Advances in electrospun skin substitutes

Abstract

In recent years, nanotechnology has received much attention in regenerative medicine, partly owing to the production of nanoscale structures that mimic the collagen fibrils of the native extracellular matrix. Electrospinning is a widely used technique to produce micro-nanofibers due its versatility, low cost and easy use that has been assuming an increasingly prominent position in the tissue engineering field. Electrospun systems have been especially investigated for wound dressings in skin regeneration given the intrinsic suitability of fibrous structures for that purpose. Several efforts have been made to combine distinct design strategies, synthetic and/or natural materials, fiber orientations and incorporation of substances (e.g. drugs, peptides, growth factors or other biomolecules) to develop an optimized electrospun wound dressing mimicking the native skin. This paper presents a comprehensive review on current and advanced electrospinning strategies for skin regeneration. Recent advances have been mainly focused on the materials used rather than on sophisticated fabrication strategies to generate biomimetic and complex constructs that resemble the mechanical and structural properties of the skin. The technological limitations of conventional strategies, such as random, aligned and core-shell technologies, and their poor mimicking of the native tissue are discussed. Advanced strategies, such as hybrid structures, cell and in situ electrospinning, are highlighted in the way they may contribute to circumvent the limitations of conventional strategies, through the combination of different technologies and approaches. The main research challenges and future trends of electrospinning for skin regeneration are discussed in the light of in vitro but mainly in vivo evidence.
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Advances in electrospun skin substitutes
J.R. Dias
a,b,c,d,
⇑
, P.L. Granja
b,c,d,e,1
, P.J. Bártolo
f,1
a
Centre for Rapid and Sustainable Product Development (CDRsp), Polytechnic Institute of Leiria, Leiria, Portugal
b
I3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal
c
INEB - Instituto de Engenharia Biomédica, Universidade do Porto, Porto, Portugal
d
ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
e
Faculdade de Engenharia da Universidade do Porto (FEUP), Departamento de Engenharia Metalúrgica e Materiais, Porto, Portugal
f
School of Mechanical, Aerospace and Civil Engineering & Manchester Institute of Biotechnology, University of Manchester, Manchester, UK
article info
Article history:
Received 15 April 2016
Received in revised form 9 September 2016
Accepted 18 September 2016
Available online 20 September 2016
Keywords:
Electrospinning
Skin
Wound healing
Electrospun nanofibers
Polymeric materials
abstract
In recent years, nanotechnology has received much attention in regenerative medicine,
partly owing to the production of nanoscale structures that mimic the collagen fibrils of
the native extracellular matrix. Electrospinning is a widely used technique to produce
micro-nanofibers due its versatility, low cost and easy use that has been assuming an
increasingly prominent position in the tissue engineering field. Electrospun systems have
been especially investigated for wound dressings in skin regeneration given the intrinsic
suitability of fibrous structures for that purpose. Several efforts have been made to com-
bine distinct design strategies, synthetic and/or natural materials, fiber orientations and
incorporation of substances (e.g. drugs, peptides, growth factors or other biomolecules)
to develop an optimized electrospun wound dressing mimicking the native skin. This paper
presents a comprehensive review on current and advanced electrospinning strategies for
skin regeneration. Recent advances have been mainly focused on the materials used rather
than on sophisticated fabrication strategies to generate biomimetic and complex con-
structs that resemble the mechanical and structural properties of the skin. The technolog-
ical limitations of conventional strategies, such as random, aligned and core-shell
technologies, and their poor mimicking of the native tissue are discussed. Advanced strate-
gies, such as hybrid structures, cell and in situ electrospinning, are highlighted in the way
they may contribute to circumvent the limitations of conventional strategies, through the
combination of different technologies and approaches. The main research challenges and
future trends of electrospinning for skin regeneration are discussed in the light of
in vitro but mainly in vivo evidence.
Ó2016 Published by Elsevier Ltd.
Contents
1. Introduction . . . . . . . . . . . . . ............................................................................... 315
2. Skin tissue and wound healing process . . . . . . . . . . ............................................................ 315
3. Skin regeneration products . ............................................................................... 317
http://dx.doi.org/10.1016/j.pmatsci.2016.09.006
0079-6425/Ó2016 Published by Elsevier Ltd.
⇑
Corresponding author at: Biomaterials for Multistage Drug & Cell Delivery Group, Instituto de Investigação e Inovação em Saúde, Universidade do Porto,
Rua Alfredo Allen, 208 4200-135 Porto, Portugal.
E-mail address: juliana.dias@ineb.up.pt (J.R. Dias).
1
These authors contributed equally to this work.
Progress in Materials Science 84 (2016) 314–334
Contents lists available at ScienceDirect
Progress in Materials Science
journal homepage: www.elsevier.com/locate/pmatsci

3.1. Autografts and allografts. ............................................................................ 317
3.2. Wound dressings. . . . . . . ............................................................................ 317
3.3. Tissue engineering-based products . . . . . . . . ............................................................ 317
3.4. Advanced skin substitutes . . . . . . . . . . . . . . . ............................................................ 318
4. Electrospun skin substitutes . . . . . . . . . ...................................................................... 318
4.1. Randomly oriented fiber meshes . . . . . . . . . . ............................................................ 320
4.2. Aligned fiber meshes. . . . ............................................................................ 321
4.3. Fibers with core/shell structure . . . . . . . . . . . ............................................................ 322
4.4. Hybrid structures . . . . . . ............................................................................ 324
4.5. Cell electrospinning. . . . . ............................................................................ 326
4.6. In situ electrospinning. . . ............................................................................ 327
5. Concluding remarks and future trends . ...................................................................... 328
Acknowledgments . . . . . . . . . . . . . . . . . ...................................................................... 329
References . . . . ......................................................................................... 329
1. Introduction
The first shield between the external environment and the human body is the skin. This tissue plays a crucial role in body
protection and, when damaged at full-size, the human life could be in risk [1,2]. According to the World Health Organization
(WHO) it is estimated that every year 265,000 deaths occurs caused by burns and, annually about 6 million people were
burned requiring medical attention [3–6]. The average length of stay in the hospital is 8.4 days, thus resulting in a consid-
erable social and economic burden for the health care systems worldwide. Therefore, innovative strategies are required to
promote skin tissue regeneration, despite the encouraging recent developments in wound dressings and tissue
engineering-based products [7]. Electrospun meshes have been gaining increasing attention through the combination of
materials and processing strategies of great potential for skin regeneration [8]. Wound dressings prepared from electrospun
nanofibers have been claimed to present exceptional properties compared to conventional dressings, such as similarity to
architecture of the natural extracellular matrix (ECM), improved promotion of hemostasis, absorption of wound exudates,
permeability, conformability to the wound, and avoidance of scar induction [9]. New processing strategies are thus being
explored in which natural and synthetic materials are combined with new design approaches allowing the incorporation
of substances that turn not electrospinnable materials into electrospinnable ones. In this review skin regeneration strategies
will be revised with a focus on electrospinning methodologies and materials.
2. Skin tissue and wound healing process
The human body comprises several organs each one with specific functions, dimensions and shapes. The largest vital
organ in the body is the skin that represents 7% of the total body weight and has the main function of protecting the human
being against the external environment. It also helps protecting the body against excessive water loss, against attacks from
chemicals and other harmful substances, and ultraviolet radiation [1,8,10–12]. In spite of the protective function of the skin,
this tissue plays other important functions namely: (i) control of body temperature, by secreting sweat through the sweat
glands, thereby lowering the temperature; (ii) sensory, through different receptors able to detect touch, pain, pressure and
temperature; and (iii) synthesis of vitamin D (after exposure to sunlight), a precursor of calcitriol hormone that is converted
in the liver and kidneys and plays an important role in the calcium absorption in the small intestine [10,13]. Although the
skin works as a barrier it is not totally impermeable: some substances are transferred across the skin, such as sweat, drugs
and biomolecules [10,14].
Skin functions are carried out by specialized cells and structures found in the two main skin layers, epidermis and dermis
(Fig. 1). Besides these two layers, beneath the dermis there is the hypodermis that provides support to the dermis [10,15].
The epidermis, the outermost skin layer, is around 120
l
m thick and is composed by numerous cells closely linked in dif-
ferent stages of differentiation, which form the stratified squamous epithelium [10]. The epidermis is avascular (nourished
through diffusion from the dermis), consists of 4 different types of cells (keratinocytes, melanocytes, Langerhans cells and
merkels cells) and presents 5 distinct cell layers (stratum basale, spinosum, granolosum, lucidum and corneum) [16,17].
The dermis layer is composed by a complex mesh of ECM material that provides structure and resilience to the skin. The
thickness of this layer varies according to the body region but is in average of 2 mm [10,17]. The dermis is composed by
a nanometer-sized network of structural proteins (collagen, which provides strength and flexibility, and elastin, which pro-
vides elasticity), blood and lymph vessels, and specialized cells (mast cells that help in the healing process and protect
against pathogenic organisms, and fibroblasts that produce collagen and elastin). This ECM network is engaged in a ground
substance that is mostly composed by glycosaminoglycans and plays an important role in hydration and in maintaining
moisture levels in the skin [10,14]. The ECM is also highly dynamic, being constantly synthesized and re-organized by the
cellular components, but in turn also having a prominent role in directing cellular behaviour through direct and indirect
signaling. For instance, ECM molecules control cell adhesion through specific cell binding sites, cell migration through
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 315

