fibers

Review
Mechanical Properties and Morphological Alterations in
Fiber-Based Scaffolds Affecting Tissue Engineering Outcomes
James Dolgin 1,2 , Samerender Nagam Hanumantharao 3 , Stephen Farias 1,2 , Carl G. Simon, Jr. 4
and Smitha Rao 3,5,6,7, *

1 Materic LLC, 1100 Wicomico St. Ste. 323, Baltimore, MD 21230, USA; stephen.farias@dipolematerials.com (S.F.)
2 Dipole Materials Inc., 1100 Wicomico St. Ste. 323, Baltimore, MD 21230, USA
3 Department of Biomedical Engineering, Michigan Technological University, 1400 Townsend Dr.,
Houghton, MI 49931, USA; samerendernh@gmail.com
4 National Institute of Standards and Technology (NIST), 100 Bureau Drive, Gaithersburg, MD 20899, USA;
carl.simon@nist.gov
5 Great Lakes Research Center, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA
6 Health Research Institute, Michigan Technological University, 1400 Townsend Dr., Houghton, MI 49931, USA
7 Department of Biological Sciences, Michigan Technological University, 1400 Townsend Dr.,
Houghton, MI 49931, USA
* Correspondence: smithar@mtu.edu

Abstract: Electrospinning is a versatile tool used to produce highly customizable nonwoven nanofiber
mats of various fiber diameters, pore sizes, and alignment. It is possible to create electrospun mats
from synthetic polymers, biobased polymers, and combinations thereof. The post-processing of the
end products can occur in many ways, such as cross-linking, enzyme linking, and thermal curing, to
achieve enhanced chemical and physical properties. Such multi-factor tunability is very promising
in applications such as tissue engineering, 3D organs/organoids, and cell differentiation. While the
established methods involve the use of soluble small molecules, growth factors, stereolithography, and
micro-patterning, electrospinning involves an inexpensive, labor un-intensive, and highly scalable
approach to using environmental cues, to promote and guide cell proliferation, migration, and
Citation: Dolgin, J.; Hanumantharao, differentiation. By influencing cell morphology, mechanosensing, and intracellular communication,
S.N.; Farias, S.; Simon, C.G., Jr.; Rao, nanofibers can affect the fate of cells in a multitude of ways. Ultimately, nanofibers may have the
S. Mechanical Properties and potential to precisely form whole organs for tissue engineering, regenerative medicine, and cellular
Morphological Alterations in agriculture, as well as to create in vitro microenvironments. In this review, the focus will be on the
Fiber-Based Scaffolds Affecting mechanical and physical characteristics such as porosity, fiber diameter, crystallinity, mechanical
Tissue Engineering Outcomes. Fibers
strength, alignment, and topography of the nanofiber scaffolds, and the impact on cell proliferation,
2023, 11, 39. https://doi.org/
migration, and differentiation.
10.3390/fib11050039

Academic Editor: Natalia Keywords: tissue engineering; scaffolds; mechanotransduction; biophysical cues; electrospinning
Mihailescu

Received: 13 March 2023

Revised: 18 April 2023
Accepted: 25 April 2023
1. Introduction
Published: 29 April 2023 1.1. Nanofiber Scaffolds in Tissue Engineering
Scaffolds are an important part of the grand challenge of creating whole organs in
the field of tissue engineering and regenerative medicine. Mechanotransduction is a
powerful tool for initiating and maintaining intercellular events, including cascades of
Copyright: © 2023 by the authors.
protein signaling, which can lead to adhesion, propagation, and differentiation. Mechanical
Licensee MDPI, Basel, Switzerland.
cues have been shown to be 40 times faster than biochemical cues in inducing signaling
This article is an open access article
in some cases [1]. While micropatterning and other microfabrication techniques [2,3] can
distributed under the terms and
recapitulate the intricately structured microenvironment of native tissue, these fabrication
conditions of the Creative Commons
techniques lack the scalability and cost effectiveness of electrospinning. More recently,
Attribution (CC BY) license (https://
there has been interest in mass production. Furthermore, there is an ever-pressing need for
creativecommons.org/licenses/by/
4.0/).
realistic models for in vitro analysis and in vivo applications. In all these applications, there

Fibers 2023, 11, 39. https://doi.org/10.3390/fib11050039 https://www.mdpi.com/journal/fibers

Fibers 2023, 11, x FOR PEER REVIEW 2 of 43

Fibers 2023, 11, 39 2 of 40

techniques lack the scalability and cost effectiveness of electrospinning. More recently,
there has been interest in mass production. Furthermore, there is an ever-pressing need
for realistic models for in vitro analysis and in vivo applications. In all these applications,
is an inherent
there need need
is an inherent to understand the relationships
to understand that exist
the relationships between
that exist scaffold
between properties
scaffold prop-
and cells.
erties and The
cells.ability to tunetothese
The ability tune properties appears
these properties to be the
appears to key to establishing
be the 3D cell
key to establishing
cultures,
3D organ-on-chip,
cell cultures, organoids,
organ-on-chip, and in and
organoids, vitroinmodels. As an example,
vitro models. a scaffold
As an example, made
a scaffold
made from poly(lactic-co-glycolic acid) for primary human bone marrow stromal cell in
from poly(lactic-co-glycolic acid) for primary human bone marrow stromal cell is shown is
Figure 1.
shown in Poly(lactic-co-glycolic acid) nanofibers
Figure 1. Poly(lactic-co-glycolic obtained from
acid) nanofibers electrospinning
obtained were used
from electrospinning
as a substrate
were used as atosubstrate
culture primary human
to culture bonehuman
primary marrow stromal
bone marrowcells.stromal
As seencells.
in Figure 1, the
As seen in
nanofibers
Figure 1, thepromote the adhesion,
nanofibers promote theelongation,
adhesion,and infiltrationand
elongation, of the cells intoofthe
infiltration thenanofibers
cells into
by providing
the nanofibersaby 3Dproviding
environmenta 3Dfor the cells. for the cells.
environment

Figure 1. Primary human bone marrow stromal cells cultured 24 h in an electrospun fiber scaffold
Figure 1. Primary human bone marrow stromal cells cultured 24 h in an electrospun fiber scaffold
made of poly(lactic-co-glycolic acid), with a nominal fiber diameter of 2.7 µm. The 3D rendering
made
was of poly(lactic-co-glycolic
created acid),
from z-stacks captured with a nominal
by confocal fiber microscopy
fluorescence diameter ofusing
2.7 µm.
a cellThe 3D render-
channel (Ore-
ing was created from z-stacks captured by confocal fluorescence microscopy using a cell
gonGreen-Maleimide 488) and a scaffold channel (Flamma Fluor FKR648). The fibers were rendered channel
(OregonGreen-Maleimide
fluorescent by spiking fluor488)
intoand
the apolymer
scaffoldsolution
channelprior
(Flamma Fluor FKR648).
to electrospinning. The Credit:
Image fibers were
Carl
rendered
Simon, fluorescent by spiking fluor into the polymer solution prior to electrospinning. Image
NIST.
Credit: Carl Simon, NIST.
The extracellular matrix (ECM) is known to play a key role in tissue homeostasis and
The extracellular
function. For example,matrix (ECM) is regulation
the mechanical known to play
of ECM a key role inplays
stiffness tissueanhomeostasis and
important role
function. For example, the mechanical regulation of ECM stiffness plays an
in cell growth, differentiation, and migration [4–8]. Much work has focused on presentingimportant role
in cell growth, differentiation, and migration [4–8]. Much work has focused
the biochemical environment surrounding the extracellular matrix around different organ on presenting
the biochemical
systems, environment
or replicating surroundingfeatures
the biomechanical the extracellular
of the ECM matrix around different
as experienced by theorgan
cells
[9]. Natural and synthetic materials have been explored and different techniques toby
systems, or replicating the biomechanical features of the ECM as experienced the
estab-
cells [9]. Natural and synthetic materials have been explored and different
lish the ECM, such as patterned substrates, hydrogels, soft biopolymers, and nanofibers, techniques
to establish
have the ECM,
been reported such Nanofiber
[10–13]. as patterned substrates,
scaffolds hydrogels, soft
are characterized biopolymers,
according and
to the me-
nanofibers, have been reported [10–13]. Nanofiber scaffolds are characterized
chanical properties of the fibers, pore size, crystallinity, and fiber alignment and morphol- according
to theEach
ogy. mechanical properties
one of these of the
attributes, byfibers,
itself pore
or in size, crystallinity,
combination, and fiber
uniquely alignment
impacts and
cell adhe-
morphology. Each one of these attributes, by itself or in combination, uniquely impacts
sion, alignment, proliferation, and differentiation. For example, the elasticity and stiffness
cell adhesion, alignment, proliferation, and differentiation. For example, the elasticity
of the substrate have been reported to impact cell migration [7,8,14]. This is attributed to
and stiffness of the substrate have been reported to impact cell migration [7,8,14]. This
“Durotaxis”, or the response to mechanical stiffness, and refers to the preferential migra-
is attributed to “Durotaxis”, or the response to mechanical stiffness, and refers to the
tion observed in cells cultured on stiffer substrates, as compared to less rigid or softer
preferential migration observed in cells cultured on stiffer substrates, as compared to less
substrates.
rigid or softer substrates.
In addition, in nanofibers, other parameters can be manipulated that directly affect
cell adhesion and proliferation. Porosity is a measurement of the percent of open space in
Fibers 2023, 11, 39 3 of 40

