(2,3) Electrospinning For Tissue Engineering

Electrospun nanofiber scaffold
applications
Regeneração de Tecidos 2022-2023

Ricardo Baptista
Content
• References
• https://doi.org/10.1016/j.msec.2021.112373
• https://doi.org/10.1016/j.smaim.2021.07.006
• Vascular tissue Engineering

• Electrospinning
• Materials
• Multilayered electrospun vascular grafts
• Fiber morphology and alignment
• Increasing porosity
• Conclusions
26-07-2023 Ricardo Baptista 2
Vascular tissue engineering
• Cardiovascular diseases remain the leading cause of mortality,
accounting for 17.8 million deaths worldwide in 2017
• In total nearly 200 million people have coronary heart disease, and
113 million people suffer from peripheral artery disease
• Graft bypass surgery is one of the most common treatments for CHD
with an estimated 371,000 surgeries performed in 2014 in the United
States
• Open revascularization surgery for the treatment of PAD is needed in
cases of critical limb ischemia or where endovascular intervention is
unsuitable
• The rise of these diseases has created an ever-present demand for
suitable vascular grafts

• Autologous vessel grafts are considered the gold standard for
bypass and revascularization surgeries, with the greater
saphenous vein considered the vessel of choice by many
• Alternative vascular conduits include internal mammary, radial,
and internal thoracic arteries as well as the lesser saphenous
and arm veins
• However, between 20 and 40% of patients lack a suitable
alternatives due to these being too small, previous removal, or
disease
• Therefore, there is a need for an alternative to autologous

vessel grafts

• Synthetic grafts composed of expanded polytetrafluoroethylene
(ePTFE) and polyethylene terephthalate (Dacron) have been used
clinically since the 1950s for the replacement of medium to large
diameter vessels
• For small diameter vessels (<6 mm in diameter), however, these
synthetic grafts are prone to failure due to rapid occlusion and acute
thrombogenicity
• This has been attributed to a mismatch of mechanical properties and
poor endothelialization
• Tissue engineering represents a promising approach to overcome the

shortcomings of currently available vessel grafts

• Tissue engineered vascular graft (TEVG)
• The TEVG would have to be biocompatible such
that it is nonimmunogenic and nonthrombogenic,
properties that are associated with a fully
developed and functional endothelium
• It would have to have similar mechanical
properties as the native vessel, able to withstand
hemodynamic forces immediately upon
implantation and over the long term
• While also not being susceptible to permanent
deformation, that could lead to an aneurysm
• A biologically active TEVG would also be
beneficial to induce remodeling, allowing
for the growth of an endothelium and the
infiltration of vascular smooth muscle
cells to successfully integrate into the
vasculature
• Finally, from a surgical perspective, the
TEVG needs to compliant enough for
handling and strong enough to hold
sutures for implantation
• Design:

• Various techniques have been employed to create TEVGs such
as casting, self-assembly, 3D bioprinting, and decellularized
scaffolds
• Many of these methods fail to mimic the structure of natural
extracellular matrix (ECM) which a key component to the
regeneration and remodeling of tissue
• Electrospinning:
• Electrospinning a polymer solution results in the production
nanofibers that recreates the natural structure of ECM
• Fiber structure, diameter, topology, and mechanical properties
are readily tunable by adjusting various operating parameters
Electrospinning
• Electrospinning was first discovered in
1887 and mathematically described in the
1960s in a series of papers by Geoffrey
Taylor
• Electrospinning involves a high voltage DC
power supply, syringe pump, spinneret
(typically a blunt hypodermic needle), and
a collector
• For vascular tissue engineering a rotating
mandrel is generally used as the collector
to produce a tubular shaped graft

Electrospinning

Electrospinning
• During electrospinning a droplet of liquid is emitted
from the spinneret
• This droplet becomes electrically charged and
deforms due to electrostatic repulsion forming a
Taylor cone
• A jet is emitted from the apex of the Taylor cone,
forming a long, thin strand of liquid
• This strand initially travels straight as it is stretched
and elongated within the electric field
• At various points the electrostatic repulsion within
the strand is great enough for bending instability to
occur causing the strand to further stretch into a
series of coils
Electrospinning
• Higher voltage results in smaller fiber
diameters although in some cases a higher
voltage may cause more liquid to be emitted
and increase fiber diameter
• Higher flow rates usually result in increased
fiber diameter
• Increasing the distance between the
spinneret and collector generally results in
thinner fibers up to a certain distance
beyond which the fiber will have solidified
and no further extension will occur

Polylactic acid (PLA)
• Polylactic acid (PLA) is a renewable, bioresorbable, low-cost manufacturable
synthetic material
• It has been wildly popular in different fields such as implantable biomedical
devices, food packaging, drug delivery system and many more
• PLA degrades naturally via hydrolysis, and its by-product lactic acid is nontoxic
to the human body
• The degradation rate of PLA is slow due to its hydrophobic methyl group (low
cell attachment)
• PLA is very brittle, which limits its ability in withstanding high stress settings
because it cannot undergo enough plastic deformation