proteolytically sensitive functionalities, and cell differentiation through bound and soluble signaling biomolecules. With rel-
evance for the present topic is the nanometer scale of the several pores and fibers (collagen, hyaluronic acid, elastin, laminin,
fibronectin, proteoglycans) that constitute the ECM, highlighting the relevance of mimicking the physical nanometer scale
fibrillar nature of this structure through electrospinning. ECM fibers are reported to exhibit diameters between 10 and
300 nm, and the minimum fiber diameter required for fibroblast adhesion and migration, and maximum interfiber distance
that fibroblasts are able to bridge, have been described as approximately 10 and 200
l
m, respectively, which lie within
parameters achievable by electrospinning but hardly achievable using alternative cell culture settings [18,19]. Furthermore,
due to their intrinsic ability to synthesize their own ECM, skin cells are known to be able to self-organize even in the absence
of molecular cues provided that an adequate 3D nucleation structure exists to enable their self-organization, thereby rein-
forcing the stimulating role of electrospun nanofibrous structures for skin regeneration [19,20].
When skin damage occurs a consecutive cascade of events called wound healing takes place to restore the skin structure
and function [17,21]. A wound can result from burns, contusion, hematoma or a disease process, causing chronic wounds. At
present, due to the increasing life expectancy, diseases with high incidence such as diabetes have been considerably increas-
ing the incidence of chronic wounds and thus making it of high social relevance [11,21].
The wound healing process consists, in general, in five different phases, namely hemostasis, inflammation, migration, pro-
liferation and maturation, occurring sequentially after damage [9,21] (Fig 1). During hemostasis, platelets suffer aggregation
to promote clotting and stop any bleeding. Delivery of important growth factors to the inflammation process occurs, which
trigger the wound healing process through attraction and activation of neutrophils, lymphocytes, macrophages and mast
cells [22,23]. The inflammation phase occurs at the same time as hemostasis. In this phase, blood neutrophils followed by
phagocytes enter and penetrate inside to the injured area to destroy bacteria and eliminate debris from dying cells and dam-
aged matrix [14,24]. The following phases, migration and proliferation, are considered by several authors as the same phase
due to their interdependence [22,25]. Migration is characterized by infiltration of new epithelial cells moving on to the dam-
aged area to replace the dead cells and during the migration the inflammation decreases. The proliferation phase consist on
covering all damaged area with epithelial cells and macrophages, while simultaneously fibroblasts and endothelial cells
move to the damaged area forming a granular tissue composed by a new matrix and blood vessels, respectively [22,25].
The last phase, maturation, comprises the remodeling process, in which fibroblasts cover all the damaged surface with a
Fig. 1. Skin structure and wound healing phases.
316 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

new skin layer and ideally leaving no evidence of scar [14,23]. Through this elaborate process of wound healing the skin has
self-regeneration ability although this capacity is strongly reduced in the case of full-thickness lesions, requiring the use of a
graft or dressing [11].
3. Skin regeneration products
3.1. Autografts and allografts
When skin lesions result in large full-thickness defects the standard clinical procedure is the autologous skin transplan-
tation based on transplanting split-thickness grafts [7,8,26]. However, this transplantation contains all of the epidermis layer
but only a small part of the dermis often leading to scar formation [15]. This process has the obvious restriction of total
amount of autologous skin that can be removed and the split-skin donor site takes one week to heal and can be used for split
skin harvesting up to 4 times. Frequent harvests also lead to scars in donor sites and hospital stays for long periods of time
[6,27,28]. Allografts are grafts removed from other individuals and constitute efficient alternatives to prevent fluid loss and
infection, reduce pain and promote the healing of underlying tissues. However, this type of graft presents several ethical
problems and is influenced by the donor’s availability and potential disease transmission [26].
3.2. Wound dressings
The first procedure when skin damage occurs consists in applying a wound dressing due their efficiency on preventing
wound infection and promoting exudate absorption, low cost and availability. The main functions of a dressing are promot-
ing a moist environment in the wound, and protecting the wound against mechanical injury and microbial contamination,
especially during the inflammatory stage [7,29]. Ideally, the dressing should be able to fit the wound shape, absorb wound
fluid without increasing bacterial proliferation or causing excessive dehydration, provide pressure for hemostasis, and pre-
vent leakage from the bandage. The dressing should also support the wound and surrounding tissues, eliminate pain, pro-
mote re-epithelialization during the reparative phase, and be easily applied and removed with minimal injury to the
wound [30].
Wound dressings can be categorized according to different characteristics. One possible classification relies in classifying
the wound dressings in passive or interactive [9,31]. The passive ones correspond to the common wound dressings and their
main function is covering the wound and allowing the regeneration beneath the dressing. Some examples are tulle dressings
(made of cotton or viscose gauze impregnated with paraffin) and low-adherence dressings (made of materials as knitted vis-
cose or polyester fabric) [32]. On the other hand, the interactive wound dressings present some advantages like the capabil-
ity to modify the wound chemical environment facing to the physiological conditions of the wound for a faster healing
process. Although in some cases this modification could take long periods of time [9,33]. Commercial available interactive
wound dressings, according the widely accept classification, are divided into hydrocolloids, hydrofibers, hydrogels, foams,
alginates and bioactive/biological dressings [32].
3.3. Tissue engineering-based products
During the past few years, the progress and evolution on tissue engineering (TE) field have been growing exponentially.
This field has been exploring the regeneration of several tissues, including skin, involving knowledge from different disci-
plines. TE includes the combination of live cells, tissues or organs, with structures and materials designed to mimic the struc-
ture of a particular tissue [34,35]. The use of TE strategies for skin tissue regeneration consists essentially in expanding skin
cells in the laboratory, cultivating them on a scaffold and applying cell-scaffold construct for restoring the barrier function
(first step in burn patients), or to promote wound healing (for instance in chronic non-healing ulcers), thereby reducing pain
and promoting optimal conditions for a correct healing [11]. Several products for skin regeneration based on TE are already
clinically available that meet the essential requirements for a clinical product, namely be safe for the patient, clinically effec-
tive and conveniently handled and applied by health care professionals [6,11].
TE skin substitutes present several advantages when compared with other available solutions including less required vas-
cularisation in the wound bed, increased dermal component of the healed wound, reduced presence of inhibitory factors and
faster and safe coverage [8]. TE skin substitutes available in the market can be classified according to different features. The
most common classification is related with the anatomical structure to be regenerated, resulting in epidermal, dermal or der-
mal/epidermal (or composite) substitutes [6,26]. An important phase in the production of epidermal substitutes is the iso-
lation of keratinocytes, obtained through a 2–5 cm
2
skin biopsy from the donor. The epidermis is separated and in vitro
cultured on top of fibroblasts [6,36]. There are several epidermal substitutes available for clinical applications using cells
either of autologous or allogenic origin, with the allogenic products presenting reduced manufacturing costs compared to
autologous substitutes. Some of the commercial epidermal substitutes available are MySkin
Ò
(CellTran ltd, UK) a synthetic
silicone support layer with surface coated and seeded with keratinocytes, Epicel
Ò
(Genzyme Biosurgery, USA), sheets of
autologous keratinocytes attached to the petrolatum gauze support, and Epidex
Ò
(Eurodern GA, Switzerland), an epidermal
equivalent from the patient’s own outer root sheath, where the keratinocytes are cultured in silicone membranes. Despite
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 317