electrospun mats, and pore size is the average diameter of these open spaces [15]. Pore size
can be controlled through various electrospinning parameters and post-processing steps,
though there is conflicting research regarding the influence of fiber size on pore size [16,17].
Pore sizes can range from nanometers to a few millimeters. While nanoscale pores have
been shown to impact the formation of collagen and ECM [18], cell density, migration, and
vascularization are impacted by macroscale pores [19–21]. Furthermore, cell migration is
reported to be linearly dependent on pore size [22]. Microenvironmental properties are
crucial to determine the fate of mesenchymal stem cells (MSCs). It has been shown that
simple alterations in the pore size of honeycomb scaffolds can change the fate of MSCs
of forming myogenic or osteogenic cells, based on osteopontin and MyoD1 staining [23].
Thus, the ability to control stiffness, porosity, and pore size has a direct impact on tissue
engineering and potential applications in regenerative medicine [24].
Nanofiber alignment not only significantly impacts the mechanical properties of
meshes [25], but also has a direct effect on actin alignment in MSCs [26]. Many studies
have shown that the fiber orientation of the substratum influences cell adhesion and
growth, [27–29], and modulates the elongated cellular patterns that are typical of native
tissues [30,31]. Nanofiber scaffolds with a topography such as native tissue have the
ability to direct the alignment of cells and subcellular structures, and successively allow
for the deposition of collagen along the electrospun nanofiber direction that will result in
an increase in the tensile properties of the new tissue [32]. Aligned nanofiber scaffolds
exhibited a higher modulus than the random nanofibers in a study reported by Pauly et al.
(2016) [9]. They also reported scaffolds with mechanical properties (modulus, yield stress,
and yield strain) within the range of native tissues. Similarly, in the muscles, the muscle
fibers are aligned along an axis and are formed by a single cell [33]. This uniaxial alignment
is the key in developing engineered muscle tissues. Such physiologically relevant models
could prove to be a suitable alternative to animal models for drug testing.
In addition to alignment, the fiber diameter directly impacts the mechanical strength of
the nanofiber scaffolds and affects the growth of various tissues. The mechanical properties
of the nanofiber scaffolds can therefore be tuned by either manipulating the alignment, fiber
morphology, or a combination of the features. Similarly, changes in crystallinity impact
mechanical properties of nanofibers, which dictates the outcomes for differentiation. The
swelling of the fibers and the degree of crystallinity, in turn, affects the behavior of cells
seeded on the scaffold. Thus, the properties can be tuned to influence outcomes such as
the differentiation of either muscle [34,35] or bone [36,37] tissues through the modulation
of mechanical properties. It has also been shown that myogenic differentiation typically
prefers increases in elasticity over the enhancement of crystallinity [38,39], while osteogenic
differentiation is greatly enhanced by crystallinity increases, despite drastic reductions in
elasticity and strength [40,41].
Three-dimensional nanofiber-based in vitro systems could be more affordable and
effective than animal models which are often costly, labor intensive, and potentially ethi-
cally controversial [42]. Hence, creating platforms that accurately recapitulate the tissue
environment for precise regenerative applications is of pressing importance.
Many techniques have been advanced for fabricating fiber scaffolds, including, but
not limited to, the following: electrospinning, forcespinning [43], meltspinning [44], pneu-
matospinning [45], blowspinning [46], melt-electrowriting [47], melt extrusion [48], wet
extrusion [49], fused deposition [50], liquid crystal deposition [51], electrochemical align-
ment [52], drawing [53], spinning, knitting [54], weaving [55], braiding [56], powder bed
fusion (laser sintering), vat photopolymerization (stereolithography) [57], binder jetting [58],
directed energy deposition [59], self-assembly (for example, fibrillogenesis) [60], and hybrid
approaches [15]. Although electrospun fibers are the primary focus, this article has bearing
on fibers made via any of these methods.
Although the characterization of fiber constructs is important, this topic is not covered
in the current review. The reader may refer to the FDA guidance on surgical meshes [61],
Fibers 2023, 11, 39 4 of 40

the ASTM standard guide for characterizing fiber-based constructs [15], and a recent
review [62] for further information of this specific topic.

1.2. Electrospinning
Electrospinning is a versatile technique invented in 1931 by Anton Formhals [63],
during which electrostatic forces are used to atomize polymer solutions for the fabrication
of nanofibers and scaffolds with complex geometries [64–68]. A typical electrospinning
setup consists of a power source, a syringe pump connected to a narrow needle via tubing,
and a collector (Figure 2). Electrospinning conventionally uses a direct current (direct
electrospinning), though alternating current electrospinning has been employed as well for
enhanced production [69]. In electrospinning, a voltage bias is applied across the needle and
the collector, on the surface of which nanostructures are obtained in the form of nanofibers
and nanoparticles, depending on the parameters used for electrospinning. A Taylor cone is
created when the electric field is greater than the surface tension of the polymer solution.
The charged polymer solution travels to the oppositely charged voltage bias (collector) in
the form of a jet. The jet elongates along a linear trajectory and then splits into multiple
streams caused by solvent evaporation and divergent electric field forces. The jet flow
becomes more turbulent as it approaches the collector, and this chaotic motion is affected by
solution properties, collector distance, and voltage. These parameters subsequently affect
the crystallinity and lattice structure of the polymeric nanostructures. Other electrospinning
parameters, such as collector properties and environmental factors, also dictate nanofiber
outcomes [70]. Environmental conditions, namely temperature, humidity and atmospheric
pressure, affect the formation of the Taylor cone and resultant nanostructures. This has been
used to make unique nanostructures [71–73]. Climate controlled setups are currently being
used to improve the reproductivity of the formation of nanostructures. The collector, which
can be a solid or liquid surface, can also lead to the formation of unique nanostructures,
depending on the type of collector used [74]. To better understand the electrospinning
processes and provide the means to theoretically determine the formation of the Taylor
cone, properties of the resultant nanostructure and ideal electrospinning conditions, we
direct the readers to these excellent references [71–81].
Electrospinning parameters and their outcomes have been studied extensively, and the
customization of these outcomes holds great promise for the field of creating diverse tissue
engineering nanomaterials. Factors such as electric field, negative electric bias, and alter-
nating voltage have been used to alter the morphology of nanostructures [69,82]. Various
collector configurations, including rotating drums, patterned collectors, liquid-containing
collectors, parallel electrodes, and magnetically charged electrodes have been used to diver-
sify nanostructure morphology [83–89]. Spinneret modifications, such as coaxial spinnerets,
multi-axle spinneret, melt, in-line polymer blending, and co-electrospinning configurations
have been used to create complex 3D patterns and multi-material structures [90–92]. The
properties of the polymeric solution, including solubility of the polymer in the solvent,
polymer flow rate, polymer viscosity, molecular weight of the polymer, boiling point of
the solvent, presence of additives, miscibility of the solution and, in certain cases, the mag-
netic properties of the solution [93,94], contribute to the structure, morphology, porosity
and other characteristics of the fibers produced. Sacrificial polymers can also be added
to electrospinning solutions, and later dissolve or thermally degrade to create porous or
mesoporous structures [95].
A large number of synthetic, natural and mixed polymer blends have been used for
electrospinning scaffolds [96–100]. The choice of material used is greatly dependent on the
intended application. It is also dependent upon the specific tissue type, although this has
not been fully explored. However, an important criterium is to ensure the biocompatibility
of the materials used to synthesize the scaffold. Biocompatibility is defined as “the ability
of a material to perform with an appropriate host response in a specific application.” The
review by Ghasemi-Mobarakeh et al., (2019), highlights some of the key concepts and
terminology in biomaterial design and biocompatibility [101]. The following sections will
Fibers 2023, 11, x FOR PEER REVIEW 5 of 43

Fibers 2023, 11, 39 ability of a material to perform with an appropriate host response in a specific applica- 5 of 40

tion.” The review by Ghasemi-Mobarakeh et al., (2019), highlights some of the key con-
cepts and terminology in biomaterial design and biocompatibility [101]. The following
sections
focus on will
somefocus onspecific
of the some of the specific
mechanical mechanical that
characteristics characteristics thatfor
are important areengineering
important
for engineering
tissues, tissues,
challenges challenges
associated with associated with it, research
it, and the current and the current
focus. research focus.

Figure
Figure 2.2. Electrospinning
Electrospinning setup. (a) An
setup. (a) electrospinning benchtop
An electrospinning modelmodel
benchtop (IME Tech
(IMEEC-Dig); (b) an
Tech EC-Dig);
illustration of the electrospinning
(b) an illustration process showing
of the electrospinning process the nozzlethe
showing tip nozzle
and rotating collector;
tip and rotating(c)collector;
an illus-
tration of the Taylor cone at the tip of the nozzle. Adapted with permission from [102].
(c) an illustration of the Taylor cone at the tip of the nozzle. Adapted with permission from [102].