• Shalumon et al. created a multilayered PLA tubular scaffold
• Initially, aligned PLA fibers were collected on a rotating mandrel after which
the aligned mat was wrapped around a tubular form and fused with a
concentrated PLA in chloroform solution
• Multilayered scaffolds were generated by wrapping additional PLA mats around
the form and fusing them
• The inner aligned PLA was selected to support ECs while the more porous
PLA/PCL layer was selected to support the growth and infiltration of VSMCs
• The bilayer graft composed of one inner PLA and one outer PLA/PCL layer had
good mechanical stability and the multilayered grafts with additional PLA layers
showed enhanced mechanical properties
Schematic representation of the fabrication of tubular
scaffolds (a) electrospinning of PLA onto rotating
mandrel (b) aligned PLA fibers on the mandrel (c)
aligned fibrous sheet removed from mandrel (d) fusing
of Bi-layer tubular scaffold from aligned PLA fibrous
mat (d1) electrospinning of nonwoven PCL/PLA
fibers on top of Bi-layer tubular scaffold (e) fusing of
multi layer tubular scaffold from aligned PLA fibrous
mat (e1) electrospinning of nonwoven PCL/PLA fibers
on top of multi layer tubular scaffold.

Represents the electron microscopy images, clearly showing the
aligned and random morphologies of fibers inside and outside the
tubular scaffolds. SEM images of (a) bi layer and (d) multi layer
tubular scaffolds are shown in the left panel of the figure and higher
magnification images of aligned PLA inner layers (b and c) and
PCL/PLA nonwoven outer layers (e and f) are shown in the right
panel of the figures.

Polyglycolic acid (PGA)
• Among many biodegradable biomaterials, polyglycolic acid (PGA) is considered
the first synthetic biodegradable polymer used for biomedical applications
• Although PGA is much like PLA in terms of structure and degradation pathway,
it lacks a methyl group thus making it hydrophilic and degrade in the presence
of water
• PGA possesses great mechanical strength due to its high crystallinity, and
excellent controlled degradation while maintaining its shape in organic solvents
due to its low solubility
• Rapid degradation and a toxic by-product are two major limitations of PGA

Poly(lactic-co-glycolic) acid
• Poly(lactic-co-glycolic) acid (PLGA), the co-polymer of PLA and PGA, has been
extensively investigated since the 1970s to better tailor the properties of PLA
and PGA
• The degradation rate of PLGA is faster than either of its co-polymers due to
morphological changes that disrupt the more crystalline structure of its pure
co-polymers and can be adjusted by varying the lactide:glycolide ratio
• The 50:50 co-polymer has been shown to be relatively unstable, degrading in
1–2 months
• Greater resistance to degradation can be found at higher co-polymer ratios at
either end of the spectrum, with the 75:25 co-polymer degrading in 4–5
months and increasing to 5–6 months for the 85:15 blend
Poly(lactic-co-glycolic) acid
• Wang et al. developed a multilayered tubular PLGA graft by rolling an
electrospun PLGA sheet coated with fibrin glue around a 2 mm diameter
mandrel
• Cells could be easily

encapsulated within
the scaffold by
seeding cells on the
PLGA sheet prior to
rolling the graft

Polycaprolactone (PCL)
• PCL has many versatile properties including excellent biocompatibility,
biodegradability, and nontoxicity
• PCL has a semi-crystalline structure
• It has a low melting point at about 60 ◦C and a glass transition temperature at
about −60 ◦C
• Therefore, under the physiological conditions, it maintains great mechanical
properties such as high toughness, strength, and elasticity
• PCL alone as a tissue engineering scaffold exhibits slow degradation rate, low
cell attachment, and poorly matched mechanical properties

Poly(L-lactide-co-ε-caprolactone)
• Poly(L-lactide-co-ε-caprolactone) (PLCL), a co-polymer of PLA and PCL, has
been investigated for its potential use in constructing TEVGs
• PLCL, an amorphous polymer with the PCL disrupting the more crystalline
nature of PLA, exhibits better mechanical properties than PLA
• Increasing the caprolactone content increases the elastomeric properties of the
polymer with a 70:30 PLCL co-polymer having a Young's modulus of 12 MPa,
significantly softer than PLA and comparable to the stiffness of native vessels
• However, PLCL copolymers do have relatively low glass transition temperatures,
making several blends metastable at temperatures ranging from room
temperature to body temperature and may undergo drastic change in
mechanical properties
• Mun et al. [60] developed an electrospun vascular graft using 50:50 PLCL. 2D mats of
electrospun PLCL were seeded on both sides with New Zealand white rabbit VSMCs and
then rolled on 4 mm diameter mandrels using fibrin to glue the layers together
• After 2 weeks, dynamically cultured grafts had
significantly higher burst pressure and collagen
content compared to the statically cultured
grafts indicating significant remodeling of the
graft was occurring
• A confluent layer of VSMCs was observed on the
outer surface and were present on all layers of
the dynamically cultured graft compared to just a
few on the statically cultured one


Multilayered electrospun vascular grafts
• Multilayered electrospun vascular grafts are a promising choice as can
replicate the natural multilayered structure of native vessels
• A multilayered design allows for the tailoring of various properties through the
choice of polymer, electrospinning parameters, or addition of bioactive
molecules to best suit the needs of the cell types found in each layer of the
graft
• Han et al. made a three-layered vascular graft designed for spatio-temporal

delivery of VEGF and platelet derived growth factor (PDGF)

An inner layer The outer layer of
composed of 75:25 PCL improved the
PLCL was chosen for mechanical strength of
the quick release of the graft, while also
VEGF to promote delaying the release of
rapid PDGF
endothelialization
A middle layer of
75:25 PLGA was
selected for a slow
release of PDGF for
the formation of a
VSMC layer