their efficiency in proving epidermal coverage, autologous and allogenic epidermal substitutes are claimed to present poor
attachment rates that can lead to blister formation [26].
The development of dermal substitutes emerged due the lack of dermal tissue in full thickness wounds and the poor qual-
ity of the scars after treatment with split thickness autografts or cultured epithelial grafts which contain little or no dermal
component, respectively [37]. There are several products available in the market that have been demonstrating great effec-
tiveness in dermal regeneration, such as Dermagraft
Ò
(Shire Regenerative Medicine, Inc, USA), a cryopreserved human
fibroblast-derived dermal substitute, generated by the culture of neonatal dermal fibroblasts onto a bioresorbable poly
(lactic-co-glycolic acid) (PLGA) mesh scaffold [38], Integra
TM
(Integra LifeSciences, USA), which is a nanofibrous bilayer mesh
specifically designed to be used in conjunction with negative pressure wound therapy, comprising crosslinked bovine tendon
collagen and glycosaminoglycan and a semi-permeable polysiloxane layer, and Karoderm
TM
(Karocell Tissue Engineering AB
company, Sweden), a human donated cell free dermis that can also be used as a biological scaffold for autologous
keratinocytes.
To mimic skin layers (dermis and epidermis) in the same construct dermal/epidermal substitutes have been explored.
Several studies have been carried out with different cell types to evaluate their performance although only autologous ker-
atinocytes were claimed effective to achieve permanent closure of skin defects [6,26]. Dermal/epidermal substitutes avail-
able in the market include PermaDerm
Ò
(Regenicin Inc., USA), composed by cultured fibroblasts and keratinocytes on an
absorbable collagen substrate, and Apligraf
Ò
(Novartis, USA), that combines two distinct nanofibrous layers, the lower der-
mal layer containing bovine type I collagen and human fibroblasts and the upper epidermal layer formed by culturing human
keratinocytes.
In spite of the great progress achieved on TE-based skin substitutes several challenges still need to be overcome to
achieve the optimal skin substitute, such as: to avoid use animal-derived materials (e.g. serum), to improve the adhesion
of cultured keratinocytes to the wound bed, to improve the rate of neovascularization of tissue engineered skin and to
enhance the scaffolds materials to resist wound contraction and fibrosis [7,8,34,39,40].
3.4. Advanced skin substitutes
Advanced skin regeneration strategies have been emerging combining cells, growth factors and scaffolds overcoming
some of the problems associated to the clinical application of skin grafts, dressings and TE-based products [7]. Scaffolds
are a crucial component because the isolated cells on their own are not able to restore the native structure of the skin with-
out support to guide the ECM growth [1]. For scaffolds fabrication, there are two main strategies: the top-down and bottom-
up approaches [41,42]. The top-down is considered the traditional approach and is based on cells seeded in a porous scaffold
generating a cellular construct, which is later subjected to the maturation process in a bioreactor. With this methodology is
expected that the cells adhere, proliferate and differentiate inside the scaffold creating an appropriate ECM stimulated by the
growth factors and mechanical or other types of stimulation [41,43]. Most TE products described before are based on this
strategy.
The bottom-up approach consists on developing biomimetic modular structures that can be created through self-
assembled aggregation, microfabrication of cell-laden hydrogels, fabrication of cell sheets or direct printing with specific
microarchitectural features [41,44,45]. The major advantages of this approach are better control over cell seeding, increasing
cell density and complexity of microarchitecture than with top-down approaches. Major disadvantages of using bottom-up
approaches include the fact that some cell types are unable to produce enough ECM, migrate or form cell-cell junctions, and
the great difficulty in developing assembly techniques able to generate engineered tissues with clinically relevant length
scales and mechanical properties [7,41,46].
4. Electrospun skin substitutes
Although the electrospinning technique is under growing development in the biomedical field its principles emerged
around 1600s. However, since 1980s, several research groups demonstrated that it is possible to produce electrospun fibers
with organic polymers increasing, since then, the number of publications exponentially [47,48]. Some of the most important
milestones are summarized on Table 1. Further details about electrospinning’s history are available elsewhere [47,49–52].
Electrospinning is a technique allowing to create submicron to nanometer scale fibers from polymer solutions or melts
and was developed from a basis of electrospraying, widely used for more than 100 years [65,68]. It is also known as electro-
static spinning, with some common characteristics to electrospraying and the traditional fiber drawing process [69].
The conventional setup for an electrospinning system consists of three major components: a high voltage power supply, a
spinneret and a collector that can be used with horizontal or vertical arrangement [47,65,70]. The syringe contains a
polymeric solution or a melt polymer, pumped at a constant and controllable rate. The polymer jet is initiated when the
voltage is turn on and the opposing electrostatic forces overcome the surface tension of the polymer solution. Just before
the jet formation, the polymer droplet under the influence of the electric field assumes the cone shape with convex sides
and a rounded tip, known as the Taylor cone [59,69,71]. During the jet’s travel, the solvent gradually evaporates, and charged
polymer fibers are randomly deposited or oriented in the collector [71].
318 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

