K. An alternative solvent system for the steady state electrospinning of polycaprolactone. Eur Polymer J 2011; 47(6): 1256-63.

[24] Munir MM, Suryamas AB, Iskandar F, Okuyama K. Scaling law on particle-to-fiber formation during electrospinning. Polymer 2009; 50(20): 4935-43.

[25] Katti DS, Robinson KW, Ko FK, Laurencin CT. Bioresorbable nanofiber-based systems for wound healing and drug delivery: Optimization of fabrication parameters. J Biomed Mater Res Part B App Biomater 2004; 70B(2): 286-96.

[26] Fridrikh SV, Yu JH, Brenner MP, Rutledge GC. Controlling the fiber diameter during electrospinning. Phys Rev Lett 2003; 90(14): 1445021-4.

[27] Tong HW, Wang M. Electrospinning of fibrous polymer scaffolds using positive voltage or negative voltage: A comparative study. Biomed Mater 2010; 5(5): 1-15.

[28] Lee KH, Kim HY, La YM, Lee DR, Sung NH. Influence of a mixing solvent with tetrahydrofuran and N, N-­‐Dimethylformamide on electrospun poly (vinyl chloride) nonwoven mats. J Polymer Sci Part B Polymer Phys 2002; 40(19): 2259-68.

[29] Rieger KA, Birch NP, Schiffman JD. Designing electrospun nanofiber mats to promote wound healing-a review. J Mater Chem B 2013; 1(36): 4531-41.

[30] Yin Z, Chen X, Chen JL, et al. The regulation of tendon stem cell differentiation by the alignment of nanofibers. Biomaterials 2010; 31(8): 2163-75.

[31] Bosworth L, Alam N, Wong J, Downes S. Investigation of 2D and 3D electrospun scaffolds intended for tendon repair. J Mater Sci: Mater Med 2013; 24(6): 1605-14.

[32] Ortega Í, Ryan AJ, Deshpande P, MacNeil S, Claeyssens F. Combined microfabrication and electrospinning to produce 3D architectures for corneal repair. Acta Biomater 2012; 9(3): 5511- 20.

[33] English A, Azeem A, Gaspar DA, et al. Preferential cell response to anisotropic electro-spun fibrous scaffolds under tension-free conditions. J Mater Sci: Mater Med 2012; 23(1): 137-48.

[34] Yang F, Murugan R, Wang S, Ramakrishna S. Electrospinning of nano/micro scale poly (l-lactic acid) aligned fibers and their potential in neural tissue engineering. Biomaterials 2005; 26(15): 2603-10.

[35] Lee YS, Livingston AT. Electrospun nanofibrous materials for neural tissue engineering. Polymers 2011; 3(1): 413-26.

[36] Teo WE, Ramakrishna S. A review on electrospinning design and nanofibre assemblies. Nanotechnology 2006; 17(14): R89- R106.

[37] De Valence S, Tille JC, Giliberto JP, et al. Advantages of bilayered vascular grafts for surgical applicability and tissue regeneration. Acta Biomater 2012; 8(11): 3914-20.

[38] Marelli B, Achilli M, Alessandrino A, et al. Collagen-reinforced electrospun silk fibroin tubular construct as small calibre vascular graft. Macromol Biosci 2012; 12(11): 1566-74.

The Multifaceted Potential of Electro-spinning in Regenerative Medicine Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 33

[39] Lee BK, Ju YM, Cho JG, et al. End-to-side neurorrhaphy using an electrospun PCL/collagen nerve conduit for complex peripheral motor nerve regeneration. Biomaterials 2012; 33(35): 9027-36.

[40] Yao L, O'Brien N, Windebank A, Pandit A. Orienting neurite growth in electrospun fibrous neural conduits. J Biomed Mater Res Part B: Appl Biomater 2009; 90(2): 483-91.

[41] Xie J, MacEwan MR, Ray WZ, Liu W, Siewe DY, Xia Y. Radially aligned, electrospun nanofibers as dural substitutes for wound closure and tissue regeneration applications. ACS Nano 2010; 4 (9): 5027-36.

[42] Vaquette C, Cooper-White JJ. Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration. Acta Biomater 2011; 7(6): 2544-57.

[43] Wang Y, Wang G, Chen L, et al. Electrospun nanofiber meshes with tailored architectures and patterns as potential tissue-engineering scaffolds. Biofabrication 2009; 1(1): 1-10.

[44] Zhang D, Chang J. Patterning of Electrospun fibers using electroconductive templates. Adv Mater 2007; 19(21): 3664-7.