(a), middle-layer PLGA/Gelatin (b) and

outer-layer PCL/Gelatin membranes (c),
and TEM images of the PELCL/
Gelatin (d) and PLGA/Gelatin (e) fibers

Appearance (a) and cross section morphology (b,c) of the prepared vascular scaffold. V and P represented the inner and
middle layer contained VEGF and PDGF, respectively

• The mechanical properties of the three-layer graft
were considerably higher than that of the porcine
coronary artery used for comparison (tensile
strength 5.2 ± 0.7 MPa vs 2.6 MPa, Young's modulus
35.9 ± 7.7 MPa vs 1 MPa, elongation at break 146.7
± 0.6% vs 100%, respectively)
• Though not seen in their 8-week in vivo study in New
Zealand white rabbits, the compliance mismatch
could lead to intimal hyperplasia and restenosis
• Wu et al. fabricated a three-layer graft with the inner

two layers tailored to ECs and VSMCs

PLGA/SF nanofibers were
collected on a rotating funnel
used to twist the fiber into
yarn
and collected on a rod
The yarn was wrapped

around the aligned PLCL
layer

• The inner layer was composed of circumferentially aligned PLCL/collagen
nanofibers
• HUVECs proliferated well on the aligned nanofibers but no difference was
observed between the aligned and random fibers
• The HUVECs were observed to orientate themselves along the nanofibers
which may help promote the growth of an organized endothelium and
functioning Ecs
• The middle layer was composed of a PLGA/SF electrospun yarn

SEM images showing the morphology of (A) the cross section of the tri-
layer tubular graft, (B) the aligned
PLCL/COL fibers in the inner layer, (C) the PLGA/SF yarns
in the middle layer, and (D) the random PLCL/COL fibers in
the outer layer

• VSMCs proliferated significantly better on the PLGA/SF yarns compared to
PLGA/SF fibers due to its more porous three-dimensional structure and were
found to orientate themselves along the yarn
• Finally, an outer layer of random PLCL/collagen nanofibers was collected to
keep the whole structure intact
• In order to mimic the structure of a native artery, Wu et al. created a three-

layered graft from PLGA and crosslinked gelatin

• The inner and outer layers were comprised of coaxial electrospun PLGA/gelatin,
with a PLGA core for strength and gelatin shell
• HUVECs proliferated well on the PLGA/gelatin fibers, nearly covering the
surface after 5 days
• The middle had electrospun PLGA and served to enhance the mechanical
strength of the graft. The radial tensile strength of the graft was comparable to
the human aorta but much higher than the coronary artery (5.13 MPa vs 5.49
vs 1.43 MPa, respectively)
• The axial tensile strength of the graft was also higher than the aorta or
coronary artery (3.14 MPa vs 1.47 MPa vs 1.30 MPa, respectively)

Fiber morphology and alignment
• Electrospun fiber morphology and mechanics play an important role in cell
growth and proliferation
• Bead-free fibers without conglutination are desired in electrospinning
applications
• Fibroblasts have been shown to exhibit increased cell area and aspect ratio in
response to larger diameter fibers while no effect on proliferation was
observed
• Fiber diameter has no effect on VSMC survival over the course of 10 days,
slower proliferation was found with increasing diameter


(A) 0.5 µm (diameter); (B) 0.7 µm (diameter); (C) 1 µm (diameter); (D) 2 µm (diameter); (E) 2.5 µm (diameter); (F) 5 µm
(diameter); (G) 7 µm (diameter); (H) 10 µm (diameter)
DAPI staining results to determine

infiltration
depth. (White dotted line: scaffold
margin. * (white): cell seeding site)

• Infiltration of VSMCs into the PCL scaffold was noted to improve with
increasing fiber diameter, associated with the increased pore size found with
the larger diameter fibers
• The number of activated macrophages was also observed to increase on
scaffolds with larger fiber diameters, which is important for the remodeling of
the scaffold
• Fiber morphology can also alter the mechanic of electrospun fibers

• Within tissues, collagen fibers have a crimped morphology that contributes to
their elasticity

• Chao et al. recreated this crimped morphology in PLA nanofibers
• It was found that by heating PLA at 85◦C for 15 min, PLA fibers developed a
stable crimped morphology hypothesized to be due to changes in its
crystallinity, going from an amorphous to semi-crystalline state
• A similar result was achieved by soaking PLA nanofibers in 95% ethanol at 37◦C
for two days and this method could also be applied to PCL and PLGA nanofibers
to achieve crimped fibers



• The stress-strain curve showed a J-shaped response that is characteristic of
native vessels
• Fibroblasts aligned themselves with the crimped fibers taking on an undulating
morphology and had significantly increased collagen type 1 expression

• Nanofiber alignment has also been identified as a method to direct the
behavior of cells on electrospun scaffolds
• ECs are aligned longitudinally with the direction of blood flow
• While VSMCs are aligned circumferentially and thus able to contract the vessel
• When electrospinning tubular scaffolds, circumferentially aligned fibers can be

obtained by rotating the mandrel at a sufficient speed
• e.g. He et al. found that a 4 mm diameter mandrel rotated at 1000 rpm
resulted in aligned nanofibers

• Longitudinally aligned fibers can be obtained by using two parallel plates as a
collector. The deposited fibers will bridge the gap between the plates,
orientated perpendicular to the plates and aligned parallel to each other
• The fibers can then be collected by rolling a mandrel over then such that they
are orientated along the length of the mandrel