Fig. 2. Electrospinning fabrication strategies, fiber orientation types and types of collectors used.
Table 1
Historical milestones of electrospinning.
Year Author Historical milestone Refs.
Around 1600s Gilbert Study of the magnetic behaviour and electrostatic phenomena [49]
Late 1800s Rayleigh Investigation of liquid jet hydrodynamic stability, with or without applied electric field [47,50]
1902 Cooley Patent registration entitled ‘‘Apparatus for electrically dispersing fluids”, considered as the
first description of a process recognizable as electrospinning
[53,54]
1914 Zeleny Study of the fluid droplets behaviour at the end of metal capillaries [51,52]
1934–1944 Formhals Publication of several patents describing important developments towards electrospinning
commercialization
[55–65]
1936 Norton Patented the use of melted polymers [49,66]
1964–1969 Taylor Development of theoretical electrospinning underpinning, which allowed the mathematical
modeling of the cone shape formed by the liquid droplet that became known as Taylor’s cone
[51,67]
Table 2
Effect of electrospinning parameters in fiber formation.
Parameter Effect Refs.
Solution Viscosity Determines the fiber formation [74,77]
Surface tension Determines the applied voltage; it must be higher than surface tension of the solution to initiate the
process
[76,78]
Conductivity Higher conductivity avoids droplet deposition in the fibers [75–77]
Dielectric effect High dielectric properties reduce bead formation and fiber diameter [77,78]
Processing Applied voltage Influences the jet stretching and acceleration and consequently fiber morphology [75,77]
Flow rate Influences the fiber diameter, its geometry and mesh porosity [75–77]
Needle diameter A small internal diameter reduces droplet formation in the fibers [77,78]
Distance needle-
collector
Influences the solvent evaporation rate [75,77]
Ambient Temperature The increase of temperature favors the solvent evaporation rate [67,77]
Humidity If too high induces morphological changes increasing the surface heterogeneity and for hydrophilic
polymers is unable form fibers and electrospraying occurs
[47,67,77,78]
Atmosphere
types
Some gases are influenced by the electrostatic field blocking the process [77]
Pressure If pressure is lower than atmospheric one the solution exists through the needle causing jet
instability
[77]
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 319

The electrospinning process can be influenced by several parameters, such as: solution parameters (viscosity, concentra-
tion, type of solvent), processing parameters (flow rate, distance between needle and collector, voltage supply, type of col-
lector) and ambient parameters (temperature and humidity), as summarized in Table 2 [47]. It should be emphasized that
the acceleration of fiber formation is up to 600 m/s
2
, which is much higher than the value of acceleration of gravitational
forces on earth (at sea level and at 45°of latitude it corresponds to 9.80665 m/s
2
), meaning that gravity does not influence
the process [72,73].
The technique is also highly versatile since, in addition to the conventional fiber configuration, it is possible to obtain a
variety of other configurations, namely core/shell (co-axial) or emulsion configurations and, according to the fiber orienta-
tion, it is possible to produce aligned or randomly oriented fibers depending the type of the collector used (Fig. 2).
The use of electrospinning to regenerate damaged tissues rose in the last decade due to its simplicity to produce meshes
and its capacity to mimic the micro-nanostructure of the natural ECM. The nanofibers produced through electrospinning
confer a high surface area to the structure, high interconnectivity that is beneficial for regenerative tissue growth and cell
migration and great potential for effective delivery of biomolecules, [47,79]. According to tissue engineering principles an
ideal scaffold should hold cellular activities, and should disappear over time while tissue regeneration occurs. To enable this,
scaffolds should mimic native tissue regarding its structure, appropriate mechanical strength, porosity for cellular infiltra-
tion and growth [35,69].
Traditional scaffolding methodologies like solvent casting and particulate leaching, gas foaming, freeze drying and gas
foaming have limited ability to form scaffolds that mimic the native tissue nanostructural architecture [80,81]. However,
electrospinning presents a unique ability to fabricate nanofiber-based scaffolds that best mimic the nanometer scale of
the native ECM as well as the mechanical properties of the native skin. Electrospun skin substitutes have been claimed to
have increased potential to promote better cellular attachment, growth and differentiation due the high surface area, high
aspect ratio and high microporosity provided by the low fiber diameter structure [35,52,82]. The versatility of this technol-
ogy further allows tuning of fibrous scaffold design in terms of mechanical properties, fiber diameter, density and orientation
to mimic the physical features of the ECM, as shown in several examples ahead.
The mechanical properties of skin in vitro and in vivo have been evaluated using different techniques (ultrasounds, inden-
tation, tensile tests, suction and torsion) [83–85]. Human skin is a complex tissue due its heterogeneity, viscoelasticity, ani-
sotropy, adhesive properties and non-linear stress-strain behaviour [83,84,86].Table 3 presents the mechanical properties of
skin tissue in comparison to electrospun meshes made by different materials and production strategies, showing that the
mechanical properties of electrospun meshes are similar to those of skin, thus demonstrating the potential of this technology
to mimic skin tissue due not only due to its similarity in terms of organization (nanofibrous mesh-like structure) but also
mechanical properties.
Additionally, the high specific surface area and porosity of electrospun meshes constitute additional functional advan-
tages by providing tunable fluid absorption and drug and biomolecule delivery, adequate oxygen, water and nutrient diffu-
sion coupled with efficient metabolic waste removal.
In spite of the significant advances in electrospinning, in the biomedical field only a few companies provide customized
nanofibers production either as single or bi-layers combining different materials. Commercially available products also
include cell culture well-plates integrating electrospun structures and meshes for stent coverage [94–96]. Specifically for
skin regeneration, a clinical trial was carried out for the treatment of diabetic foot ulcers using a multilayer polyurethane
electrospun transdermal patch releasing nitric oxide [97]. However no clinical trials using the electrospinning technique
for skin regeneration are ongoing [98].
4.1. Randomly oriented fiber meshes
Conventional electrospinning set-up configuration consists in fibers randomly deposited over the grounded collector,
which is usually a metal plate [47,79,99]. The random deposition is a consequence of the jet instability resulting from the
electric field applied to overcome the polymeric solution surface tension [51,100].
There are several studies comparing random and aligned deposition strategies in terms of nanofibers morphology,
hydrophilicity, mechanical properties and cell adhesion and proliferation [101,102].
In terms of biological response numerous studies demonstrated that aligned fibers usually exert a more relevant influence
on cellular behaviour including cell morphology, cellular density and gene expression. In terms of mechanical properties the
elongation at break presents better results when fibers are randomly oriented [101–104]. Although both strategies allow
producing structures with suitable properties to promote skin regeneration, skin is generally characterized by a meshlike
random orientation of fibrils, making random meshes the electrospun structures the more suited to mimic native skin’s
ECM [18]. Jha and colleagues explored the application of randomly oriented fiber meshes to improve wound healing, in
which they assessed skin regeneration promoted by collagen electrospun fibers crosslinked with glutaraldehyde on adult
guinea pigs [105].In vitro and in vivo results showed that the created wounds closed after 16 days of implantation and no
adverse inflammatory reactions or other antigenic complications were observed, showing the great potential of this strategy
for dermal reconstruction. Said and co-workers [106,107] also investigated randomly oriented electrospun fibers for wound
healing by combining PLGA with different substances as antimicrobial wound dressing. In vivo results after application of
fusidic acid (FA)-loaded PLGA electrospun ultrafine fibers showed high efficacy of this strategy to promote wound healing
and reduced infection (Fig. 3)[106,107]. A study performed by Coskun et al. evaluated the performance of randomly oriented
320 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