2. Nanofiber
Nanofiber Structure and Properties Affecting Affecting Outcomes
Outcomes in in Engineered
Engineered Tissues
Tissues
The Morphology
2.1. The Morphology of Nanofibers Influences Tissue Growth
2.1.1. The
2.1.1. The Alignment
Alignment and and Pore
Pore Size
Size ofof Nanofiber
Nanofiber Scaffolds
Scaffolds Positively
Positively Influence
Influence Myogenic
Myogenic
Differentiation
Differentiation
The in vivo myogenic environment is comprised of both nanoscale collagen fibrils
The in vivo myogenic environment is comprised of both nanoscale collagen fibrils
and aligned microscale basal lamina tracks. Spatial cues, such as nanofiber align-
and aligned microscale basal lamina tracks. Spatial cues, such as nanofiber alignment and
ment and porosity, are significant in determining the fate of stem cells toward muscle.
porosity, are significant in determining the fate of stem cells toward muscle. The positive
The positive influence of alignment on muscle differentiation has been widely ex-
influence of alignment on muscle differentiation has been widely explored [3,70–77]. Mus-
plored [3,70–77]. Muscle fibers are aligned along an axis and are formed by a single
cle
cellfibers are aligned
[33]. Aligned along an
scaffolds axis the
mimic andmicroenvironment
are formed by a single cell [33].
of native Aligned
muscle tissue,scaffolds
as well
mimic the microenvironment
as increase bulk modulus andofimprove native muscle
surfacetissue,
wetting as[34].
well Nanofibers
as increase canbulkbemodulus
aligned
and improve
to create surfacedifferentiation
myogenic wetting [34]. Nanofibers can be to
outcomes similar aligned
that oftomicropatterned
create myogenicsubstrates.
differen-
tiation outcomes similar to that of micropatterned substrates.
Myofibers grown on aligned polycaprolactone (PCL) nanofibers, for example, show Myofibers grown on aligned
align-
polycaprolactone
ment analogous to that of micropatterned PDMS, with muscle fibers aligning within of
(PCL) nanofibers, for example, show alignment analogous to that 20mi-
◦ of
cropatterned PDMS, with muscle fibers aligning within 20° of nanofibers,
nanofibers, exactly as in micropatterned PDMS [3]. Such scaffolds create muscle fibers with exactly as in
micropatterned
a myosin-heavyPDMS [3]. Such molecule
chain (MyHC) scaffolds create
contentmuscle10-foldfibers with
greater a myosin-heavy
than randomly oriented chain
(MyHC) molecule content 10-fold greater than randomly oriented
fibers. Aligned nanofibers increase the fusion index and myotube length of randomly fibers. Aligned nano-
fibers
oriented increase the fusion index and
poly(hydroxybutyrate) PHB myotube
in H9c2length of randomly
rat myoblasts, andoriented poly(hydroxy-
C2C12 myoblasts form
butyrate)
600 µm myotubes in aligned PHB [103]. MyHC increased 8-fold in C2C12myotubes
PHB in H9c2 rat myoblasts, and C2C12 myoblasts form 600 µm cells differ-in
aligned PHB [103]. MyHC increased 8-fold in C2C12 cells differentiated
entiated on aligned 2:1 PCL, i.e., chitosan nanofibers compared to 2D films of the same on aligned 2:1
PCL, i.e., chitosan
composition [35]. nanofibers
Fujie et al. compared
(2015) showed to 2Dthatfilms of the same
myoblasts cancomposition
be spatially [35]. Fujie et
coordinated
al.
and(2015) showed on
differentiated that myoblasts
aligned can be [104].
nanoribbons spatially coordinated
Alignment can also and differentiated
create scaffolds with on
aligned
moduli,nanoribbons
more appropriate[104].for
Alignment
myogenesis can than
also create scaffolds with
their randomly moduli,
oriented more appro-
counterparts [35].
priate
Studies for myogenesis than
recapitulating their and
the nano randomly oriented
microscale counterparts
features of native [35].
muscleStudies
with recapitulat-
electrospin-
ing
ning and lyophilizing have directed myogenic differentiation and created denselyophiliz-
the nano and microscale features of native muscle with electrospinning and myotube
ing have [105].
bundles directed myogenic differentiation and created dense myotube bundles [105].
A high porosity
porositypositively
positivelyaffects
affectsmyogenic
myogeniccell cell infiltration,
infiltration, andand subsequent
subsequent differ-
differenti-
entiation.
ation. Recent Recent studies
studies have have
shownshown
the the
need need
for atforleast
at least
80%80% porosity,
porosity, as well
as well as align-
as alignment,
for directing
ment, muscle
for directing growth,
muscle especially
growth, in thick
especially scaffolds
in thick [106].[106].
scaffolds Porosity as high
Porosity as 97%
as high as
has facilitated
97% aligned
has facilitated myogenesis
aligned [107]. [107].
myogenesis Large-pore-size
Large-pore-sizescaffolds have treated
scaffolds volumetric
have treated vol-
muscle loss
umetric (VML)
muscle loss defects in vivo in
(VML) defects [30,31]. Kawano
vivo [30,31]. et al. (2013)
Kawano showed
et al. (2013) that pore
showed that sizes
pore
similar to cell size leads to the myospecific differentiation of hMSCs, and even further,
myodifferentiation can be achieved with a fiber size smaller than the cell size [29]. If pores
increase in size beyond 20 µm and the fiber diameter increases as well, cells may grow
 ther, myodifferentiation can be achieved with a fiber size smaller than the cell size [2
pores increase in size beyond 20 µm and the fiber diameter increases as well, cells
grow along fibers instead of in a 3D configuration, which could hinder growth and d
Fibers 2023, 11, 39 entiation [30]. The structure of pores can also positively influence myogenesis, 6 of 40 with
allel pores of 20 µm to 50 µm width, allowing for the parallel arrangement of myo
within pore structures. These parallel pore scaffolds achieved in vivo compatibility
along fibers instead
force generation of in a 3D configuration,
in regenerated muscle [31]. which could hinder
Porosity can aid growth and differenti-
in promoting self-align
ation [30]. The structure of pores can also positively influence myogenesis, with parallel
of thepores
muscle tissue, as well as aid in nutrient and waste transport in cell culture.
of 20 µm to 50 µm width, allowing for the parallel arrangement of myotubes within
pores pore
can structures.
be achieved in parallel
These electrospun mats through
pore scaffolds achieved in salt-leaching,
vivo compatibility laser-ablation
with force or sa
cial polymer addition. Porosity can also be altered through processing ofchanges
generation in regenerated muscle [31]. Porosity can aid in promoting self-alignment the vi
muscle tissue, as well as aid in nutrient and waste transport in cell
influence of the fiber diameter. Evidence of correlation between fiber diameter and mculture. Such pores can
be achieved in electrospun mats through salt-leaching, laser-ablation or sacrificial polymer
differentiation is conflicting,
addition. Porosity can also bewhich
altered could
throughbe due to the
processing unpredictable
changes ways
via the influence in which
of the
size isfiber
influenced
diameter.by the fiber
Evidence diameterbetween
of correlation [30,32,108,109].
fiber diameter Narayanan et al. (2020) [30] sh
and muscle differentiation
is conflicting, which could be due to the unpredictable ways
that on aligned matrices, larger fiber diameter led to greater alignment, in which pore size is influ- elonga
enced by the fiber diameter [30,32,108,109]. Narayanan et al. (2020) [30] showed that on
spreading, and differentiation of myoblasts (Figure 3). Further research controlling f
aligned matrices, larger fiber diameter led to greater alignment, elongation, spreading, and
ber diameter to relate
differentiation myogenesis
of myoblasts (Figure 3).and pore
Further size is
research needed.forThe
controlling fiberdevelopment
diameter to of
neered muscle
relate tissues
myogenesis and
and poreinsize
vitro muscleThe
is needed. analogs for the
development repair and
of engineered modeling
muscle tissues of mus
and in vitro muscle analogs for the repair and modeling of muscular
wounds, muscular dystrophy, and degenerative diseases could serve as suitable m wounds, muscular
dystrophy, and degenerative diseases could serve as suitable models for drug testing and
for drug testing and in vitro studies.
in vitro studies.

FigureFigure
3. Confocal images
3. Confocal imagesofofC2C12 cells
C2C12 cells showing
showing elongation
elongation 2 h post-seeding
2 h post-seeding on matrices on matrices
of (a) 335 of (
nm and nm(b)
and3013 nm
(b) 3013 nmaverage fiber
average fiber diameter,
diameter, demonstrating
demonstrating the
the ability of ability toofrespond
myoblasts myoblasts
to fiberto respo
fiber diameters (red,
diameters (red, actin
actin cytoskeleton;
cytoskeleton; blue,green,
blue, nuclei; nuclei; green,Scale
vinculin). vinculin). Scale
bar: 10 µm. bar: 10 µm. Quan
Quantification
of (c) alignment angle, (d) elongation ratio, and (e) cell spreading area
tion of (c) alignment angle, (d) elongation ratio, and (e) cell spreading area of seededof seeded myoblasts 2 hmyobla
post-seeding, showing enhanced myoblast alignment, elongation ratio, and cell spreading area on
post-seeding, showing enhanced myoblast alignment, elongation ratio, and cell spreading ar
matrices spun from 20% (Matrix 1), 30% (Matrix 2), and 40% (Matrix 3) PLGA w/v solutions. n ≥ 4
matrices spun from 20% (Matrix 1), 30% (Matrix 2), and 40% (Matrix 3) PLGA w/v solutions
images for each sample. * p < 0.05 compared to Matrix 1. Image reproduced with permission from
imagesNarayanan
for eachetsample. * p < 0.05 compared to Matrix 1. Image reproduced with permission
al. (2020) [30].
Narayanan et al. (2020) [30].

2.1.2. Fiber Morphology Directs Osteogenesis

It has been widely shown that bone cell maturation can be controlled by chang
Fibers 2023, 11, 39 7 of 40

2.1.2. Fiber Morphology Directs Osteogenesis

It has been widely shown that bone cell maturation can be controlled by changes to
nanofiber morphology [88–96,110–112]. Simon et al. (2011) and Kumar et al. (2011) showed
that nanofiber matrices are unique in their ability to direct osteogenesis of stem cells by
providing fibrous adhesion sites and forcing elongated and highly branched morpholo-
gies similar to that of mature osteocytes [113,114]. Such studies showed that nanofibers
alone induced morphological changes in a manner similar to osteoinduction cell culture
media. Kumar et al.’s study (2011) particularly showed that of the many scaffold types,
including salt-leached, gas-foamed, phase-separated, 3D-printed and electrospun, only
electrospun nanofiber scaffolds were able to differentiate the human bone marrow stromal
cells (hBMSCs) down an osteogenic lineage without osteogenic supplements, irrespective of
polymer chemistry. Fibers may drive osteogenic differentiation through the control of cell
morphology, whereby the 3D microenvironment of the fibers enables cells to attain a more
3D morphology [115]. Fiber morphology also affects the shape and function of intracellular
organelles, including the nucleus, mitochondria, and peroxisomes [116]. There is evidence
that changes in substrate topography that change the cell shape and nuclear shape lead
to changes in chromosome positioning, gene expression and protein synthesis [117,118].
Further, cell morphology may serve as an early indicator of an osteogenic response to fiber
scaffolds [119,120].
It has been shown that aligned nanofibers cause higher osteoinduction than random
microfibers (Figure 4) [121]. It has also been shown that a larger micron-scale fiber diame-
ters of both poly L-lactic acid (PLLA) and Poly D,L-lactic acid (PDLLA) lead to a higher
proliferation, aspect ratio and alkaline phosphatase (ALP) production of a clonal murine
cell line of immature osteoblasts derived from mice (MC3T3-E1) compared to those cultured
on nanofibers [36]. Aligned fibers have been shown to significantly upregulate calcium
deposition of bone marrow stromal cells and alkaline phosphatase of preosteoblasts [122].
Efforts to recapitulate the native 10–300 nm aligned collagen fibers, upon which osteoblasts
adhere and mineralize, were made by electrospinning aligned bio-based materials and
showed high osteoconductivity [123]. Chen et al. (2013) created 3D multilayered con-
structs in which five layers of aligned and randomly oriented nanofibers, pre-seeded with
pre-osteoblasts, were overlaid to mimic the structure of in vivo tissues [23]. These struc-
tures were successful in inducing osteogenesis compared to randomly oriented scaffolds.
Aligned nanofibers can also increase stiffness and toughness, which can be beneficial to
osteogenesis and will be explored in further sections [37]. However, the underlying mecha-
nisms responsible for aligned fiber-related osteoinduction remains unclear. Some studies
have shown no influence of aligned nanotopography on osteogenic stem cell fate [124–126],
while others have shown that aligned nanotopography upregulates osteogenic stem cell
fate [23,123,127].
Ultimately, mimicking hierarchical native conditions with morphological changes to
nanofibers positively influences osteogenesis. Whether this must be through aligned fibers,
random fibers, or a combination of both, requires further investigation. There have also
been conflicting studies on nanofiber diameter impact on osteogenesis. Difficulties arise in
linking nanofiber morphology properties to osteogenesis due to the many other variables
influencing osteogenesis. For example, intercellular force, cell shape, and cell density
heavily influence osteogenesis [108,128]. Other material properties, such as mechanical
properties and surface characteristics, also play a significant role in influencing osteogenesis,
which will be explored in the later sections.
Fibers 2023, 11, x FOR PEER REVIEW 8 of 4
Fibers 2023, 11, 39 8 of 40