[45] Obata A, Hotta T, Wakita T, Ota Y, Kasuga T. Electrospun microfiber meshes of silicon-doped vaterite/poly (lactic acid) hybrid for guided bone regeneration. Acta Biomater 2010; 6(4): 1248-57.

[46] Liang D, Hsiao BS, Chu B. Functional Electrospun nanofibrous scaffolds for biomedical applications. Adv Drug Deliv Rev 2007; 59(14): 1392-412.

[47] Baker BM, Shah RP, Silverstein AM, Esterhai JL, Burdick JA, Mauck RL. Sacrificial nanofibrous composites provide instruction without impediment and enable functional tissue formation. Proc Natl Acad Sci 2012 109(35): 14176-81.

[48] Sun D, Chang C, Li S, Lin L. Near-field electrospinning. Nano Lett 2006; 6(4): 839-42.

[49] Sill TJ, Von Recum HA. Electrospinning: Applications in drug delivery and tissue engineering. Biomaterials 2008; 29(13): 1989-2006.

[50] Zeng J, Xu X, Chen X, et al. Biodegradable electrospun fibers for drug delivery. J Control Release 2003; 92(3): 227-31.

[51] Luong-Van E, Grøndahl L, Chua KN, Leong KW, Nurcombe V, Cool SM. Controlled release of heparin from poly(ε-caprolactone) electrospun fibers. Biomaterials 2006; 27(9): 2042-50.

[52] Ji W, Sun Y, Yang F, et al. Bioactive electrospun scaffolds delivering growth factors and genes for tissue engineering applications. Pharm Res 2011; 28(6): 1259-72.

[53] Heunis TDJ, Smith C, Dicks LMT. Evaluation of a Nisin-eluting nanofiber scaffold to treat Staphylococcus Aureus-induced skin infections in mice. Antimicrob Agents Chemother 2013; 57(8): 3928-35.

[54] Tseng YY, Kao YC, Liao JY, Chen WA, Liu SJ. Biodegradable drug-eluting poly[lactic-co-glycol acid] nanofibers for the sustainable delivery of vancomycin to brain tissue: In vitro and in vivo Studies. ACS Chem Neurosci 2013; 4(9): 1314-21.

[55] Gilchrist SE, Lange D, Letchford K, Bach H, Fazli L, Burt HM. Fusidic acid and rifampicin co-loaded PLGA nanofibers for the prevention of orthopedic implant associated infections. J Control Release 2013; 170(1): 64-73.

[56] Vargas EAT, Do Vale Baracho NC, De Brito J, De Queiroz AAA. Hyperbranched polyglycerol electrospun nanofibers for wound dressing applications. Acta Biomater 2010; 6(3): 1069-78.

[57] Ignatova M, Starbova K, Markova N, Manolova N, Rashkov I. Electrospun nano-fibre mats with antibacterial properties from quaternised chitosan and poly(vinyl alcohol). Carbohydr Res 2006; 341(12): 2098-107.

[58] Prabhakaran MP, Nair AS, Kai D, Ramakrishna S. Electrospun composite scaffolds containing poly(octanediol-co-citrate) for cardiac tissue engineering. Biopolymers 2012; 97(7): 529-38.

[59] Sell SA, McClure MJ, Barnes CP, et al. Electrospun Polydioxanone-elastin blends: Potential for bioresorbable vascular grafts. Biomed Mater 2006; 1(2): 72-80.

[60] Fon, D, Zhou K, Ercole F, et al. Nanofibrous scaffolds releasing a small molecule bdnf-mimetic for the re-direction of endogenous neuroblast migration in the brain. Biomaterials 2014; 35(9): 2692-712.

[61] Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, Nasr-Esfahani MH, Ramakrishna S. Electrospun poly(ɛ-Caprolactone)/ gelatin nanofibrous scaffolds for nerve tissue engineering. Biomaterials 2008; 29(34): 4532-9.

[62] Rajzer I, Menaszek E, Kwiatkowski R, Chrzanowski W. Bioactive nanocomposite PLDL/nano-hydroxyapatite electrospun membranes for bone tissue engineering. J Mater Sci: Mater Med 2014; 25(5): 1239-47.

[63] Goonoo N, Bhaw-Luximon A, Rodriguez IA, et al. Poly (ester-ether)s: II. Properties of electrospun nanofibres from polydioxanone and poly (methyl dioxanone) blends and human fibroblast cellular proliferation. Biomater Sci 2014; 2(3): 339-51.