electrospun poly (vinyl alcohol)/sodium alginate as wound dressing in vivo. This study compared commercially available
wound dressings (tulle grass, Eczacibasi), woven cotton antibacterial bactigras (Smith & Nephew) and nonwoven
Suprasorb-A (Lohmann) made from calcium alginate fibers to the electrospun meshes during 21 days. In the early time-
points (4 and 6 days) no significant differences were observed, although after the following time-points (15 and 21 days)
important differences were observed. Electrospun meshes presented the best healing performance as shown through epithe-
lization, epidermis characteristics, vascularization and formation of hair follicles (Fig. 4)[108].
These studies demonstrate the importance of the nanostructure provided by randomly oriented electrospun meshes to
promote wound healing. In fact the electrospinning technique allows production of nanostructures with similar diameter
(native range between 10 and 300 nm), porosity and random orientation similar to the collagen fibrils in the ECM of skin
[18,108].
4.2. Aligned fiber meshes
In the TE field one of the most important criteria to design the optimal scaffolds relies in mimicking the tissue ECM, which
may involve a considerable degree of orientation, depending on the tissue type and ECM. Hence, random fiber deposition
may not be adequate when mimicking tissues where specific fiber orientation is required [72,79]. Therefore several alterna-
tive set-ups to conventional electrospinning have been developed to achieve optimized architectures. To obtain aligned
Fig. 3. Effect of plain and fusidic acid loaded PLGA ultrafine fibers on the healing of wounds in rats [107].
Table 3
Mechanical properties of skin tissue and electrospun meshes.
Structure Young’s modulus (MPa) Tensile strength (MPa) Elongation at break (%) Refs.
Human skin 2.9–150 1–32 17–207 [87–90]
PCL 21.42 ± 0.04 6.87 ± 0.25 116.0 ± 6.53 [91]
PCL/collagen 82.08 ± 17.86 8.63 ± 1.44 24.0 ± 7.16 [91]
PLCL 47.66 ± 2.24 7.24 ± 0.16 158.54 ± 66.67 [90]
CA/pullulan 2.91 ± 0.21 0.13 ± 0.08 22.2 ± 0.01 [92]
HA/PLGA core/shell 28.0 1.52 60.07 [93]
CA – cellulose acetate; HA - hyaluronic acid; PCL – poly (Ɛ-caprolactone); PLCL – poly(Ɛ-caprolactone-co-lactide); PLGA – poly(lactic-co-glycolic acid).
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 321

fibers by electrospinning several collectors with varied configurations have been designed to match the desired orientation
(Fig. 2). Fiber alignment can also be achieved using near electrospinning or melt electrospinning. In both cases the collector is
moving in X and Y directions to induce filament orientation and the process is characterized by short distances between the
tip of the needle and the collector. To achieve a stable jet region for controllable deposition the average distance of near elec-
trospinning lies between 500
l
m and 3 mm and for melt electrospinning between 3 and 5 cm [109–111].
Compared to randomly oriented fibers, aligned fibers present significantly higher resistance to tensile stress, when tested
parallel to fiber alignment, and also exert a distinct influence on cell behaviour [70]. Since a variety of tissues are constituted
by oriented fibers the development of support structures capable of influencing cellular behaviour at the right orientation is
of significant importance. These tissues include ligaments, tendons, brain, muscles, cardiac and vascular tissues [112]. Recent
studies demonstrated the influence of aligned fibers over cell organization and function [113–115].
Despite the general random orientation of native skin tissue, Annaidh and colleagues have reported the relevance of the
orientation of collagen fibers in the dermis due to the correlation between their orientation and Langer lines [85]. In the past,
Cox and Stark already concluded that the Langer lines have an anatomical basis, since they remained after removal of skin
from the body and after tension tests [116,117]. A few studies have investigated the use of aligned fiber meshes to promote
wound healing. Patel et al. developed aligned and bioactive nanofibrous scaffolds by immobilizing extracellular matrix pro-
tein and growth factor onto poly(
L
-lactide) (PLLA) nanofibers, which simulated the physical and biochemical properties of
native matrix fibrils. The aligned nanofibers significantly induced neurite outgrowth and enhanced skin cell migration during
wound healing compared to randomly oriented nanofibers. Furthermore, the immobilized biochemical factors (as efficient as
soluble factors) synergized with aligned nanofibers to promote highly efficient neurite outgrowth but had less effect on skin
cell migration [118]. Kurpinski and co-authors showed that aligned PLLA nanofibers enhanced bovine aortic endothelial cells
(BAECs) infiltration as a result of a high pore openness, which facilitated cell migration across the structure. In vitro and
in vivo tests on a dermal wound healing model showed the importance of nanofiber alignment coupled with the effect of
added heparin as effective biophysical and biochemical cues, respectively, to regulate the cellular behaviour and tissue
remodeling [119]. In terms of wound size reduction no significant improvements were observed after 7 days. However, his-
tological findings revealed that in aligned fibers the epidermal layer grew, migrating from the wound edge towards nanofi-
brous graft [119].
4.3. Fibers with core/shell structure
The core/shell technology emerged among the most promising set-ups in the field of electrospinning since it is based on
the combination of two different materials or substances. Using this approach, the same filament may have distinct inner
Fig. 4. Histological cross-sections of tissues obtained from regions covered by wound dressings on the 21st postoperative day. (a) Tulle grass. (b) Bactigras.
(c) Suprasorb-A. (d) Electrospun poly (vinyl alcohol)/sodium alginate mesh. Arrows: hair follicies (reprinted from [108], with permission from IOS Press)
[108].
322 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

and outer layers, allowing different compositions such as a material surrounded by another material or by a matrix loaded
with dispersed particles [79,120]. This design was developed to incorporate substances (e.g. drugs, enzymes, growth factors
or other biomolecules) inside the nanofibers. It presents two main advantages [52]: (i) substances can be incorporated in the
inner layer being protected from environmental factors, such as the organic solvents usually used in the electrospinning
technique and (ii) and the incorporated substance can be released from the inner layer and past the outer shell layer in a
more controlled and sustained pattern [70]. The design parameters, selected materials, thickness and microstructure of
the shell will directly influence the release pattern of the substance contained inside of the fibers. The core/shell design is
also being widely explored to improve the surface properties of nanofibers, such as the hydrophilicity, which in turn will
influence the biological response [70].
There are two different processes to produce core/shell fibers: co-axial electrospinning and emulsion electrospinning. Co-
axial electrospinning consists on a capillary concentrically inserted inside the other capillary, resulting in a co-axial config-
uration in which each capillary is connected to a reservoir containing a given material. Similarly to the conventional elec-
trospinning set-up this approach can work in the vertical or horizontal positions [120]. Through this process, several
structures can be produced, such as bicomponent fibers, hollow fibers and fibers with microparticles (Figs. 2 and 5). Bicom-
ponent fibers with core/shell configuration can be obtained from two electrospinnable materials or the combination of a
spinable material with other non-spinable. This approach presents as major advantages the obtention of a final fiber present-
ing unique properties and the use of materials that on their own could not be used in the electrospinning process. Using this
approach, the range of materials used in electrospinning considerably increases, overcoming the limitations to obtain elec-
trospun fibers from specific materials due their low molecular weight, limited solubility, unsuitable molecular arrangement,
or lack of required viscoelastic properties [120]. For instance, Nguyen and co-workers (2011) developed electrospun meshes
of chitosan (CS) (core) and poly(lactic acid) (PLA) (shell) although CS, due to its high molecular weight, high viscosity and
polycationic nature, cannot be electrospun on its own, and demonstrated their antibacterial activity and the high potential
of these composite nanofibers for applications in the biomedical field [121].
The combination of fibers with drugs, growth factors and other substances or biomolecules also provides novel function-
alities to the produced fibers [120]. The entrapment of substances inside the fibers allows controlling the release rate, which
is dependent on the degradation rate of the outer fiber polymer, thus smoothing the sudden release [52]. Several research
groups have been showing interesting results from substances encapsulation. Maleki et al. reported an easier control of drug
release profile through core/shell fibers compared to monolithic fibers using tetracycline hydrochloride (TCH) as core and
poly(lactide-co-glycolide) (PLGA) as shell [124]. Despite their interesting properties the wide application of liposomes in
regenerative medicine is difficult due to their short half-life and inefficient retention at the site of application. Mickova
and co-workers claim that these disadvantages could be significantly reduced by their combination with nanofibers
[123]. They demonstrated the incorporation potential of liposomes into nanofibers by coaxial electrospinning of poly(vinyl
alcohol) (PVA) (core) and polycaprolactone (PCL) (shell). The study validated that the enzymes encapsulated on liposomes
dispersed into PVA fibers survived intact to the process fabrication. The potential of this system was also proved by the
enhancement of mesenchymal stem cell proliferation, indicating its promising characteristics as drug delivery system [123].
Not many studies are available reporting the use of core-shell nanofibers for skin regeneration. Jin et al. demonstrated
that nanofibers composed of gelatin (core)/poly(
L
-lactic acid)co-poly-(e-caprolactone) (PLLCL) (shell) and epidermal induc-
tion medium embedded in the core promoted the desired sustained release of the medium without burst release and further
induced the differentiation of adipose-derived stem cells into epidermal lineages [125]. According to Xu et al. an efficient
delivery system is critical for the success of cellular therapies. To deliver cells to a dynamic organ, the biomaterial vehicle
should mechanically match the non-linear elastic behaviour of the host tissue. In this study, non-linear elastic biomaterials
have been fabricated from a chemically crosslinked elastomeric poly(glycerolsebacate) (PGS) (core) and the thermoplastic
poly(
L
-lactic acid) (PLLA)(shell) using the core/shell electrospinning technique. Mechanical tests demonstrated values com-
parable to skin tissue (Table 3) and ex vivo and in vivo trials shown that the elastomeric mesh supports and fosters the
growth of enteric neural crest (ENC) progenitor cells.
Fig. 5. Core/shell fibers (a) Transmission electron microscopy image of: Core/shell chitosan/poly(lactic acid) electrospun composite nanofibers produced
using a co-axial approach [121]. (b) Scanning electron microscopy image of a cross-section of a polycaprolactone (PCL) hollow fiber in water coagulation
bath [122]. (c) Confocal microscopy image of a poly(vinyl alcohol) (core)/PCL (shell) nanofiber mesh with encapsulated liposomes (in the core) stained with
fluorescein [123].
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 323