(a) Macroscopic appearance and (b) cross-
sectional SEM image of scaffolds after PEG was
removed. (c) High-magnification SEM image of
part of panel (b). (d) SEM image of the surface of
a densely packed longitudinally aligned internal
layer. (e) SEM image of the surface of
conventional monolayer PCL vascular grafts
(Type A, control). (f) High-magnification SEM
image of part of panel (e). (g) SEM image of the
surface of an external layer of Type-B scaffolds
with PCL and PEG (relatively thick fibers). (h)
SEM image of the surface of an external layer of
Type-B scaffolds after PEG was dissolved. (i)
High-magnification SEM image of part of panel
(h)

PCL fibers are stained in green, and PEG fibers are stained in red: (a) surface of Type-A (monolayer)
scaffolds; (b) internal layer of Type-B (bilayer) scaffolds; (c) PEG fibers inside the external layer of
Type-B (bilayer) scaffolds; (d) external layer of Type-B (bilayer) scaffolds. (e) Tensile strength as a
function of tensile strain for Type-A (monolayer, control) and Type-B (bilayer) electrospun tubular
grafts
• One method for altering the mechanical behavior of an electrospun is to alter
the shape of the mandrel
• Of particular interest is grafts with a wavy cross section that allow
circumferential expansion of the graft to better replicate the nonlinear
mechanical behavior of native vessels
• Yu et al. and Mi et al. both used a round mandrel surrounded by thin (0.8 mm
diameter) rods
• At sufficient rotational speed the thin rods flexed in a manner analogous to a
jump rope
• As nanofibers were collected on the satellite rods, they would become
constrained and shrink back against the mandrel resulting in a wavy structure


• Both used elastic PU due to its ability to mimic
the behavior of elastin
• In order to mimic the stiffer collagen Yu et al.
used SF and Mi et al. used PCL
• In both cases an intermediate blend of the
elastic and stiff polymers best held the wavy
shape
• With pure stiff polymer unable to contract the
satellite rods and pure elastic polymer
contracting to a round shape after removal of
the mandrel
Increasing porosity
• One major limitation of electrospinning in tissue engineering is the inherently
small pore size and porosity as electrospun scaffolds are essentially 2D mats
• The lack of porosity hinders cellular infiltration of the grafts, which limits
remodeling and integration of the graft into native tissue, as well as the
exchange of nutrients and waste products
• Porosity can be changed by adjusting the electrospinning parameters including
voltage, flow rate, distance to collector, and rotational speed
• Thicker fibers are also associated with larger pore size and increased porosity

Increasing porosity
• Wang et al. obtained thicker PCL fibers by increasing PCL concentration while
using a higher flow rate and reducing the voltage and collector distance
• The thick fibers were 5.59 ± 0.67 μm in diameter compared to 0.69 ± 0.54 μm
for the thinner fibers
• This resulted in a nearly 8-fold increase in pore size and marked increase in
porosity
• The increased porosity influenced macrophage polarization and allowed for the
remodeling of the grafts and the formation of a tunica media after 100 days in
vivo

Increasing porosity
SEM images of electrospun PCL mats with thicker fibers (A)
and thinner fibers (B). Cross-sections of tubular thickerfiber
grafts (DeF) and thinner-fiber grafts (GeI). The representative
tensile strain-stress curve was shown in C.
SEM images show the attachment on the thinner-fiber PCL

mat (A) and the thicker-fiber PCL mat (B). Fluorescent
images show the infiltration and distribution of the cells
within the thinner-fiber mat (C) and the thicker-fiber mat
(D).

Increasing porosity
SEM images show the endothelialization process of the thicker-fiber PCL
grafts over time in rat aorta implantation. The explants were
longitudinally cut into two pieces. A, graft of day 14; E, graft of day 28;
F, graft of day 100. B, C and D are three selected areas in A with higher
magnification.
Histological analysis and deposition of extracellular matrix in the

regenerated grafts at day 100 in comparison with native aorta. Cross-
sectional images of the regenerated grafts (A,C and E) and native artery
(B, D and F) were immunochemically stained to identify the endothelial
cells, smooth muscle cells and elastin. H&E staining show the structure of
the explanted grafts (G) and native aorta (H). Masson’s trichrome staining
show the presence of collagen (green) in the explanted grafts (I) and
native aorta (J). Verhoeff’s staining show the presence of elastin (black)
in the explanted grafts (K) and native aorta (L). Scale bar: 100 mm.

Increasing porosity

Increasing porosity

Increasing porosity
- SEM micrographs of scaffold fiber morphology. (A) Fibers deposited
on the solid mandrel. ((B)–(F)) Fibers deposited on perforated mandrel
with an applied air pressure of 0, 50, 100, 200, 300 kPa, respectively.
(G) Fiber diameter results on the specified scaffolds produced. (H)
Interfiber distance results for different scaffolds. ((A)–(F) 2000×
magnifications, scale bar = 10 m. (B) The inserted white arrow area
indicates that fibers are crossed or knotted).
- Burst pressure (A) and compliance (B) results of the P(LLA-CL)/SF
scaffolds with different processing conditions and mandrels. * indicates
a significant difference from the solid grafts (p < 0.05), # indicates a
significant difference from solid and air impedance grafts (0, 50, 300
kPa) (p < 0.05).