[64] Nguyen TH, Lee BT. The Effect of cross-linking on the microstructure, mechanical properties and biocompatibility of electrospun polycaprolactone-gelatin/PLGA-gelatin/PLGA-chitosan hybrid composite. Sci Technol Adv Mater 2012; 13(3): 2381-97.

[65] Liu X, Lin T, Gao Y, et al. Antimicrobial electrospun nanofibers of cellulose acetate and polyester urethane composite for wound dressing. J Biomed Mater Res Part B: Appl Biomater 2012; 100(6): 1556-65.

[66] Hong Y, Fujimoto K, Hashizume R, et al. Generating elastic, biodegradable polyurethane/poly (lactide-co-glycolide) fibrous sheets with controlled antibiotic release via two-stream electrospinning. Biomacromolecules 2008; 9(4): 1200-7.

[67] Wang C, Wang M. Dual-source dual-power electrospinning and characteristics of multifunctional scaffolds for bone tissue engineering. J Materi Sci: Mater Med 2012; 23(10): 2381-97.

[68] Ji Y, Ghosh K, Li B, Sokolov JC, Clark RAF, Rafailovich MH. Dual-syringe reactive electrospinning of cross-linked hyaluronic acid hydrogel nanofibers for tissue engineering applications. Macromol Biosci 2006; 6(10): 811-7.

[69] Thakur RA, Florek CA, Kohn J, Michniak BB. Electrospun Nanofibrous polymeric scaffold with targeted drug release profiles for potential application as wound dressing. Int J Pharmaceutics 2008; 364(1): 87-93.

[70] Baker BM, Gee AO, Metter RB, et al. The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers. Biomaterials 2008; 29(15): 2348-58.

[71] Whited BM, Whitney JR, Hofmann MC, Xu Y, Rylander MN. Pre-osteoblast infiltration and differentiation in highly porous apatite-coated PLLA electrospun scaffolds. Biomaterials 2011; 32(9): 2294-304.

[72] Ladd MR, Lee SJ, Stitzel JD, Atala A, Yoo JJ. Co-electrospun dual scaffolding system with potential for muscle-tendon junction tissue engineering. Biomaterials 2011; 32(6): 1549-59.

[73] Samavedi S, Guelcher SA, Goldstein AS, Whittington AR. Response of bone marrow stromal cells to graded co-electrospun scaffolds and its implications for engineering the ligament-bone interface. Biomaterials 2012; 33(31): 7727-35.

[74] Ji W, Yang F, Van den Beucken JJJP, et al. Fibrous scaffolds loaded with protein prepared by blend or coaxial electrospinning. Acta Biomater 2010; 6(11): 4199-207.

[75] Park JH, Braun PV. Coaxial Electrospinning of self-­‐healing coatings. Adv Mater 2010; 22(4): 496-9.

[76] Zhao Y, Cao X, Jiang L. Bio-mimic multichannel microtubes by a facile method. J Am Chem Soc 2007; 129(4): 764-5.

[77] Han D, Steckl AJ. Triaxial electrospun nanofiber membranes for controlled dual release of functional molecules. ACS App Mater Interfaces 2013; 5(16): 8241-5.

[78] Hu W, Huang ZM, Liu XY. Development of braided drug-loaded nanofiber sutures. Nanotechnology 2010; 21(31): 315104.

[79] Hu C, Liu S, Zhang Y, et al. Long-term drug release from electrospun fibers for in vivo inflammation prevention in the prevention of peritendinous adhesions. Acta Biomater 2013; 9(7): 7381-8.

[80] Yang Y, Xia T, Zhi W, et al. Promotion of Skin regeneration in diabetic rats by electrospun core-sheath fibers loaded with basic fibroblast growth factor. Biomaterials 2011; 32(18): 4243-54.

[81] Nguyen TTT, Ghosh C, Hwang SG, Chanunpanich N, Park JS. Porous core/sheath composite nanofibers fabricated by coaxial electrospinning as a potential mat for drug release system. Int J Pharmaceutics 2012; 439(1): 296-306.

[82] Liu JJ, Wang CY, Wang JG, Ruan HJ, Fan CY. Peripheral nerve regeneration using composite poly (lactic acid-­‐caprolactone)/nerve growth factor conduits prepared by coaxial electrospinning. J Biomed Mater Res Part A 2011; 96(1): 13-20.

34 Pharmaceutical Nanotechnology, 2014, Vol. 2, No. 1 Fuller et al.

[83] He CL, Huang ZM, Han XJ. Fabrication of drug-loaded electrospun aligned fibrous threads for suture applications. J Biomedi Mater Res Part A 2009; 89A(1): 80-95.