Co-axial electrospinning can also be used to produce hollow fibers without the need of a template to be coated, as in the
chemical vapor deposition method [126]. In this strategy the core material is dissolved by a specific solvent, at the end of the
process or the core interacts with shell forming a hollow fiber during the processing. Wei et al. showed the potential of hol-
low fibers to act as a drug delivery system using core/shell fibers of PVA (core) and polyethersulfone (PES) (shell) with the
core material containing the drug (curcumin). During the fabrication process the core and shell wall interacted forming a
hollow fiber bilayer containing the drug on the fiber inner wall [127].
Core/shell fibers can also be produced using emulsions. This approach does not require a special needle with a physical
separation between the core and the shell solutions neither such a careful selection of operation parameters as in the co-
axial approach. In this case the dispersed drop in the emulsion turns into the core and the continuous matrix become the
shell [120,128,129]. Ma et al. reported the formation of core/shell fibers through the emulsion using sodium alginate as core
and poly(ethylene oxide) (PEO) as shell. However, when crosslinking occurs it induces changes on fiber morphology and the
electrospun mesh looses the configuration and becomes a film. The water-in-oil (w/o) emulsion is being widely explored to
encapsulate hydrophilic drugs or bioactive molecules in the core to avoid burst release and prolong the release time [128].
Zhang et al. prepared bovine serum albumin (BSA) entrapped in a water-in-oil emulsion as the core, encapsulated in the shell
polymer (methoxy polyethylene glycol-b-poly(
L
-lactide-co-e-caprolactone) (PELCL) and poly(
L
-lactide-co-glycolide)(PLGA))
via emulsion-core (EC) coaxial electrospinning. The fibrous membranes reduced the initial burst release of BSA, which can
be tailored by changing the composition to PLGA in the core emulsion. The results showed that EC electrospinning performed
better than conventional co-axial electrospinning with respect to protein delivery for tissue engineering applications [130].
4.4. Hybrid structures
The characteristic small pore size of nanofibrous meshes produced by electrospinning and the lack of specific groups to
interact with cells on the commonly used polymers limits the cellular migration into the scaffold and could results in 2D
tissue formation becoming a hindrance to the success on 3D tissue regeneration [131,132]. To overcome these limitations
several promising approaches have been developed, either combining different variants of electrospinning or through com-
bination with additive technologies [133,134].
The combination of different electrospinning set-ups allows the fabrication of hybrid structures. Several research works
explored the development of hybrid structures combining different fiber diameters [135–137], different materials to improve
the properties of the structure [138–140] or combining aligned/random fibers [141]. For skin regeneration most of the avail-
able works only explore the combination of materials and different fiber diameters, building structures without gradients.
The droplet formation phenomena, initially considered an handicap to fiber production, is a consequence of the low vis-
cosity of the polymeric solutions used in electrospinning [50,142]. However, exploration of particle formation under influ-
ence of an electric field, a new technique was developed, called electrospraying, ensuring that with one entanglement per
chain it is possible to obtain particles from micro to nano scale [143]. Particle electrospraying is of great interest for tissue
engineering applications by providing encapsulation of biomolecules due its high encapsulation efficiency and increase in
the surface area [144,145]. Only a few works are available combining electrospraying with electrospinning, aimed at devel-
oping hybrid structures mimicking native tissues. It has been previously demonstrated that combining both techniques it is
possible obtain hybrid structures with potential for tissue regeneration due its capacity to promote cell adhesion and pro-
liferation (Fig. 6a) [146–149]. The combination could be an interesting approach to produced meshes for skin regeneration
although it has not been explored in that sense yet. As previously explained nanofiber meshes have unique properties to pro-
mote skin regeneration although, especially coupled with controlled delivery of relevant therapeutic molecules. This could
be achieved using the electrospraying technique that provides advantages over the more widespread use of nanoparticles
prepared through conventional techniques, since no emulsion nor high temperatures are required, no further drying step
is necessary and it provides an enhanced control over particle size distribution [145,150,151].
Hybrid structures produced through the combination of solution and melt electrospinning is another interesting emerg-
ing approach. The use of a molten polymer with electrostatic field was reported for the first time by Larrondo and Mandley
[154–156]. However, only recently the use of melt polymers has received attention again rather than polymers dissolved in
organic solvents. Although fibers obtained through melt electrospinning usually present relative high diameters, the tech-
nology presents important advantages namely not using organic solvents, thus avoiding solvent accumulation and the need
to subsequently eliminating them to decrease sample toxicity [109,157]. The combination of solution with melt electrospin-
ning also contributes to solve the problems associated with low cellular infiltration as a consequence of high-density packing
of nanofibers, with the microfibers increasing the pore size and porosity required for cell infiltration, and nanofibers con-
tributing to promote cell attachment and growth [145,158]. However, despite the potential of this kind of combination, only
few works are available explored this approach, demonstrating that 3D hybrid structures made from PLGA micro and nano-
fibers show improved mechanical properties, cell attachment and growth than structures composed only by microfibers,
thus representing a greater potential for tissue engineering applications, namely for skin regeneration (Fig. 6b) [134,152].
Another strategy to obtain hybrid structures involves additive manufacturing technology combined with electrospinning.
Additive manufacturing (AM) techniques have been widely studied for scaffold fabrication due their ability to produce por-
ous structures with high reproducibility, tailored external shape and internal morphology [159–161]. However, the pro-
duced scaffolds present lack of nanometer-sized details to mimic the native ECM of tissues. To improve cellular
behaviour the combination with electrospun nanofibers is a possibility [131,160,162,163]. The initial works combining both
324 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