Increasing porosity
• Using a novel electrospinning technique, Yin et al. used a porous mandrel through
which pressurized air was pumped
• The pressurized air impeded the deposition of PCLC/SF nanofibers resulting in
larger pore sizes
• Interfiber distance was largest at 50 kPa and decreased with higher pressures
• Another common approach to increase scaffold porosity is to utilize sacrificial

fibers
• With this technique, a soluble polymer such as noncrosslinked gelatin, polyvinyl
alcohol (PVA), polyethylene glycol (PEG), or PEO is co-electrospun with a
nonsoluble polymer
Increasing porosity
• Composite grafts utilizing electrospinning and another method of graft formation
have also been used to generate porous grafts
• In these grafts the electrospun layer typically provides increased mechanical
support that the porous polymer structure is unable to provide
• The simplest method to produce a porous scaffold is freeze drying a hydrogel
which leaves behind a highly porous scaffold
• Norouzi and Shamloo co-electrospun a PCL/gelatin inner layer around which a

gelatin hydrogel was cast
• After freeze drying and crosslinking with glutaraldehyde a porous outer layer was
generated
Increasing porosity

Increasing porosity
• The graft displayed good mechanical properties due to the PCL nanofiber
• VSMC proliferation was supported by the outer porous layer while inner
electrospun PCL/gelatin layer successfully allowed adhesion and proliferation of
VSMCs

Conclusions
• In summary, electrospinning shows great potential to develop a successful
TEVG that meets all the needs of a synthetic graft, with number of approaches
showing great promise
• However, there is a clear need for more research to advance the field and
develop a TEVG with similar performance to that of the gold standard
autologous vessels

Content
• References
• https://doi.org/10.1016/j.msec.2021.112373
• https://doi.org/10.1016/j.smaim.2021.07.006
• Nerve tissue Engineering

• Electrospinning
• Materials for electrospun nanofibrous scaffolds
• Modification of functionalized electrospun nanofibrous scaffolds
• Mechanical properties
• Bioelectricity
• Conductive macromolecules
• Conclusions
Nerve tissue engineering
• The nervous system regulates many physiological functions,
including breathing, muscle contraction, and sensory
• However, severe nerve injury is usually difficult to recover from
• Nerve injury includes central nerve injury (CNS) and peripheral
nerve injury (PNI)
• The CNS mainly refers to the nerve injury of the brain and spinal
cord, whereas the nerve injury outside the central nervous system
is called PNI
• Degenerative nerve diseases and injuries of the CNS may result in

the breakdown of neural circuits and a loss of neuronal function
• 3% of injuries are accompanied by PNI, and there are more than
five million new cases of PNI each year
Nerve tissue
engineering
• In the repair of PNI, several strategies have
been investigated to guide nerve fiber
regeneration to the distal nerve, thereby
improving axonal regeneration
• Conventionally, autogenous nerve
transplantation is most commonly used for
nerve repair
• However, it suffers from secondary injury
and donor nerve deficiency
• Therefore, tissue engineering scaffolds
have been studied as alternative methods
for guiding nerve regeneration

• Various methods for preparing nanofibers have been reported, such as
template synthesis, self-assembly, and phase separation
• As one of the most widely-used techniques to fabricate tissue engineering
scaffolds, electrospinning has attracted much attention because of its
advantages compared with other methods
• Template synthesis technology can well control the fiber diameter, while it is a
time-consuming process and can not produce continuous nanofibers
• Self-assembly technology makes it possible to make successive and uniformly-
shaped nanofibers, but its main drawback lies in the uncontrolled nanofiber
size
• Phase separation technology widely used in the preparation of nanofibers is simple
and inexpensive, and it enables successive nanofibers one after another and mass
production
• However, it has some major limitations, such as time-consuming, lack of structural
stability, difficulty in maintaining porosity, and therefore is not suitable for all polymers
• Electrospinning possesses many virtues including easy operation, governable scaffold

structure and fiber diameter, and is suitable for all sorts of materials
• Scaffolds own a high surface area to volume ratio for cell adhesion, porous structure
for cell infiltration, and adjustable mechanical properties to guide optimal
mechanoresponses of residing cells

Electrospinning
• First, polymer solution is entered through a thin needle
and then a suspended conical droplet is formed
• The surface tension of the droplet is balanced with the
applied electric field between the spinneret and collector
• Subsequently, a tiny jet is ejected from surface of the
droplet when the electrostatic field is strong enough to
overcome surface tension of the liquid
• As the solvent evaporates and the nanofibers move
towards the collecting plate, a thin film eventually forms
on the collecting plate
• Thus, the polymer solution in the syringe is converted
into fibers

• Electrospun scaffolds still cannot optimize the efficient healing of large-scale
nerve defects, mainly due to the following challenges: disordered cell
migration, absence of electrical signal stimulation, …
• Electrospinning fibers with controlled alignment were especially fit for neural
tissue engineering owing to their spatial guidance of neurite growth and axon
elongation
• Aligned electrospun nanofibers were found to promote the migration of
Schwann cells (SCs) for the regeneration of axon growth cones
• Conductive materials were often used to transmit electrical signals and
stimulate neuron growth

Materials for electrospun nanofibrous scaffolds
• Scaffolds: The high specific surface area enables the scaffold to carry and
release a variety of biochemical substances such as drugs, proteins, and nucleic
acids, and at the same time increases the contact area between cells and fibers
so that cells fully absorb these substances, thereby regulating cell behavior
• Nanofibrous scaffolds are porous, which is permeable to nutrient and O2
penetration, cell uptake, and metabolite excretion
• Materials: Natural materials that simulate ECM composition generally have
good biocompatibility and biodegradability
• Synthetic materials are engineered for better mechanical strength and key
material properties can be fine-tuned to deliver reproducible results