[84] Zhao P, Jiang H, Pan H, Zhu K, Chen W. Biodegradable fibrous scaffolds composed of gelatin coated poly(ϵ-caprolactone) prepared by coaxial electrospinning. J Biome Mater Res Part A 2007; 83A(2): 372-82.

[85] Liao I, Chew S, Leong K. Aligned core-shell nanofibers delivering bioactive proteins. Nanomedicine 2006; 1(4): 465-71.

[86] Saraf A, Baggett LS, Raphael RM, Kasper FK, Mikos AG. Regulated non-viral gene delivery from coaxial electrospun fiber mesh scaffolds. J Control Release 2010; 143(1): 95-103.

[87] Wang C, Yan KW, Lin YD, Hsieh PC. Biodegradable core/shell fibers by coaxial electrospinning: Processing, Fiber characterization, and its application in sustained drug release. Macromolecules 2010; 43(15): 6389-97.

[88] Lee YJ, Lee JH, Cho HJ, Kim HK, Yoon TR, Shin H. Electrospun fibers immobilized with bone forming peptide-1 derived from BMP7 for guided bone regeneration. Biomaterials 2013; 34(21): 5059-69.

[89] Pişkin E, İşoğlu İA, Bölgen N, et al. In vivo performance of simvastatin-­‐loaded electrospun spiral-­‐wound polycaprolactone scaffolds in reconstruction of cranial bone defects in the rat model. J Biomed Mater Res Part A 2009; 90(4): 1137-51.

[90] Tavakoli-Darestani R, Kazemian G, Emami M, Kamrani-Rad A. Poly (lactide-co-glycolide) nanofibers coated with collagen and nano-hydroxyapatite for bone tissue engineering. Novelty Biomed 2013; 1(1): 8-15.

[91] Bölgen N, Vargel İ, Korkusuz P, Menceloğlu YZ, Pişkin E. In vivo performance of antibiotic embedded electrospun pcl membranes for prevention of abdominal adhesions. J Biomed Mater Res Part B: Appl Biomater 2007; 81(2): 530-43.

[92] Yan D, Jones J, Yuan X, et al. Plasma treatment of random and aligned electrospun pcl nanofibers. J Med Biol Eng 2013; 33(3): 171-8.

[93] Zeugolis DI, Panengad PP, Yew ESY, Sheppard C, Phan TT, Raghunath M. An in situ and in vitro investigation for the transglutaminase potential in tissue engineering. J Biomed Mater Res Part A 2010; 92A(4): 1310-20.

[94] Kim HS, Yoo HS. MMPs-responsive release of dna from electrospun nanofibrous matrix for local gene therapy: In vitro and in vivo evaluation. J Control Release 2010; 145(3): 264-71.

[95] Kim HS, Yoo HS. In vitro and in vivo Epidermal growth factor gene therapy for diabetic ulcers with electrospun fibrous meshes. Acta Biomater 2013; 9(7): 7371-80.

[96] Park BJ, Seo HJ, Kim J, et al. Cellular responses of vascular endothelial cells on surface modified polyurethane films grafted electrospun plga fiber with microwave-induced plasma at atmospheric pressure. Surf Coat Technol 2010; 205(Supplement 1): S222-6.

[97] Sharma S, Mohanty S, Jassal M, Agrawal AK, Tandon R. Surface modified electrospun poly (ε-caprolactone) scaffold with improved optical transparency and bioactivity for damaged ocular surface reconstruction. Invest Ophthalmol Visual Sci 2014; 55(2): 899-907.

[98] Mouthuy PA, El-­‐Sherbini Y, Cui Z, Ye H. Layering PLGA-based electrospun membranes and cell sheets for engineering cartilage-bone transition. J Tissue Eng Regener Med 2013; Epub Ahead of Print. (DOI: 10.1002/term.1765).

[99] McClure MJ, Sell SA, Simpson DG, Walpoth BH, Bowlin GL. A three-layered electrospun matrix to mimic native arterial architecture using polycaprolactone, elastin, and collagen: A preliminary study. Acta Biomate 2010; 6(7): 2422-33.

[100] Fu S, Ni P, Wang B, et al. In vivo Biocompatibility and osteogenesis of electrospun poly (e-caprolactone))-poly (ethylene glycol)-)-poly (e-caprolactone)/nano-hydroxyapatite composite scaffold. Biomaterials 2012; 33(33): 8363-71.

[101] Krishnan L, Touroo J, Reed R, Boland E, Hoying JB, Williams SK. Vascularization and cellular isolation potential of a novel electrospun cell delivery vehicle. J Biomed Mater Res Part A 2013; Epub ahead of print. (DOI: 10.1002/jbm.a.34900).