techniques were reported in 2008, demonstrating improved mechanical properties, cell attachment and proliferation of the
hybrid structures and their potential for tissue engineering (Fig. 6c) [153,164,165]. Since then, only few additional works
have been reported, most of them combining additive techniques based on fused deposition modeling (FDM) with electro-
spinning, and often using PCL [160,166–172]. Although no works exploring this approach for skin regeneration are available
Fig. 6. Hybrid structures produced by combination of: (a) Electrospinning and electrospraying [146,147]. (b) Solution electrospinning and melt
electrospinning [152]. (c) 3D printing (FDM) and electrospinning [153].
Table 4
Critical analysis of the essential characteristics of scaffolds produced by additive manufacturing (AM) in comparison with nanofibers produced by
electrospinning and their influence on tissue regeneration.
Type of
structure
Characteristics Influence Refs.
AM scaffolds Controlled microstructure Facilitates oxygen and nutrients transport across the structure by
increasing the diffusion efficiency
[161,164,173]
Suitable mechanical properties Maintains scaffold structural integrity and stability and matches
native tissue’s mechanical characteristics to expose cells to the
correct stress environment
[164,165,174,175]
Large pore size Limits cell seeding efficiency [166,168,171,176]
Smooth filaments Inhibits initial cell attachment [164,166,171]
Electrospun
nanofibers
High surface area Mimics the hierarchical structure of ECM that is critical for cell
attachment, spreading and proliferation, as well as for nutrient/
waste transportation
[52,79,82,168]
High porosity Favors cell attachment, differentiation and mimics the native
ECM, facilitating nutrient and waste exchange and vascularization
[70,75,177–179]
Fibers with low diameter Fiber diameters match structural properties of the ECM and confer
high surface area to volume specific ratio
[50,70,142,180]
Low mechanical properties Limits structural and functional integrity and does not provide the
correct stress environment to produce neotissues
[70,99,164,167]
High packing required to obtain 3D
structures
Restricts cellular infiltration across the mesh [160,181,182]
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 325

it would be interesting to integrate the advantages of AM (control of pore size, pore size distribution, interconnectivity and
mechanical properties) with electrospun nanofibers. The use of hybrid structures allows combining the advantages of both
techniques and reducing or eliminating the disadvantages resulting of the separate use of each technique, as explained in
Table 4.
Despite recent advances towards the development of hybrid structures for tissue engineering applications, several chal-
lenges still remain. Most of the hybrid structures produced are based on the combination of solution electrospinning
together with electrospraying, melt electrospinning or additive manufacturing technologies. Combinations with other
techniques, although yet little explored, represent equally exciting potential, even if for specific applications. Pateman
and colleagues explored the potential of combining the stereolithography and electrospinning to create channels with ori-
ented fibers supporting the regeneration of injured nerves and guide Schwann cell growth [183]. This approach could be
equally interesting for wound dressing development by, for instance, allowing to produce fibers with photocrosslinkable
hydrogels that combine the advantages of wound dressings composed by nanofibers (promoting hemostasis, semiperme-
ability, no scar induction, among others) with photocrosslinkable hydrogels that, beyond the advantages of hydrogels in
the wound healing process, allow precise control over the diffusion rate of bioactive substances across the structure
[9,181,184].
4.5. Cell electrospinning
Scaffolds are critical to support, promote and guide cell growth, thus making the development of structures mimicking
the ECM a subject of intense research. To recreate the complex tissue nano-microstructure, modular structures are
required providing precise control over the architecture, biomechanical behaviour, cell density and degradation rate
[1,7,41]. At present, two main approaches are available to integrate cells into the scaffolds: cell seeding and cell print-
ing/bioprinting, correlated with top-down and bottom-up approaches, respectively. Cell seeding is the most widely used
method to integrate cells into 3D structures and consists on seeding cells on scaffolds. However, this approach presents
limited control over cell density, localization and spreading, resulting in low seeding efficiency, minimal cell penetration
of scaffold walls and not mimicking the cellular organization of native tissues [185–187]. Although different approaches
exist for cell seeding, cell printing been attracting great attention due to the possibility of integrating cells directly into the
filaments that compose the 3D structure [188,189]. Different cell printing technologies allow the production of 3D struc-
tures, in which cells and biomaterials can be positioned in pre-determined places due to the precise control over the inter-
nal/external architecture and layer-by-layer fabrication [161,186,189]. The most widely used technologies for cell printing
are the inkjet [45,190–192] extrusion [189,193,194], laser [195–197], valve-based [188,198] and acoustic ones [199,200]
(Fig. 7).
The biological performance of electrospun meshes, similarly to other structures designed for tissue engineering applica-
tions, depends on their ability to incorporate the desired cell types and to promote the intended functionality of the incor-
porated cells [201]. As previously mentioned, the most common procedure relies in incorporating cells after scaffold
production, although recent works have been exploring the combination of electrospinning with bioelectrospraying to seed
cells during the production of the structure. The earlier incorporation of a high number of cells was reported to improve
structural stability and biochemical composition of engineered tissues [202,203].
Similarly to the previous cell printing technologies mentioned, the cell electrospinning methodology intends to signifi-
cantly reduce the time needed to generate complex cellularized structures, the non-uniformity in the seeded cells and
the time required for cells to fully infiltrate the entire architecture [204]. This concept was pioneered in 2006 by Jayasinghe’s
research team, when they proposed cell electrospinning with different cell concentrations and using a core-shell set-up and
varying flow rates [205]. The cell line used belongs to the neuronal lineage, and hence is more suitable to function under the
influence of electric impulses without damage. Since cell suspensions by themselves are not electrospinnable the core-shell
approach was used, in which the inner part was composed by a cellular suspension and the outer part by polydimethylsilox-
ane (PDMS). The outer solution should act as a shield for cells and provide a matrix for cell growth. The cellular viability
in vitro post electrospinning was evaluated through flow cytometry and showed that cell growth and 100% of confluence
in all samples was reached after 3 weeks of culture. In vivo tests were performed using real-time bioluminescent imaging
in which results showed that the processing did not compromise the ability of the electrospun cells to proliferate [205–
207]. However, according to Townsend-Nicholson and colleagues, when the bicomponent filaments (PDMS/cell in suspen-
sion) were submerged into the cell growth medium the nanofiber mesh configuration was lost, thus indicating that the cell
viability mentioned before corresponded to the cell suspension alone and not to the performance of electrospun meshes
incorporating cells. Nevertheless, the study showed the possibility of electrospinning living organisms. The same research
team updated the previous work by using the same polymer (PDMS) but increasing the cell concentration from 10
6
to 10
7
-
cell/mL and using primary porcine vascular smooth muscle cells and rabbit aorta smooth muscle cells [206]. Although cell
viability was described as not affected by the electric field and fibers were electrospun containing high cellular concentra-
tions, no evidence of remaining scaffolds after cell culture was provided. Recently, Sampson and co-workers reported in vitro
and in vivo studies using cell electrospun meshes produced with modified matrigel as shell to the cell suspension. According
to their results the cells submitted to the electric discharge showed a similar behaviour to the control ones (not submitted to
any discharge), although the matrix was dissolved and cells disassociated from the scaffold before analysis [207].
326 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