Collagen
• Collagen (COL) is the most abundant component in the ECM
• Due to the excellent biocompatibility of COL, it is commonly used in the
fabrication of tissue engineering scaffolds
• Guzman et al. made nonwoven nanofiber mats with 100-nm-thick irregular and
relatively discontinuous fiber for nerve repair
• Culture of embryonic chick dorsal root ganglia (DRG) explants demonstrated
that the fabricated nanofiber layer supported explant attachment, and
promoted the elongation of neurites and migration of satellite SCs in a similar
fashion, compared with electrospun COL-I fibers

Collagen
Nanofibers were finally observed when the
solute protein was increased from 0.5 to 2%

Chitosan
• Chitosan (CS) is a kind of polysaccharide biomaterial obtained by partial deacetylation of chitin
• CS shares similar properties with polysaccharide and glycosaminoglycan components of the
ECM, which promotes fetal mouse cerebral cortex cell adhesion, migration, and ultimately tissue
incorporation
• Moreover, since its degradation product, chitooligosaccharide, can promote nerve repair, it is
been widely used in nerve tissue engineering
• A bilayered CS tube composed of an outer layer of CS membrane and an inner layer of

electrospun CS nonwoven nano/microfiber mesh was used for nerve repair
• The CS membrane in the outer layer improved the mechanical properties of the tube
• The nanofibers of inner layer resembled the native ECM due to their porous tubular structure
and had a high surface area to volume ratio

Chitosan
(A) SEM micrographs of the bilayered chitosan tube. F –
chitosan film, M – electrospun nano/microfiber mesh. (B)
Enlargement of the electrospun nano/microfiber mesh.
The nano/microfiber structure comprises randomly
oriented fibers. Three-dimensional pores formed among
fibers are interconnected and distributed throughout the
structure.
The nano/microfibrous structure was inserted into the

chitosan film tube and immersed in distilled water at room
temperature for 30 min. The nano/microfibrous structure in the
inner layer swelled up to adhere to the outer film tube.

Polycaprolactone
• Electrospun PCL nanofibers with a microstructure similar to the peripheral
nerve ECM have been widely used in the fabrication of nerve guide conduits
(NGCs)
• Electrospun PCL fibers also support embryonic stem cells differentiation into
the neural lineage
• For neuron tissue engineering, aligned PCL fibers were produced to provide
topographical cued to promote axon elongation and guide axon growth better
than random fibers

Polycaprolactone
(A, E) random PCL scaffold; and (B, F)
highly aligned PCL scaffold; (C) and (D) are
orientation analysis of random and aligned
electrospun fibers with OrientationJ plug-in
for imageJ; and SEM images of PCL
nanocomposite scaffold with core-shell
PLGA
nanospheres at (G) low magnification and
(H) at high magnification.

Polycaprolactone
(A, E) random PCL scaffold; and (B, F)
highly aligned PCL scaffold; (C) and (D) are
orientation analysis of random and aligned
electrospun fibers with OrientationJ plug-in
for imageJ; and SEM images of PCL
nanocomposite scaffold with core-shell
PLGA
nanospheres at (G) low magnification and
(H) at high magnification.

Conducting polymers
• Polymers with loose electrons in their skeletons are often called CPs
• A neutral polymer chain is oxidized or reduced to a positive or negative charge,
resulting in a highly conductive state
• It promotes the adhesion, proliferation, infiltration,
directional migration of nerve-derived cells, and the
outward growth of nerve processes
PLGA-doped PHT nanofibers

Conducting polymers

Conducting polymers
• The aligned PLGA–PHT blend scaffold exhibited one order increase in the
conductivity from its respective random counterpart
• The cell adhesion and proliferation studies demonstrated the potential of
oriented scaffold for glial cell adhesion and proliferation
• Results have confirmed that longitudinally aligned 3D scaffolds have better
elastic property, good electrical property, slow degradation, and comparable
porosity with respective random 2D scaffolds and seem to be potential
candidates as scaffolds for neural regeneration

Modification of functionalized electrospun nanofibrous
scaffolds
• According to the topographic structures, electrospun fibers can be classified as
nonwoven structures, aligned structures, helical structures, crimping
structures, micropatterned structures, rope structures, and tube structures
• Aligned topologies of ECM provide contact guidance for neurite growth and
axon elongation in vitro, thereby regulating cell orientation, migration,
proliferation, and differentiation for nerve regeneration
• Cultured on parallel arrays of nanofibers, neurites tended to extend
preferentially along the long axis of the fibers
• At the boundary between the aligned and the random nanofibers, neurons
assumed both polarized and random morphology
scaffolds

scaffolds
• Alvarez et al. compared 18 mm PLA-based electrospun scaffolds with aligned
and random topography
• The Young's modulus of random nanofibers was isotropic, whereas the

aligned nanofibers showed almost four times higher anisotropic Young's
modulus
• On the random scaffold, neurons and glial cells assumed random orientation
whereas on the aligned scaffold, they were polarized, orientated in the
direction of the fibers and soon infiltrated into the scaffold

scaffolds
• Micropatterned fibers with controllable shape and orientation also provide
enhanced contact guidance for nerve cell alignment and neurite growth
• Grooves of the micropatterned fibers further facilitate the attachment and
growth of nerve cells
• The highly aligned micropatterned structure accelerates the directional growth
of new tissue and neurite exogenesis
• Wu et al. reported a simple method based on coaxial electrospinning to

fabricate directional microfibers with nanoscale grooves that promoted the
polarization and directional migration of neurites

scaffolds
The mechanism of this method depended on
the immiscibility of PCL and poly(vinyl
pyrrolidone) (PVP) in 2,2,2-trifluoroethanol
(TFE) to produce PVP/TFE bags on the PCL
jet surface.
With the efflux, the bag was stretched and
elongated, eventually forming nanoscale
grooves after the removal of PVP.