[102] Gui-­‐Bo Y, You-­‐Zhu Z, Shu-­‐Dong W, De-­‐Bing S, Zhi-­‐Hui D, Wei-­‐Guo F. Study of the electrospun PLA/silk fibroin-­‐gelatin composite nanofibrous scaffold for tissue engineering. J Biomed Mater Res Part A 2010; 93(1): 158-63.

[103] Zhang H, Jia X, Han F, et al. Dual-delivery of VEGF and PDGF by double-layered electrospun membranes for blood vessel regeneration. Biomaterials 2013; 34(9): 2202-12.

[104] Yin H, Wang XH, Zhu XD, Han H, Guo WY, Fu ZR. A tissue engineered renovascular graft composed of proteins, polymers, smooth muscle and endothelial cells for renal artery stenosis. J Biomed nanotechnol 2013; 9(8): 1345-53.

[105] Okuda T, Tominaga K, Kidoaki S. Time-programmed dual release formulation by multilayered drug-loaded nanofiber meshes. Journa Control Release 2010; 143(2): 258-64.

[106] Miao YE, Zhu H, Chen D, Wang R, Tjiu WW, Liu T. Electrospun fibers of layered double hydroxide/biopolymer nanocomposites as effective drug delivery systems. Mater Chem Phys 2012; 134(2-3): 623-30.

[107] Reneker DH, Yarin AL. Electrospinning jets and polymer nanofibers. Polymer 2008; 49(10): 2387-425.

[108] Ekaputra AK, Prestwich GD, Cool SM, Hutmacher DW. Combining electrospun scaffolds with electrosprayed hydrogels leads to three-dimensional cellularization of hybrid constructs. Biomacromolecules 2008; 9(8): 2097-103.

[109] Sridhar R, Ramakrishna S. Electrosprayed nanoparticles for drug delivery and pharmaceutical applications. Biomaterials 2013; 3(2): 1-12.

[110] Chakraborty S, Liao IC, Adler A, Leong KW. Electrohydrodynamics: A facile technique to fabricate drug delivery systems. Adv Drug Deliv Rev 2009; 61(12): 1043-54.

[111] Hong Y, Huber A, Takanari K, et al. Mechanical properties and in vivo behavior of a biodegradable synthetic polymer microfiber-extracellular matrix hydrogel biohybrid scaffold. Biomaterials 2011; 32(13): 3387-94.

[112] Guo X, Elliott CG, Li Z, Xu Y, Hamilton DW, GuanJ. Creating 3D angiogenic growth factor gradients in fibrous constructs to guide fast angiogenesis. Biomacromolecules 2012; 13(10): 3262-71.

[113] Stankus JJ, Guan J, Fujimoto K, Wagner WR. Microintegrating smooth muscle cells into a biodegradable, elastomeric fiber matrix. Biomaterials 2006; 27(5): 735-44.

[114] Thayer PS, Dimling AF, Plessl DS, et al. Cellularized cylindrical fiber/hydrogel composites for ligament tissue engineering. Biomacromolecules 2013; 15(1): 75-83.

[115] Ramier J, Grande D, Bouderlique T, et al. From design of bio-based biocomposite electrospun scaffolds to osteogenic differentiation of human mesenchymal stromal cells. J Materi Sci: Mater Med 2014. Epub ahead of print. (DOI: 10.1007/s10856-014-5149-9): 1-13.

[116] Bock N, Woodruff MA, Steck R, Hutmacher DW, Farrugia BL, Dargaville TR. Composites for delivery of therapeutics: Combining melt electrospun scaffolds with loaded electrosprayed microparticles. Macromol Biosci 2013; 14(2): 202-14.

[117] Gupta D, Venugopal J, Mitra S, Giri Dev V, Ramakrishna S. Nanostructured biocomposite substrates by electrospinning and electrospraying for the mineralization of osteoblasts. Biomaterials 2009; 30(11): 2085-94

[118] Yang L, Fitié CFC, Van Der Werf KO, Bennink ML, Dijkstra PJ, Feijen J. Mechanical properties of single electrospun collagen type I fibers. Biomaterials 2008; 29(8): 955-62.

[119] Zeugolis DI, Khew ST, Yew ESY, et al. Electro-spinning of pure collagen nano-fibres - just an expensive way to make gelatin? Biomaterials 2008; 29(15): 2293-305.

Received: February 11, 2014 Revised: March 17, 2014 Accepted: March 18, 2014