Although a few publications already exist on the topic of cell electrospinning, the issues related to cell behaviour in a high
electric field, namely the in depth assessment of cellular damage, has not been reported yet. Jayasinghe and his team
reported that cells survive the electrospinning process without any major damage, although enough evidence is still missing
showing that the 3D structures encapsulating the cells maintain their architecture over time. Another limitation of this pro-
cess is related with the fiber size. One major advantage of electrospun nanofibers in wound healing is the relatively small
fiber diameters (in the order of nanometers), which mimics the native ECM. In suspension cells assume a size of around
10–20
l
m, which considerably limits the fiber diameter achievable with the cell electrospinning approach to the micrometer
size range, thus compromising the natural advantage of electrospinning to mimic the native fibers of ECM (10–300 nm) com-
pared to other competing technologies such as cell printing.
Therefore, additional studies are required to further address the issues described above. However, in the field of skin
regeneration this new approach brings enormous potential, with the possibility of incorporating cells into the core of poly-
mer fibers, thus eventually decreasing the problems associated with low cell infiltration as a consequence of small pore size
and high packing associated to electrospun 3D meshes.
4.6. In situ electrospinning
In situ electrospinning is, a new concept that intends to produce appropriate substitutes for tissue repair and regeneration
directly on the patient’s lesion [7]. To fabricate the adequate substitutes this approach is associated to real-time imaging tech-
niques and path-planning devices for the digitalization of the damaged area and definition of the path for the deposition of
biomaterials with or without cells that can be combined with encapsulated cells [7]. The main goal of this approach is to pro-
vide a tool to directly create a customized wound dressing to the wound bed, with easy and quick application, painless to
remove and at a low cost [208,209]. Xu et al. recently patented an easily handled and portable e-spinning battery-operated
apparatus for in situ electrospinning (Fig. 8a) [210], allowing the deposition of electrospun meshes, with similar characteristics
Fig. 7. Cell printing technologies.
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 327

to the ones obtained by the conventional electrospinning technique, directly to the skin and using varied polymeric
micro/nanofibers (Fig. 8b) [208]. More recently, the same team explored the effect of in situ electrospinning on the wound
healing process. They deposited in situ mesoporous silica nanoparticles (Ag-MSNs) dispersed in PCL electrospun fibers and
evaluated the antimicrobial activity and biological efficacy in wistar rats. The in vitro and in vivo results confirmed the antimi-
crobial activity and bioavailability of 5% Ag-MSNs/PCL electrospun fibers (average diameter of 658 nm). The results showed
efficient antibacterial properties against predominant pathogenic bacteria (gram negative Escherichia coli) responsible for sev-
eral burn wound infections. In vivo studies clearly showed the improvement of in situ deposited nanofibers on wound healing
compared to the control groups. After four weeks of post-treatment it was possible to observe significant wound closure and
complete re-epithelialization (Fig. 8c) [209]. This new approach can bring considerable advances in the wound care field,
allowing a quick deposition of skin substitutes independently of wound size and depth, although some issues still remain
to addressed, such as the decrease of fiber diameter (from ca. 650 to 10–300 nm, the average diameter of fibers in native
ECM), and the matching of mechanical properties between structures developed and native skin.
5. Concluding remarks and future trends
Nanoscale constructs, and electrospun meshes in particular, have been receiving great attention from the scientific and
medical communities for skin regeneration. In the past few years, important advances have been achieved in terms of
nanofiber fabrication strategies, and related material synthesis and functionalization, and in vitro cell culture procedures.
The developments in the field reported so far have been considerably contributing for a more efficient mimicking of the
ECM through the combination of materials, growth factors, proteins and biomolecules which, associated to the novel
advanced processing strategies, making possible the production of wound dressings with a remarkable potential for skin
regeneration. However, recent advances in the specific topic of skin regeneration have been mainly focused on materials
rather than in sophisticated fabrication strategies to generate biomimetic and complex constructs that resemble the
mechanical and structural properties of skin. Research efforts have been focused on both the development of novel material
combinations and the improvement of the biochemical properties of existing materials, for instance, through the use of func-
tionalization procedures and surface modification processes. In general, improved skin substitutes need to be developed to
avoid the use of animal-derived materials, improve the adhesion of cultured keratinocytes to the wound bed, improve the
rate of neovascularization of tissue engineered skin and enhance the scaffolds materials to resist to the wound contraction
and fibrosis.
Fig. 8. In situ electrospinning concept, (a) portable electrospinning system, (b) SEM of electrospun meshes obtained with portable system, (c) in vivo
evaluation of in situ electrospun mesh, PCL-polycaprolactone, Ag-MSNs -mesoporous silica nanoparticles [208,209].
328 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334

Several advances on the evolving field of electrospinning can be foreseen with specific application in wound healing,
namely:
(i) Development of combined and functional structures using different deposition strategies, such as the multimaterial
approach, where two or more materials are deposited at the same time on a single collector, or create a multilayer struc-
ture with sequential production. Other interesting strategy is the use of hybrid structures through the combination of
filaments produced using different methodologies, such as using emulsions, copolymers, or the core/shell approach.
(ii) Integration of different technologies with electrospinning and different electrospinning approaches to obtain hybrid
structures with tunable gradients, properties and functionalities. Combining different materials and fiber composi-
tions, different fiber diameters and nano/microarchitectures to achieve the most suitable mimetic structure to regen-
erate the skin tissue;
(iii) Cell electrospinning of skin cells (keratinocytes and fibroblasts) needs to be explored to evaluate the electric field influ-
ence on the cells viability, proliferation and gene expression. The integration of cells into electrospun fibers will bring
forward a new generation of skin substitutes and solve problems of cell infiltration associated to electrospun meshes;
(iv) The in situ electrospinning is a promising technology providing the possibility of direct deposition of electrospun
nanofibers on the wound with no restriction due to wound size or depth. This technology open considerable possibil-
ities, especially combined with previously mentioned developments, namely the deposition of multilayer structures to
build hybrid structures or the integration with cell electrospinning.
Acknowledgments
This work was financed by European Regional Development Fund (ERDF) through the COMPETE 2020 - Operational Pro-
gramme for Competitiveness and Internationalization (POCI), Norte Portugal Regional Operational Programme (NORTE
2020), under the PORTUGAL 2020 Partnership Agreement, and by Portuguese funds through Portuguese Foundation for
Science and Technology (FCT) in the framework of the project Ref. PTDC/BBB-ECT/2145/2014. Juliana Dias is also grateful
to FCT for the doctoral grant SFRH/BD/91104/2012.
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Juliana R. Dias is currently Ph.D. candidate on Biomedical Sciences at the i3S-Instituto de Investigação e Inovação em Saúde,
University of Porto, Portugal. Her main research focus is on hierarchical electrospun nanostructures for skin regeneration. Her
main interests focus is on biomaterials, biofabrication, electrospinning and tissue engineering.
J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334 333

Pedro L. Granja is presently Scientific Coordinator of Instituto de Engenharia Biomédica (INEB), group Leader at Instituto de
Investigação e Inovação em Saúde (i3S, University of Porto-UP), Associate Professor at Instituto de Ciências Biomédicas Abel
Salazar (ICBAS, UP) and Invited Auxiliary Professor at Faculty of Engineering of UP (FEUP). He is also Editor-in-Chief of the
Biomaterials Network (Biomat.net), and Editor-in-Chief of the recently launched journal Biomatter.
Paulo J. Bártolo is Chair Professor in Advanced Manufacturing at the School of Mechanical, Aerospace and Civil Engineering,
Director of the Manchester Biomanufacturing Centre, Principal Investigator at the Manchester Institute of Biotechnology,
University of Manchester (UK). He is further Visiting Professor at the Nanyang University (Singapore) and fellow of the Inter-
national Academy of Production Engineering. He is also Editor-in-Chief of both Virtual and Physical Prototyping Journal and
biomanufacturing Reviews.
334 J.R. Dias et al. / Progress in Materials Science 84 (2016) 314–334