Figure 4. ALP staining and semi-quantitative analyses at day 7 (A,B) and day 10 (C,D), showing a
Figure 4. ALP staining and semi-quantitative analyses at day 7 (A,B) and day 10 (C,D), showing
higher ALP activity using aligned nanofibers, compared to randomly oriented fibers and microfibers
higher ALP activity using aligned nanofibers, compared to randomly oriented fibers and microfi
(** p < 0.01, * p < 0.05); and ARS staining and semi-quantitative analysis at day 21 (E). Image
bers (** p < 0.01, * p < 0.05); and ARS staining and semi-quantitative analysis at day 21 (E). Imag
reproduced with permission from Xie et al. (2021) [121].
reproduced with permission from Xie et al. (2021) [121].
2.2. Surface Characteristics of Nanofibers Influence Tissue Growth
2.2.2.2.1.
Surface Characteristics
Smooth of Nanofibers
Nanotopography Influence
and Biological CuesTissue Growth
Influence Myogenesis
For the differentiation
2.2.1. Smooth Nanotopography of muscle
and cells, it has been
Biological Cuesshown that smooth
Influence features at the
Myogenesis
nanoscale level positively influence myogenesis [98,99]. It has also been shown that biolog-
For
ical theboth
cues, differentiation
through surface of muscle cells,and
modification it has been shown
the addition that smooth
of biopolymers, features at th
positively
nanoscale
influencelevel positively
myogenesis. In 2Dinfluence
films withmyogenesis
random nano- [98,99]. It has also been
and microtopography, lowshown
surface that bio
logical cues, positively
roughness both through surface
influences modification
myogenesis [129]. Hu and the
et al. addition
(2011) showed ofsensitivity
biopolymers,of pos
C2C12 mouse muscle cells to nanometer-scale surface roughness
tively influence myogenesis. In 2D films with random nano- and microtopography, low differences caused by
the addition of tropoelastin to silk. Shin et al. (2015) showed that micron-level decreases
surface roughness positively influences myogenesis [129]. Hu et al. (2011) showed sens
in surface roughness through the addition of graphene oxide and collagen to randomly
tivity of C2C12 mouse muscle cells to nanometer-scale surface roughness difference
oriented PLGA fibers caused increases in attachment, proliferation, and myogenic expres-
caused byC2C12
sion of the addition ofWhile
cells [130]. tropoelastin
randomlytooriented
silk. Shin et al. (2015)
nanorough featuresshowed that micron-leve
on 2D substrates
decreases in surface
show a negative roughness
correlation through the
with myogenesis, in addition of graphene
aligned micropatterns, oxide
there is a and collagen t
positive
correlation
randomly betweenPLGA
oriented roughness andcaused
fibers myogenesis [131]. in
increases Nanoroughness
attachment, may interrupt the
proliferation, and myo
controlled alignment of muscle fibers in 2D and randomly oriented
genic expression of C2C12 cells [130]. While randomly oriented nanorough features on 2D microenvironments.
However, nanoroughness also decreases contact angles with cells, which can positively in-
substrates show a negative correlation with myogenesis, in aligned micropatterns, ther
fluence differentiation. The contact angle effect of nanoroughness may dominate in aligned
is amicroenvironments,
positive correlation thusbetween roughness
assisting in myogenic and myogenesis
differentiation [131]. scaffolds.
on aligned Nanoroughness
This ma
interrupt the controlled alignment of muscle fibers in 2D and randomly
would explain the synergistic effect of alignment and nanoroughness in a study conducted oriented micro
environments.
by Patel et al. However, nanoroughness
(Figure 5) (2020) also
[132]. Yeo et al. decreases
(2019) used alignedcontact angles to
nanofibers with cells, whic
directly
enhance surface topology of 3D-printed PCL struts, and further
can positively influence differentiation. The contact angle effect of nanoroughness maused poly vinyl alcohol
(PVA) leaching
dominate to create
in aligned fibrillation mimicking the
microenvironments, thus hierarchical
assistingtopography
in myogenic of native muscle,
differentiation o
significantly enhancing myogenesis compared to less realistic controls [133]. The influence
aligned scaffolds. This would explain the synergistic effect of alignment and nanorough
of nanorough nanofiber surfaces on myogenesis has not been studied, but may show a
ness in a study positive
synergistically conductedeffectby Patel etgrowth.
on muscle al. (Figure
Though 5)graphene
(2020) [132].
oxide hasYeobeen
et al. (2019) use
shown
aligned nanofibers to directly enhance surface topology of 3D-printed
to enhance surface roughness for myogenesis on hydrogels, its use as a surface roughness PCL struts, an
further used
modifier waspoly vinyl alcohol
overlooked in Jo et(PVA) leaching
al.’s study (2020)to ofcreate fibrillation
incorporating mimicking
nanoscale graphene the hierar
oxide or nGOs into polyurethane nanofibers [39,134].
chical topography of native muscle, significantly enhancing myogenesis compared to les
realistic controls [133]. The influence of nanorough nanofiber surfaces on myogenesis ha
not been studied, but may show a synergistically positive effect on muscle growth
Though graphene oxide has been shown to enhance surface roughness for myogenesis o
hydrogels, its use as a surface roughness modifier was overlooked in Jo et al.’s study (2020
of incorporating nanoscale graphene oxide or nGOs into polyurethane nanofibers [39,134
Fibers 2023, 11,
Fibers 2023, 11, 39
x FOR PEER REVIEW 99of
of 43
40

Figure 5. Effect
Figure 5. Effect of
of fiber
fiber alignment
alignment on on myoblasts
myoblasts differentiating
differentiating into
into myotubes.
myotubes. Schematic
Schematic showing
showing
the formation of multinucleated myotubes over a 14-day period on pristine vs. CNT fibers (A). Mag-
the formation of multinucleated myotubes over a 14-day period on pristine vs. CNT fibers (A).
nification 10× (B) and 20× (C) images showing the formation of continuous multinucleated myotubes
Magnification 10× (B) and 20× (C) images showing the formation of continuous multinucleated
similar to muscle fiber bundles in cells grown on CNT fibers. Myotube fusion index (cells containing
myotubes
two or more similar to muscle
nuclei/total cellsfiber bundles
in image) in number
(D), cells grown on CNT
of nuclei perfibers. Myotube
myotube, fusion
(E) and indexmatu-
myotube (cells
ration index (number of myotubes containing five or more nuclei/total number of myotubes)and
containing two or more nuclei/total cells in image) (D), number of nuclei per myotube, (E) (F)
myotubeamaturation
showing significantlyindex
higher(number of myotubes
fusion and maturation containing fiveseeded
in myoblasts or more onnuclei/total
CNT fibers (*number of
indicates
pmyotubes)
< 0.05). Image reproduced
(F) showing with permission
a significantly higherfrom Patel
fusion andetmaturation
al. (2016) [132].
in myoblasts seeded on CNT
fibers (* indicates p < 0.05). Image reproduced with permission from Patel et al. (2016) [132].
Biological cues also enhance the surface characteristics of scaffolds, promoting myo-
Biological
genesis. cues also
Biopolymers are enhance the surface
often added to PCL characteristics
to enhance the of scaffolds,
adhesion andpromoting myo-
differentiation
genesis.
of Biopolymers
precursors. are most
PCL is the oftenused
added to PCLfor
polymer to enhance the adhesion
tissue engineering dueand differentiation
to its ease of elec-
of precursors.
trospinning, PCL is stability,
chemical the mostandused polymer forproperties
bioresorbable tissue engineering
[135,136]. due
Otherto polyesters,
its ease of
such as PLLA, poly lactic-co-glycolic acid (PLGA) and poly D-lactic acid (PDLA), have
Fibers 2023, 11, 39 10 of 40

electrospinning, chemical stability, and bioresorbable properties [135,136]. Other polyesters,

such as PLLA, poly lactic-co-glycolic acid (PLGA) and poly D-lactic acid (PDLA), have
been employed for enhanced hydrophilicity and biocompatibility [137]. An addition of
chitosan to PCL increases MyHC in differentiated human embryonic stem cells (hESCs)
relative to pure PCL [138]. Myogenic factor 6 (MyF6) expression also doubled when chi-
tosan was added. This is important as MyF6 is implicated in regulating skeletal muscle
myogenesis and muscle regeneration. The benefits of biopolymer addition are mirrored in
lyophilized chitosan/PCL samples, where increases in chitosan concentration also increase
the myotube diameter [139]. This may be due to the increase in surface roughness imparted
by chitosan addition, though chitosan addition at 12% and beyond may have compromised
tensile strength to an extent unsuitable for muscle cell growth. Nanofibrous biopolymers
can also be used to create biomimetic structures for the myogenic induction of C2C12s, as
in Yeo et al.’s study (2019) [133]. In this study, nanofibrous alginate was electrospun onto
micropatterned PCL struts to mimic the hierarchical structure of the basal lamina. Myotube
length increased from 58.8 µm to 83.0 µm, the fusion index increased 3 fold, and the MyHC
expression increased 14.6 fold on nanofiber-coated struts compared to nanofiber-free struts.
Combinations of PCL with decellularized bovine skeletal muscle ECM electrospun into
aligned fibers were shown to triple the proliferation of primary rat satellites when com-
pared to pure PCL, as well as double the expression of the myoblast determination protein,
MyoD [27], known to play a major role in muscle differentiation. PCL:ECM blended
scaffolds were then shown to support MyHC regeneration in mice with VML [28]. Pure
skeletal muscle ECM was highly successful in producing fully formed myotubes with
dimensions of 25 µm width and 200 µm length [140]. Collagen, too, is a widely used bioma-
terial in electrospinning scaffolds for myogenesis. Collagen is integrated into electrospun
myogenesis systems for a variety of reasons, including mechanical property optimization
for compliance matching of smooth muscle [141], enhanced cytocompatibility [142], and
adhesion [143] and enhanced myotube formation [144]. Biopolymers, such as chitosan and
collagen, have potential as myogenesis-promoting additives to nanofiber scaffolds for their
unique ability to enhance hydrophobicity, while decreasing nanoroughness [145]. Bioactive
surface additions, such as platelet-rich plasma [146], arginine–glycine–aspartic acid (RGD)
motifs [147], and recognition peptides [148] positively influence muscle differentiation by
decreasing the cell contact angle, and activating integrin-mediated differentiation pathways.
In conclusion, smooth adhesion sites on nanofibers play a role in upregulating myogenic
differentiation. Rough sites on nanofibers, however, may be beneficial to myogenesis
if such roughness is aligned. This is based on evidence of a positive influence of such
roughness seen on other 3D and 2D substrates. In general, additives such as biopolymers
which increase hydrophilicity will upregulate myogenesis, though further studies are
needed to analyze how roughness may affect myogenesis purely through the modulation
of hydrophilicity.