scaffolds
• In contrast to the smooth surface of the microfibrous
scaffold, the presence of nanoscale grooves greatly
enhanced the growth of neurites from PC12 cells and
the DRG body, as well as inducing the directional
migration of SCs
• By controlling the size of the groove, it could be
potentially optimized for controlled neurite extension
and accelerated wound closure
• It turned out that the micropatterned scaffold
significantly promoted axonal elongation and
elongation of PC12 cells in the controlled direction

scaffolds
• In the process of electrospinning, the fiber diameter will be reduced as the
voltage increases or the collection distance increases
• In general, the higher the speed or the right voltage, the better the fiber
alignment
• Various approaches can be used to improve the alignment of fibers during
electrospinning, including rotary receiving devices, auxiliary electrodes,
magnetic fields, and water baths
• The rotary collector has been shown to create aligned fibers due to the
stretching effect as the jet touches surface of the drum

scaffolds

scaffolds
• Matthews et al. demonstrated the influence of rotating speed of the drum on COL fiber
alignment during electrospinning
• Collected COL fibers were randomly oriented at speeds below 500 rpm
• However, as the rotational speed of the axle was increased to 4500 rpm, the COL fibers assumed
aligned orientation along the axis of rotation
• Copper wires wound around the insulated cylinder were used as electrodes to collect highly
aligned bundles of fibers at a speed below 2000 rpm
• The use of a negative high-voltage power supplied with alternated electrospinning receivers
forced positively charged fibers to line up on a rotating stainless-steel rod, thus forming an
orientated polymer conduit
• This method ensured the order of aligned fiber structure and also increased the tunability of
mechanical properties
Mechanical properties
• NGCs should have mechanical properties similar to those of peripheral nerves, so that
they can withstand the pressure of surrounding tissues and be able to flexibly maintain
continuity of the original lumen as the limbs are bent
• In addition to tensile and compression properties, NGCs should also have the same
rigidity and flexibility with internal and external healthy nerves
• The mechanical properties of electrospun nanofiber NGCs are primarily controlled by

factors such as material composition and production method
• Pure polyacrylamide hydrogel generally has high brittleness, which is not conducive to
its further application in tissue engineering
• The addition of sodium alginate with good softness and graphene oxide (GO) with high
strength can well regulate the tensile properties of the hydrogel
Mechanical properties
• These biomechanical properties have a significant impact on the behavior of
SCs and even can influence the recovery of peripheral nerve function
• Stiffness of the composite hydrogel affected biological functions of SCs,
including proliferation and differentiation
• Electrospun membrane collected on a static aluminum foil, the resultant

nanofibers are randomly distributed and present homogeneous mechanical
properties
• Oriented electrospun fibers always show significantly different mechanical
properties in two opposite directions

Bioelectricity
• In addition to the alignment of electrospun nanofibrous scaffolds, bioelectricity
is also important for NSCs migration, proliferation, and differentiation into
neurons
• Applying electrical stimulation to the biomaterial can change the local electric
field of ECM protein, which may change the adsorption of protein and then
affect the growth of neurites
• It has been shown that the external electric field can accelerate the growth and
modulate the direction of axons of neurons in vitro
• Yao et al. studied the effect of an electric field on neuronal migration
• Their work demonstrated that external electric fields induced directional
migration of rat hippocampal neurons
Bioelectricity
B: EF-exposed: neurons show

robust cathodal neuronal
migration in an EF (300
mV/mm) for 1 h.

Bioelectricity
• Applied electrical stimulation has also been shown to induce the neurogenic
differentiation of stem cells
• Neurogenic differentiation of mesenchymal stem cells (MSCs) can be induced
by chemical growth factors, ECM signaling, as well as electrical or mechanical
cues
• Self-powered electrical stimulation systems can enhance the neurogenic
differentiation of MSCs without biological/chemical cues
• Yan et al. showed enhanced neurogenic differentiation of NSCs in rats by

combining electrical stimulation and micropatterned conductive
electrospinning nanofiber as topographic guidance
Bioelectricity
simple electrospinning rotational electrospinning

process process

Bioelectricity

Bioelectricity
• Electrical stimulation could significantly promote the activity of nerve-derived cells
• Neurons were activated by electrical stimulation via membrane depolarization, which
ultimately accelerated controlled sensory and motor axon regeneration
• Compared with the cells without electrical stimulation, the cells in random electrospun
RE-PCAT10/NGF with electrical stimulation showed better cell adhesion
• The neurite length and the percentage of differentiated NSCs on electrically stimulated
RE-PCAT10/NGF nanofibers were significantly increased
• The results confirmed that there was a significantly greater number of cell
attachments on the micropatterned electrospun (ME)-PCAT10/NGF nanofibers with
electrical stimulation and aligned micropatterned structures, suggesting that
electrospinning networks with electrical stimulation synergetically promoted nerve
repair
Conductive macromolecules
• In addition to CPs, inorganic carbon nanotubes (CNTs) and their allotropes have
attracted great attention in neural tissue engineering due to their excellent mechanical
properties, thermal stability, electrical conductivity, biodegradability, and inherent
cellular cytocompatibility
• CNTs are cylindrical carbon nanostructures with extremely high aspect ratios
• Single-walled CNTs are typically about 1 nm in diameter, but multi-walled CNTs are
about 100 nm in diameter
• Multi-walled CNTs coated fibrous PLCL scaffolds enhanced neurite growth in DRG in
rats. Compared with the PLCL control group that was cultured in vitro for nine days,
the expression of focal adhesion kinases in PC12 cells was significantly increased in the
PLCL/CNTs group, resulting in an increase in neurite length