2.2.2. Surface Characteristics and Bone

Micro- and Nano-Rough Surfaces Positively Influence Osteogenesis
The importance of micro- and nanotopographical property influences on osteogenesis
has been previously explored in several studies [145–150]. Generally, roughness can direct
osteogenic differentiation, with higher nanoroughness upregulating osteopontin and ALP
activity [149,150]. While nanoroughness upregulates osteopontin according to Jahanmard
et al. (2020), microroughness has a positive effect on osteocalcin (Figure 6) [149]. Wang
et al. (2012) showed that on crosshatched TiO2 nanofiber scaffolds produced through sacri-
ficial co-spinning with polyvinylpyrrolidone (PVP), microroughness, as well as a higher
nanofiber diameter caused an increase in osteocalcin [151]. ALP activity, on the other
hand, was more prominent on non-patterned low-roughness lower diameter nanofiber
substrates. In this study, the roughness and nanofiber diameter were altered without
affecting chemistry, suggesting that morphological properties of scaffolds, such as micror-
oughness and nanofiber diameter alone, may influence osteogenesis. The downregulation
Fibers 2023,
Fibers 11, x39FOR PEER REVIEW
2023, 11, 1111ofof43
40

of osteopontin
as early osteogenic
(OPN)marker ALP versus
and osteocalcin the upregulation
(OCN) on micro and of late markers,
nano-rough such suggests
surfaces, as osteo-
pontin (OPN) and osteocalcin (OCN) on micro and nano-rough surfaces, suggests
that roughness drives the maturation of osteoblasts. In another study, nanoroughness wasthat
roughness drives the maturation of osteoblasts. In another study, nanoroughness was
shown to selectively upregulate the expression of osteo differentiation markers [152]. For
shown to selectively upregulate the expression of osteo differentiation markers [152]. For
example, OCN, bone sialoprotein (BSP), and collagen A (Col1A) were upregulated by
example, OCN, bone sialoprotein (BSP), and collagen A (Col1A) were upregulated by
smoother nanofibers (Ra~14.3 nm), while OPN, BMP2, RUNX2, and ALP were upregu-
smoother nanofibers (Ra~14.3 nm), while OPN, BMP2, RUNX2, and ALP were upregulated
lated by rougher (Ra~71 nm) nanofibers [152].Tissue engineering scaffolds for optimal
by rougher (Ra~71 nm) nanofibers [152].Tissue engineering scaffolds for optimal bone mat-
bone maturation may benefit from using combined micro and nano-rough surfaces in
uration may benefit from using combined micro and nano-rough surfaces in nanofibers, or
nanofibers, or hierarchical nanoroughness. Such combined surfaces mimic bone structural
hierarchical nanoroughness. Such combined surfaces mimic bone structural hierarchy and
hierarchy and have previously been explored for osteogenesis on non-nanofiber scaffolds,
have previously been explored for osteogenesis on non-nanofiber scaffolds, with certain
with certain substrates able to exhibit osteoconductivity without osteogenic supplements
substrates able to exhibit osteoconductivity without osteogenic supplements [153,154].
[153,154].

Figure
Figure 6.
6. Addition
Addition ofof functionalized
functionalized multiwalled
multiwalled carbon
carbon nanotubes
nanotubes (F-WMNCTs)
(F-WMNCTs) allows
allows for
for the
the
tunability of both stiffness and roughness of nanofibers, which separately control spreading and
tunability of both stiffness and roughness of nanofibers, which separately control spreading and
elongation, ultimately leading to the expression of various osteo-specific differentiation markers.
elongation, ultimately leading to the expression of various osteo-specific differentiation markers.
Image reproduced with permission from Jahanmard et al. (2020) [149].
Image reproduced with permission from Jahanmard et al. (2020) [149].
Hydrophilic
Hydrophilic Surface
Surface Chemistry
Chemistry Improves
Improves Osteoconductivity
Osteoconductivity of of Nanofibers
Nanofibers
Hydrophilic substrates allow cells to spread, adhere,
Hydrophilic substrates allow cells to spread, adhere, and flatten,and flatten, inducing
inducing osteogen-
osteoge-
esis
nesis [155]. Generally, surface chemistry which improves surface wetting properties will
[155]. Generally, surface chemistry which improves surface wetting properties will
enhance
enhanceosteogenesis.
osteogenesis.However,
However, more
moreresearch
researchis needed
is needed to evaluate
to evaluatethe influence of sur-
the influence of
face-bound
surface-bound functional groups
functional groupsononosteogenesis.
osteogenesis. ForForexample,
example,Sarkar
Sarkaretetal.al.(2016)
(2016)showed
showed
that
that in
inthe
theNaOH-based
NaOH-based hydrolysis
hydrolysis of of PCL
PCL nanofiber
nanofiber scaffolds,
scaffolds, thethe carboxyl
carboxyl and andhydroxyl
hydroxyl
groups
groupswere wereexposed
exposedonon the nanofiber
the nanofiber surface, causing
surface, causing a significant increase
a significant in water
increase con-
in water
tact angleangle
contact and decrease in osteogenic
and decrease expression
in osteogenic in hBMSCs.
expression Ultimately,
in hBMSCs. it was the
Ultimately, it cellular
was the
curvature, tortuosity,
cellular curvature, solidity, solidity,
tortuosity, and aspect andratio thatratio
aspect mostthatsignificantly correlatedcorrelated
most significantly with os-
teogenic differentiation. The area, perimeter, and circularity of cells were
with osteogenic differentiation. The area, perimeter, and circularity of cells were not sig- not significantly
correlated with osteogenic
nificantly correlated differentiation,
with osteogenic though in 2D
differentiation, culture
though in environments, the spread
2D culture environments,
and roundness
the spread and were significantly
roundness correlated correlated
were significantly with osteogenic commitment.
with osteogenic This suggests
commitment. This
that the 3D
suggests architectures
that of nanofiber
the 3D architectures scaffolds are
of nanofiber likely are
scaffolds the likely
dominating factor in deter-
the dominating factor
mining the cell shape
in determining and
the cell differentiation,
shape as opposed
and differentiation, to surfacetochemistry
as opposed [156]. Though
surface chemistry [156].
exposing carboxyl carboxyl
Though exposing groups may interfere
groups with osteogenesis
may interfere in vitro, in
with osteogenesis studies
vitro, have
studiesshown
have
that
shown negatively chargedcharged
that negatively functional groupsgroups
functional interactinteract
with calcium and phosphate
with calcium and phosphate ions
Fibers 2023, 11, 39 12 of 40

ions present in bodily fluids and facilitate hydroxyapatite crystallization, which may aid in
osteogenesis [157]. Interestingly, studies involving plasma treatment cite resultant exposure
of carboxyl and hydroxyl groups as adhesion promoters, and thus osteogenesis promot-
ers [158]. It is more probable that the hydrophilic effect from creating oxygen functional
groups is what drives osteogenesis. Cold atmospheric plasma treatment of PLGA drives
osteogenesis through increased hydrophilicity of nanofibers, as well as the formation of
micro/nano roughness. Plasma treatment also leads to improved viability through im-
proved adhesion. Atyabi et al. (2016) showed a 2× improvement in cell viability via cold
atmospheric plasma treatment of the scaffolds [159]. Plasma treatment may also improve
adhesion of additional surface modifiers. Kooshki et al. (2019) used a plasma treatment
followed by a lipopolysaccharide coating to enhance osteoconductivity of mesenchymal
stem cells (MSCs) on PLLA [160]. Lipopolysaccharide promotes osteogenic differentiation
by fortifying the activity of TAZ (transcriptional coactivator with a PDZ-binding motif), a
transcriptional co-regulator which plays a key role in osteoblast differentiation, as mediated
by the Wnt/β-catenin pathway [161]. The conjugation of amino acids aspartic acid (ASP)
and glutamic acid (GLU) enhances hydrophilicity more than cold atmospheric oxygen [162].
Conducting both plasma treatment and the conjugation of GLU/ASP to PLGA nanofibers
synergistically improves osteoconductivity of the scaffolds. ASP induces cell proliferation
and osteogenic differentiation more than GLU, due to the role of ASP in integrin binding
recognition and higher binding affinity for calcium ions by ASP. Surface wettability was
enhanced through the addition of chitosan and a polydopamine layer on PLLA nanofibers,
leading to improved cell spreading and more accurate recapitulation of in vivo conditions,
leading to enhanced osteogenesis [163]. Similar synergistic results were observed with
the addition of gelatin and hydroxyapatite to PLGA [164]. At 60% concentrations, medici-
nal Cissus quadrangularis extract in PLLA increased hydrophilicity of MSCs and doubled
ALP activity and calcium deposition [165]. Nano-hydroxyapatite (nHA)-coated nanofibers
mimic the bone extracellular matrix, which is composed of collagen type I nanofibers miner-
alized with nanosized calcium phosphate crystals. In experiments with polydopamine and
nHA-coated PCL nanofibers, the water contact angle was reduced to 0◦ and osteoinduction
was enhanced [166]. Similar results were seen in nHA-coated PLLA nanofibers, which
induced bone formation 10 weeks after subcutaneous implantation in mice, showing that
nHA can induce bone formation in absence of exogenous agents or cells [167].
The addition of peptides and hydrophilicity enhancers significantly upregulate os-
teoinduction. The formation of nanorough or microrough features for cell adhesion is
essential for promoting bone cell differentiation. Studies exploring how roughness can be
dynamically tuned to fit the needs for biological cues throughout bone cell differentiation
are needed to further optimize osteoinductive nanofibers.

2.3. Mechanical Properties and Crystallinity

2.3.1. Muscle Differentiation Is Supported by Elastic, Conductive, and Crystalline Scaffolds
Scaffold crystallinity and mechanical properties play an important role in regulating
myogenesis. Crystallinity directly influences mechanical properties of scaffolds and may
also have an independent role in enhancing the conductivity of scaffolds for improved
myogenesis. Myogenic differentiation depends highly on the microenvironment elasticity
being 8–17 kPa [168]. The subsequent proliferation, elongation, alignment and myofibrillo-
genesis of myoblasts [169] occurs in an even smaller range of microenvironmental stiffness
of 11–15 kPa, with the striation of cells decreasing significantly outside this range [170]. It
is important to note that this mechanical testing was conducted on individual nanofibers
using AFM nanoindentation, while more conventional bulk tensile testing of nanofiber
material often yields stiffness in the megapascal (MPa) range. In differentiating stem cells
into muscle tissue on nanofibers, scaffolds which most successfully induce differentiation
fall within the bulk modulus range of 10–15 MPa [27,38,134,138]. The elastic modulus
of 17 kPa has been shown to dictate the differentiation of mesenchymal stem cells into
muscle cells positive for alpha-actin [148], replicating the modulus effect seen in Engler
Fibers 2023, 11, 39 13 of 40

et al.’s study (2004). Patel et al. (2020) were able to mix high-modulus (75 MPa) PCL
and low-modulus decellularized muscle ECM (1 MPa) to achieve scaffold mechanical
properties ideal for myogenesis, with the added benefit of including ECM pro-myogenic
biological cues [9]. High elasticity, high strain at break, fast stress relaxation rate, and
tensile strength of 5–15 MPa generally favor myogenesis [38,39,171]. Exceptions to certain
rules may occur as in, for example, Xu et al.’s study (2014) employing multiwalled carbon
nanotubes [171]. In this study (Figure 7), an improved conductivity of scaffolds was able
to enhance myogenesis, despite moduli in the range of 70–100 MPa. This highlights the
Fibers 2023, 11, x FOR PEER REVIEW importance of scaffold crystallinity properties in dictating myogenesis, as conductivity
14 ofand
43
crystallinity are intimately linked.

Figure
Figure 7.
7. Young’s
Young’s moduli
moduli(A)
(A) and
and conductivity
conductivity(B)
(B)properties
propertiesof
ofPLGA
PLGAnanofibers,
nanofibers,and and(C)
(C)corre-
corre-
sponding quantitative analysis of morphological indexes of myotubes on days 3 and 5, including
sponding quantitative analysis of morphological indexes of myotubes on days 3 and 5, including
myotube length, width, aspect ratio, surface coverage, and fusion index (n = 10; *, #, &: p < 0.05; *:
myotube length, width, aspect ratio, surface coverage, and fusion index (n = 10; *, #, &: p < 0.05;
compared to PLGA; &: compared to day 3; #: compared to PLGA/0.1%MWNTs). Adapted from
*: compared to PLGA; &: compared to day 3; #: compared to PLGA/0.1%MWNTs). Adapted
[171].
from [171].
Scaffolds can be optimized for myodifferentiation through an enhancement of elas-
Through nanofiber crystallinity changes, conductivity, elasticity, and strength can
ticity, crystallinity, and conductivity. The interplay and hierarchy of these variables, with
be altered to create materials appropriate for muscle stem cell differentiation. Surface
regards to influencing myogenesis, is yet to be confirmed empirically. A summary of re-
wettability is also an important material parameter which positively influences myogenic
cent innovations relating nanofiber scaffold materials and respective mechanical, electri-
differentiation. Bloise et al. (2018) altered wetting and mechanical properties of electrospun
cal, and crystallinity properties studied for muscle differentiation, is listed in Table 1.
poly(butylene 1,4-cyclohexanedicarboxylate) through the addition of PEG-like co-units,
decreasing crystallinity and increasing amorphous regions, leading to increased surface
Table 1. List of the recent innovations relating fiber-based scaffolds for engineering muscle tissue
based on biophysical properties of the scaffold.

Type of Mechanical Electrical/Crystal

Material Used Result of Tissue Growth Reference
Electrospinning Properties linity Properties
Increased myotube
Fibers 2023, 11, 39 14 of 40

wetting and elasticity, and decreased stiffness. These changes made the scaffolds suitable
for successful regeneration in murine models [38]. Jana et al. (2014) showed that the addi-
tion of chitosan from 4% to 8% to PCL increased the scaffold crystallinity and resulted in an
increased myotube diameter of cultured C2C12 cells [139]. In a separate study, the addition
of chitosan from 0% to 4% decreased the crystallinity in scaffolds, but improved myogene-
sis [105]. Myogenesis is controlled by many factors, and the biopolymer concentration can
alter crystallinity to optimize the scaffold systems for myogenesis. Increased crystallinity of
graphene oxide nanoparticles through APTES ((3-Aminopropyl)triethoxysilane) treatment
had a positive effect on myogenesis, improving conductivity, wetting, and elasticity, as well
as the stress relaxation capacity of polyurethane nanofibers [39]. In this study, a significant
improvement in myogenic differentiation was observed on fibers containing 8% nano-
graphene oxide, the only observably conductive sample in the experiment. Crystallinity,
it is apparent, can play an important role in supporting myodifferentiation if the impact
on mechanical elasticity and recovery is not too great. In general, preliminary properties
of solution stability must first be met by nanofibers to support myogenesis. Uribe-Gomez
et al. (2021) showed, for example, that of the fourteen PCL-PU (polyurethane) block copoly-
mers synthesized, only one medium-crystallinity polymer was able to maintain stability in
phosphate-buffered saline or PBS, and support myogenesis [172]. Once such stability is
achieved, a polymer with both elasticity and conductivity is the most ideal for optimized
myodifferentiation.
Scaffolds can be optimized for myodifferentiation through an enhancement of elasticity,
crystallinity, and conductivity. The interplay and hierarchy of these variables, with regards
to influencing myogenesis, is yet to be confirmed empirically. A summary of recent
innovations relating nanofiber scaffold materials and respective mechanical, electrical, and
crystallinity properties studied for muscle differentiation, is listed in Table 1.

Table 1. List of the recent innovations relating fiber-based scaffolds for engineering muscle tissue
based on biophysical properties of the scaffold.

Type of Elec- Mechanical Electrical/Crystallinity

Material Used Result of Tissue Growth Reference
trospinning Properties Properties
Increased myotube
PCL nanofibers Direct electro- Aligned and
growth, alignment, and
and PEG spinning randomly oriented [3]
nuclear aspect ratio from
hydrogel (rotating drum) scaffolds studied
aligned fibers
Aligned nanofibers
Direct electro- Higher stiffness and
decrease proliferation, but
PHB spinning strength in aligned [78]
increase differentiation
(rotating drum) nanofibers
markers of muscle cells
Direct electro-
spinning Higher stiffness and Increased
2:1
(rotating drum strength in aligned myodifferentiation in [79]
PC–chitosan
and parallel nanofibers aligned nanofibers
electrodes)
Aligned nanoribbon
Multilayer, parallel biolayers upregulated
Micropatterning
and orthogonal myogenic markers and led
PLGA and [80]
nanoribbons to muscle maturation
spincoating
fabricated moreso than random or
single-layer nanoribbons
Aligned nanofibers
Direct Nanofiber/
perpendicular to aligned
5:2 eletrospinning microchannel
microchannels caused the [71]
PCL–chitosan (parallel layered structures
highest degree of
electrodes) fabricated
myodifferentiation
Fibers 2023, 11, 39 15 of 40

Table 1. Cont.

Type of Elec- Mechanical Electrical/Crystallinity

Material Used Result of Tissue Growth Reference
trospinning Properties Properties
Addition of ECM
1:1 Bovine Direct electro- Higher stiffness and enhanced
muscle spinning strength in aligned, myodifferentiation, [73]
ECM–PCL (rotating drum) non-ECM nanofibers despite the loss of
mechanical properties
Direct Mechanical
Larger fiber diameter
eletrospinning properties similar for
PLGA upregulates [83]
(parallel scaffolds
muscle-specific markers
electrodes) 300–3000 nm
Aligned CNTs showed
Dry drawing PAX7 and MyF5 were
Carbon higher conductivity than
from aligned upregulated in aligned [92]
nanotubes randomly oriented and
CNT forest CNT substrates
flat substrates
Increased strength, Enhanced attachment,
Direct electro-
PLGA/graphene decreased stiffness Enhanced crystallinity proliferation, and MyHC
spinning [99]
oxide/collagen and elongation with with GO addition expression in fibers with
(rotating drum)
collagen and GO GO and collagen
Direct electro- ~2 MPa alginate
Myotube length and
spinning nanofiber-coated
MyHC expression
Alginate/PCL (parallel PCL strut with a [102]
improved dramatically in
rotating high roughness
hierarchal strut structures
drums) fabricated
Increased strength Collagen and GO addition
Direct electro- Conductivity enhanced
Polyurethane/graphene and stiffness from increased adhesion,
spinning with APTES and GO [103]
oxide graphene oxide proliferation, and
(rotating drum) addition at 8%
addition myogenic differentiation
Decreased stiffness Increased PEG units led to
Direct electro-
PBCE–PEG and strength, more muscle-like elastic
spinning [135]
copolymer increased elasticity, properties and increased
(rotating drum)
with more PEG units myodifferentiation
Increased myotube length,
Decrease in elasticity
PLGA/multiwalled 1.3 × 10−2 s/m diameter, and
Direct electro- and strength, with
carbon conductive fibers proliferation, with [136]
spinning increased MWCNT
nanotubes fabricated increased MWCNT
addition beyond 0%
concentration
Recovery and elastic PCL–urethane–
Polyester Crystallinity customized
modulus increased, butanediol chain extender
polyurethane Touch- with urethane segments
with increased ratio necessary to optimize [137]
with glycol spinning and glycol chain
urethane segment for successful myocyte
chain extenders extenders
addition growth on nanofibers
Abbreviations: PCL, polycaprolactone; PLLA, poly L-lactic acid; PEG, polyethylene glycol; GO, graphene
oxide; PHB, polyhydroxybutyrate; PC, phosphatidylcholine; PLGA, poly lactic-co-glycolic acid; ECM, extra-
cellular matrix; CNT, carbon nanotubes; PAX7, paired box 7; MyF5, myogenic factor 5; PBCE, poly(butylene
1,4-cyclohexanedicarboxylate).

2.3.2. Bone Differentiation Prefers Crystallinity and Conductivity, Followed by Stiffness

and Strength
It has been shown in nanofiber scaffolds that a high tensile modulus of 20 MPa
to 60 MPa is beneficial for osteogenesis [149,173,174]. For example, Nam et al. (2011)
created PCL nanofibers with and without a polyether sulfone (PES) core, thereby forming
nanofibers of 7.1 MPa modulus and 30.6 MPa modulus for the PCL and PES-PCL core-shell
nanofibers, respectively. Murine MSCs were seeded, and the softer pure-PCL fibers led
to the upregulation of chondrocyte-specific Sox9, Co12a1, and Acan, and increased GAG
Fibers 2023, 11, 39 16 of 40