• Carbon-based materials, including graphene, have good conductivity to deliver
electrical stimulation to neuron cells as well as glial cells in surrounding nerves
• One reported preparation method was to conduct surface ammonolysis and
electrostatic adsorption on GO nanosheets
• Then, the “double copy template method” described was used to prepare PLCL
micrographic membranes
• The GO modified micropattern significantly promoted the collective migration
of SCs and the migration of SCs from their spheres

The micropattern was introduced
onto the PLCL membrane by
polydimethylsiloxane template hot pressing
method, and further adsorption was carried out
with GO nanosheets after
ammonolysis.

• A 10-mm sciatic nerve defect was generated in rats and bridged by NSCs made
of a 3/3 μm PLCL membrane (the ridges and grooves modified with GO
nanosheets were linear micropatterns of 3 μm, respectively), autologous grafts,
and other control NSCs
• Wound healing rates did not reach 10 % on all flat and undecorated
micropatterned PLCL surfaces
• Wound healing percentage was expressly improved to 50 % on the
micropatterned PLCL surfaces modified with GO nanosheets
• The healing rate of 3/3-GO membrane reached more than 75 %, which was
significantly higher than that of other surfaces

• SCs migrated a larger distance with GO nanosheets absorption, indicating that
the migration effect of stem cells from the sphere can be expressly enhanced
by surface modification of GO nanosheets
• Cell adhesion on the original PLCL membrane enhanced after GO modification
and was further improved on the micropatterned/GO surface
• Sezer et al. developed and studied an electrospun fiber mat containing zero-
valent iron nanoparticles, which improved electrical conductivity and
mechanical properties, and could be degraded at controlled doses




• A direct and generally applicable electrospinning/leaching method has been developed to prepare
the highly porous PLA/ CNT fibers with self-closing functionality. First, samples with different
PLA/PEO weight ratios were prepared
• The composition of 5:5 led to the formation of the largest pores with an average size of 260 ± 5 nm,
much larger than those of 7:3 (120 ± 9 nm) and 3:7 (90 ± 11 nm)
• Considering that the continuous porous structure with large pore dimensions could afford great
opportunity for the encapsulation of biomacromolecules, fibers fabricated from PLA/PEO weight
ratio of 5:5 were chosen for the subsequent experiments
• The effects of CNT concentrations at 0, 0.2, 0.4, and 0.8 mg mL−1 on fiber morphology, structure,
thermal/mechanical, degradation, cell viability, and biomacromolecule encapsulation/release were
further investigated
• It was found that, the fibers containing 0.4 mg mL−1 of CNTs exhibited a rapid self-closure of the
nanopore on the surface of the fibers, the highest encapsulation efficiency, as well as an extended-
release profile

Conclusions
• In the nerve tissue-engineering field, electrospun nanofibrous scaffolds have
the following advantages:
(1) The highly porous structure with high surface-to-volume ratio is suitable for the loading
and release of biologically active molecules (including proteins, drugs, and nucleic acids);
(2) By adjusting the electrospun device setups, it can produce fibers with diameters
ranging from micrometers to several nanometers. Such fibrous structure well mimics
natural ECM and thus guides the adhesion and migration of adherent cells;
(3) The versatility of electrospinning allows the use of various polymers or polymer
mixtures for co-electrospinning. The fabrication parameters can be finetuned to produce
scaffolds with optimal surface wettability, mechanical property, patterned structure, and
degradability to meet the requirements of different tissue engineering applications.

Conclusions
• The multifunctional nanofibrous scaffolds combining aligned topological
ultrastructure, electrical stimulation, bioactive molecules, and antioxidant
drugs will be able to improve the efficiency of nerve tissue repair.

(2,3) Electrospinning For Tissue Engineering

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(2,3) Electrospinning For Tissue Engineering

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Electrospun nanofiber scaffold

Regeneração de Tecidos 2022-2023

• Vascular tissue Engineering

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• Therefore, there is a need for an alternative to autologous

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• Tissue engineering represents a promising approach to overcome the

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• Cells could be easily

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• Han et al. made a three-layered vascular graft designed for spatio-temporal

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(a), middle-layer PLGA/Gelatin (b) and

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• Wu et al. fabricated a three-layer graft with the inner

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The yarn was wrapped

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• The middle layer was composed of a PLGA/SF electrospun yarn

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• In order to mimic the structure of a native artery, Wu et al. created a three-

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DAPI staining results to determine

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• Fiber morphology can also alter the mechanic of electrospun fibers

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• When electrospinning tubular scaffolds, circumferentially aligned fibers can be

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SEM images show the attachment on the thinner-fiber PCL

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Histological analysis and deposition of extracellular matrix in the

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