production, while stiffer PES-PCL core-shell nanofibers led to the upregulation of osteocyte-
specific RUNX2, Alp, and Oc [173]. Jahanmard et al. (2020) showed that functionalized
multiwalled carbon nanotubes (F-MWCNTs) increase nanofiber stiffness at loadings of
0.5% to 1% F-MWCNTs. A higher stiffness increased the expression of osteocalcin while
a higher nanoroughness increased the expression of osteopontin, indicating that unique
signaling pathways can be independently controlled by material stiffness and material
topography [149]. Chitosan and polydopamine have been used to synergistically enhance
tensile strength and Young’s modulus to the Gigapascal (GPa) range, as well as surface
wetting conditions, to enhance the osteogenesis of MC3T3 cells [163]. Hydroxyapatite and
graphene oxide (GO) incorporated into PLGA showed synergistic increases in proliferation,
calcium deposition, adhesion, and expression of ALP, osteopontin and RUNX2 in MC3T3-
E1 murine preosteoblasts by increasing the tensile strength and modulus [174]. With the
tensile strength of cancellous bone being 10.4 GPa and cortical bone being 18.6 GPa, a stiff
microenvironment will promote osteogenesis over a less stiff one with other properties
held constant [175].
Nanofiber scaffolds loaded with magnetic nanoparticles, bioactive ingredients, or
additives to enhance material crystallinity can often increase osteogenesis, despite drasti-
cally hindering mechanical properties [40,144,165,176,177]. Crystallinity has been shown
to be a significant driver of osteogenesis in many studies [117,139,147,148], but few have
successfully decoupled their effect on mechanical properties. The addition of graphene
oxide (GO) to nanofibers, for example, was shown to decrease tensile strength more than 10
fold, as well as decrease stiffness, but positively influence osteogenesis (Figure 8 [40]. Using
almost equal parts of GO and polymer, Saburi et al. (2019) saw more of an improvement
in the proliferation and ALP expression of hiPSC cells than studies using both GO and
hydroxyapatite, despite negatively impacting mechanical properties [40,174].
Graphene oxide has been shown to increase the crystallinity of polyvinylidene fluoride
(PVDF) nanofibers, and introduce crystalline regions, which would explain this impact on
osteogenesis [178]. The addition of nanoclay to PCL nanofibers similarly decreased the
strength and stiffness, but increased crystallinity, as evidenced by X-ray diffractometer
(XRD) and differential scanning calorimetry (DSC) measurements [150]. Nanoclay and
graphene oxide do not always decrease the strength and stiffness of electrospun scaffolds;
studies have shown the opposite effect when other base polymers are employed [179,180].
Yao et al. (2020) showed that the addition of graphene oxide to PVDF drastically up-
regulated osteodifferentiation compared to pristine PVDF. These matrices produced such
outcomes despite having a relatively low strength and stiffness, likely due to their increased
crystallinity. Studies such as these demonstrate the need for further investigation into the
mechanisms involved in crystallinity-modulated osteoinduction. Studies have shown the
positive impact of crystallinity on osteogenesis in 2D films, but have not been repeated
for 3D and nanofiber systems [181]. Little is known about the influence of crystallinity
on osteogenesis, and some studies have shown amorphous morphology, not crystalline
morphology, to be responsible for bone cell growth [182,183]. The maturation of reinforc-
ing bone starts with an amorphous precursor, amorphous Ca-phosphate (ACP), which
converts to crystalline hydroxyapatite. Recent studies by Muller et al. (2020) showed that,
due to this, the amorphous polyphosphate stimulated osteogenesis more than crystalline
polyphosphate [183]. These contradictions are likely due to present limitations on scaffold
technology. Scaffolds capture only a snapshot of cell maturation microenvironments. More
research is needed on scaffolds with dynamic properties to correspond to the morphological
changes of tissue over time.
Osteo-differentiation is highly sensitive to mechanical properties, with stiffness and
strength positively correlated with osteogenic markers. However, this research has shown
that osteoconductive additives such as hydroxyapatite, graphene oxide, polydopamine,
and carbon nanotubes will promote bone growth despite negatively impacting mechanical
properties. This is likely due to increases in hydrophilicity, conductivity, or combinations of
both. Thus, newly fabricated electrospinning materials should pursue integration with such
 Fibers 2023, 11, 39 17 of 40

additives or maximization of wetting and electrical conductivity properties. Fiber-based

Fibers 2023, 11, x FOR PEER REVIEW constructs may be useful for musculoskeletal applications. A device for rotator cuff repair
17 of 43
that is made from electrospun fibers is undergoing testing in humans [184]. A summary of
recent innovations for nanofiber scaffold materials and respective mechanical, electrical
and crystallinity properties studied for bone differentiation, is listed in Table 2.

Figure
Figure Osteogenicdifferentiation
8. 8.Osteogenic differentiation ofofiPSCs,
iPSCs,asasshown
shownviavia
ALPALP(A) (A)
and and
calcium (B) expression,
calcium (B) expression,
with evidence of significant (* indicates p < 0.05) upregulation due to the addition of graphene
with evidence of significant (* indicates p < 0.05) upregulation due to the addition of graphene oxide,oxide,
despite a reduction in strength and stiffness (C). Adapted from
despite a reduction in strength and stiffness (C). Adapted from [40]. [40].

Graphene oxide has been shown to increase the crystallinity of polyvinylidene fluo-
ride (PVDF) nanofibers, and introduce crystalline regions, which would explain this im-
pact on osteogenesis [178]. The addition of nanoclay to PCL nanofibers similarly de-
creased the strength and stiffness, but increased crystallinity, as evidenced by X-ray dif-
fractometer (XRD) and differential scanning calorimetry (DSC) measurements [150].
Nanoclay and graphene oxide do not always decrease the strength and stiffness of elec-
trospun scaffolds; studies have shown the opposite effect when other base polymers are
employed [179,180]. Yao et al. (2020) showed that the addition of graphene oxide to PVDF
drastically upregulated osteodifferentiation compared to pristine PVDF. These matrices
produced such outcomes despite having a relatively low strength and stiffness, likely due
to their increased crystallinity. Studies such as these demonstrate the need for further in-
vestigation into the mechanisms involved in crystallinity-modulated osteoinduction.
Fibers 2023, 11, 39 18 of 40

Table 2. List of the recent innovations relating fiber-based scaffolds for engineering bone tissue based
on biophysical properties of the scaffold.

Type of Elec- Mechanical Electrical/Crystallinity Result of Tissue

Material Used Reference
trospinning Properties Properties Growth
Flat surfaces
Aligned CNTs showed
Dry drawing upregulated
higher conductivity than
Carbon nanotubes from aligned osteogenic markers [92]
randomly oriented and
CNT forest (RUNX2,
flat substrates
osteopontin)
Osteogenesis
PCL and
Increased stiffness at induced by
functionalized
Direct electro- 0.5–1% F-MWCNT CNTs known to increase F-MWCNTs, with
multiwalled carbon [116]
spinning loading, decreased at conductivity osteocalcin
nanotubes
2–3% loading upregulated by
(F-MWCNTs)
increased stiffness
Decreased stiffness Increased
Crystallinity increased
Direct electro- and strength with mineralization and
PCL and nanoclay with the addition of [117]
spinning 1–a 10% addition of ALP activity with 1%
nanoclay
nanoclay addition nanoclay
Coaxial Direct Increased stiffness Increased
PCL shell with PES
Electrospin- with the addition of osteoinduction with [138]
core
ning a PES core a stiffening PES core
Increased stiffness Increased
and strength osteoinduction
PLLA/chitosan/ Direct electro-
through the addition through a synergistic [129]
polydopamine spinning
of chitosan and addition of chitosan
polydopamine and polydopamine
Decreased stiffness
and strength with Increased
PLGA/graphene ox- Direct electro- HA addition, Conductivity increased osteoinduction with
[139]
ide/hydroxyapatite spinning increased stiffness due to HA and GO addition of HA and
and strength with GO
GO addition
Decrease in peak
stress, strength, and Increased
PVDF/graphene Direct electro- Piezoelectric properties
stiffness with osteoinduction with [144]
oxide spinning exhibited by PVDF
graphene oxide addition of GO
addition
Abbreviations: PCL, polycaprolactone; RUNX2, runt-related transcription factor 2; PES, polyethersulfone; HA,
hydroxyapatite; PLLA, poly L-lactic acid; PEG, polyethylene glycol; GO, graphene oxide; PLGA, poly lactic-co-
glycolic acid; CNT, carbon nanotubes; PVDF, polyvinylidene fluoride.

3. Biological Requirements Affecting the Choice of Nanofiber Scaffolds

3.1. Skin and Wounds
Skin tissue is the largest organ of the human body, with a total surface area of more than
15,000 cm2 and accounting for 5% of the total body weight. The skin, an ectodermal tissue,
is the first line of defense and protects internal organs and tissues, such as muscle, bone
from damage such as pathogens and chemical hazards, maintain isothermal conditions,
and provides mechanical support for the optimal functioning of the body. The skin also acts
as the primary sensory organs and an immunological mediator. Wound healing, skin tissue
regeneration and restoration of the skin is a vast field with particular interest in scaffolds.
Different materials have been demonstrated for use as scaffolds for skin tissues [82,185].
An example of PCL-PANi used as a scaffold for adult human dermal fibroblasts is shown
in Figure 9 [82].
 bone from damage such as pathogens and chemical hazards, maintain isothermal condi-
tions, and provides mechanical support for the optimal functioning of the body. The skin
also acts as the primary sensory organs and an immunological mediator. Wound healing,
skin tissue regeneration and restoration of the skin is a vast field with particular interest
Fibers 2023, 11, 39
in scaffolds. Different materials have been demonstrated for use as scaffolds for skin tis-
19 of 40
sues [82,185]. An example of PCL-PANi used as a scaffold for adult human dermal fibro-
blasts is shown in Figure 9 [82].

Figure 9. Adult
Adult human
human dermal
dermal fibroblast
fibroblast (HDFa)
(HDFa) cells
cells after
after 77 days
days of
of culture
culture on
on polycaprolac-
polycaprolac-
tone/polyaniline
tone/polyanilinescaffolds.
scaffolds.Blue: DAPI
Blue: nuclei
DAPI stain;
nuclei green:
stain; Alexa
green: Fluor
Alexa
® 488
Fluor ® Phalloidin F-actin
488 Phalloidin fila-
F-actin
ments. Image
filaments. courtesy
Image of Samerender
courtesy of SamerenderN. Hanumantharao,
N. Hanumantharao, Michigan Technological
Michigan University.
Technological University.

The
The skin
skin is composed of
is composed three types
of three types of
of tissue
tissue layers,
layers, each
each having
having its own distinct
its own distinct
morphological
morphological andand mechanical
mechanical properties.
properties. The
The epidermis
epidermis isis the
the outermost
outermost layer
layer of skin
that receives its nutrients from the dermis layer. The
The deepest
deepest layer
layer ofof the
the Stratum
Stratum Basale,
Basale,
also known as stratum germinativum, is one which divides and attaches to the basement
membrane through
throughhemidesmosomes.
hemidesmosomes.The The epidermis
epidermis renews
renews itselfitself constantly
constantly and isand is
being
replaced every 28 days due to the activity of keratinocytes present in this layer. This
layer also contains melanocytes responsible for producing melanin. Above this layer
lies a lining of spine-like cells of eight to ten cell layers called the stratum spinosum
containing the dendritic cells. The stratum granulosum is three to five cell layers and
contains keratohyalin granules, which are the keratin precursors and lamellar granules
that help in cell–cell attachment. The stratum lucidum is a thin layer that contains the
transformation of keratohyalin (eleidin). The stratum corneum is the outermost layer
that is twenty to thirty cell layers thick made up of anucleate squamous cells from dead
keratinocytes. This layer is present at differing thicknesses, especially in callused skin.
This layer is embedded in a lipid matrix composed of cholesterol, and other free fatty
acids, which helps water retention and acts as the first barrier. It also produces defensins
through the Langerhans cells. The basal layer also contains the Merkel cells, oval shaped
type I mechanoreceptors that are slow-adapting and found attached to the layer through
desmosomes, Merkel disks which sense light touch, Pacinian corpuscles which sense
deep pressure, Meissner’s corpuscles which sense low-frequency stimulation, and Ruffini
corpuscles which sense pressure. The dermis is connected to the epidermis through the
basal membrane and has hair follicles, sweat glands, and somatic and autonomic nerves.
The hair follicles also have smooth muscle fibers that are attached to the connective tissue
sheath called the arrector pili muscles. This helps in controlling the sebaceous glands. The
dermis is 1 mm to 2 mm thick and consists of a system of filamentous connective tissue with
vasculature and nerve cells. The dermis provides elasticity to the skin tissue primarily due
to the high elastin amount present in the tissue and mechanical support due to the loose
collagen networks. The final layer is the hypodermis, which is composed of fibroblasts and
adipocytes, providing mechanical and thermal insulation.
Fibers 2023, 11, 39 20 of 40

Fiber scaffolds could be useful as skin substitutes and wound dressings. An elec-
trospun nanofiber dressing for wound care has been cleared by the Food and Drug Ad-
ministration (FDA) for marketing in the USA, but clinical results have not yet been pub-
lished [186,187]. A handheld electrospinning device has been developed for creating
spray-on wound dressings that custom fit the patient’s wound bed. This system is currently
being tested on patients [188]. A summary of recent innovations relating nanofiber scaffold
materials and respective mechanical, electrical and crystallinity properties studied for skin
differentiation, is listed in Table 3.

Table 3. List of the recent innovations relating fiber-based scaffolds for engineering skin tissue based
on biophysical properties of the scaffold.

Type of Electrical
S No. Material Used Mechanical Properties Cell Response Reference
Fabrication Properties

Nanocomposite PU/PCL Direct • 3D porous structure Increased hydrophilicity and

1 • Increased fiber diameter - [189]
scaffolds with GO electrospinning biocompatibility

Tunable
PCL, PGS and regenerated Nozzle-free
2 hydrophilicity/hydrophobicity - Increased fibroblast attachment [190]
silk fibroin electrospinning
based on PGS

• Increased hydrophilicity • Increased cellular

due to HA attachment and proliferation
Polyurethane/starch Coaxial • Complete degradation of of mouse fibroblasts in vivo
3 - [191]
(hyaluronic acid) electrospinning scaffolds by week 3 • Presence of sebaceous
• Adequate tensile strength glands in mice on day 14

Addition of GRGO increased

PVA/glucose–reduced Direct Increased hemocompatibility
4 hydrophobicity and the - [192]
graphene oxide (GRGO) electrospinning and biocompatibility
diameter of fibers

• Highly hydrophilic • In vitro drug release of

Santa Barbara amorphous • Large surface area due to curcumin
Direct
5 (SBA)-15–incorporated the presence of mesopores - • Increased wound healing [193]
electrospinning
PVA with curcumin • Mesh-like topography activity in vivo

Direct
Increased reinforcement due to
electrospinning
presence of Kefiran improving Increased biocompatibility and
6 Core-shell PLA/kefiran followed by air - [194]
tensile strength and collagen production
plasma
crystallinity.
treatment

• Accelerated healing of
Environmentally wounds in mice,
Maltodextrin mixed with
controlled Nanofibrous morphology Negative zeta vascularization of tissue
7 argi- [195]
direct Elastic and high breaking point potential in fluids • Antioxidant activity aided
nine/lysine/polylysine
electrospinning in healing

Increased
Uniform fibrous structure conductivity due
Polypyrrole/chitosan/ Direct Increase in polypyrrole-reduced to polypyrrole in Increased cell adhesion, growth,
8 [196]
collagen electrospinning diameter of fibers the and proliferation
Adequate mechanical strength semiconducting
polymer range
Increase in mechanical
The release of Mg ions and the
PCL/gelatin/MgO properties due to gelatin and
Direct structure of scaffolds aided in
9 preseeded with MgO - [197]
electrospinning full thickness skin wound
endometrial stem cells Increased porosity
closure in mice.
Release of Mg ions

• Increase in hydrophilicity
• Smooth fibrous morphology
• Hybridization and Increased cell adhesion
Chitosan–PVA and silk
Co- co-electrospinning processes The MSC-derived keratinocytes
10 seeded with differentiated [198]
electrospinning increased mechanical stimulated wound healing in
keratinocyte
characteristics of the mice
scaffold
Fibers 2023, 11, 39 21 of 40

Table 3. Cont.

Type of Electrical
S No. Material Used Mechanical Properties Cell Response Reference
Fabrication Properties

• Increase in fiber diameter

Electrospun due to nanocrystalline
chitosan/nanocrystalline Direct cellulose-graft-poly(N- Increase in cytocompatibility
11 vinylcaprolactam) - [199]
cellulose–graft-poly(N- electrospinning and cell proliferation
vinylcaprolactam) • Increased thermal stability
• Increase in hydrophilicity

Core-shell structure aids in

Collagen–graphene oxide
release of bFGF, increased
(Col–GO) scaffolds loaded Coaxial Increased healing and skin
12 mechanical strength and - [200]
with basic fibroblast electrospinning regeneration in rats
degradation conducive to
growth factor (bFGF)
wound healing
Increase in ultimate tensile
PCL/gelatin and modified
Direct strength and reduction in Increased cell adhesion and
13 acetylated cellulose - [201]
electrospinning degradation rates due to the proliferation
nanofibers
addition of cellulose nanofibers
Direct
electrospinning Control over topography aided Increased stratification and
14 Poly(ε-caprolactone) with micro- in fabrication of ridge-like - skin-like formation due to the [202]
stereolithography structures such as native tissue topography of scaffold
(µSLA)
Loading with RCSP and Ag-NP
Janus nanofibers, rana
improved the hydrophilicity Accelerated wound healing
chensinensis skin peptides Uniaxial
15 and mechanical properties, - characterized by [203]
(RCSPs), and silver electrospinning
while also providing re-epithelization
nanoparticles (Ag-NPs)
anti-bacterial activity
Increased hydrophilicity, Improved wound healing ratio
Corn peptides (CPs) with Coaxial biocompatibility, mechanical with enhanced fibroblast
16 - [204]
Janus nanofibers electrospinning strength, and free-radical proliferation and formation of
scavenging capabilities hair follicles and capillaries

• Increased hydrophilicity,
and mechanical properties Cytocompatibility studies using
Fish collagen/PCL
of the bilayer scaffold human fibroblasts and
bio-composite with Direct Electro-
17 • The porosity of the scaffold - keratinocytes demonstrated an [205]
covalently cross-linked spinning
is also improved due to the effective scaffold–cell
chitooligosaccharides
electrospinning process penetration and proliferation

Increased elasticity and strain,

Antibacterial ZnO hydrophilicity, and Increased vascularization and
Direct Electro-
18 quantum dots with biodegradability of the - promoted wound healing in [206]
spinning
PCL/collagen fibers scaffolds. ZnO quantum dots early stages of wound healing
provided antibacterial activity
Ulvan–cellulose blended
Direct Electro- The fibrous scaffolds improved In vivo angiogenesis
19 with polylactide and - [207]
spinning mechanical stability demonstrated in rats
polydioxanone
Increased mechanical strength
Coaxial due to fiber morphology and Improved adhesion and cell
20 PMMA/silk fibroin - [208]
electrospinning structure Highly porous spreading on scaffolds in vitro
scaffolds
Melt electro writing process
The precision fabrication of the
Melt enables the precise position of
21 PCL fiber constructs enabled tight [209]
electro-writing each individual fiber in the
control of cell morphology
constructs
Abbreviations: PU, polyurethane; PGS, poly(glycerol sebacate); PCL, polycaprolactone; PLLA, poly L-lactic acid;
GO, graphene oxide; PLA, poly lactic acid; PMMA, poly(methyl methacrylate); PVA, polyvinyl alcohol.

3.2. Vascular
The vascular system plays an important role in infiltrating the body’s tissue to provide
nutrients to the tissues. They can be classified roughly as capillaries, arterioles, veins,
and arteries based on the function of these vessels. The arteries are the largest and carry
oxygenated blood to the distant organs, while veins transport back the deoxygenated
blood. The capillaries and arterioles act intermediates between the veins and arteries
and help during the efficient transport of nutrients and waste within the organs and
tissues. Depending on the function of the vessel, the shape and structure of the vessel
is different. The arteries are composed of three layers of concentric tissues: the intima,
media, and adventitia, each with its own unique function. The tunica intima consists
Fibers 2023, 11, 39 22 of 40

of an endothelium, a basal lamina that separates it from tunica media and an acellular
endothelial layer composed of tightly bound extracellular matrix proteins, such as collage,
laminin, and fibronectin. The basal lamina is elastic and composed of elastin and type IV
collagen molecules. It is porous, with varying pore densities, and regulates the elasticity of
the vascular wall to regulate blood pressure. Porosity also helps provide control over the
diffusion of nutrients toward the surrounding tissue regions. The endothelium interacts
with the blood and plays a key role in several physiological processes, such as coagulation,
maintenance of vascular homeostasis, barrier, inflammation, maintenance, and modification
of ECM components. The tunica media, or the middle layer, is composed of smooth muscle
cells and collagen (Type I and III) in a concentric manner along the axis of the blood vessel
and aid in vasodilation. The outermost layer of the blood vessel, the tunica adventitia, is
composed of fibroblasts, collagen (Type I and II), mast cells, nerve endings, and different
types of vasa vasorum. This layer protects the vessels from overextending or over retracting,
helps in cellular trafficking and aids in cell signaling. This layer is actively involved in tissue
repair and remodeling by changing the medial smooth muscle tone. Fiber-based constructs
may have application in repairs to vasculature (Figure 10) [210]. Here, similarities are shown
between a native artery and an implanted hyaluronic acid (HA)/collagen nanofibrous graft.
In another study, a coronary artery stent that is coated with a thin electrospun nanofiber
membrane has been developed for the treatment of coronary artery perforation and is
being tested in human [211,212]. A summary of recent innovations relating nanofiber
scaffold materials and respective mechanical, electrical and crystallinity properties studied
for vascular differentiation, is listed in Table 4.

Figure 10. Vascular grafts fabricated with cellularized tubular HA/collagen nanofibrous scaffold
facilitate vascular reconstruction. H&E (hemotoxylin and eosin) staining (a), VVG (Verhoeff–Van
Gieson) staining (c) and fluorescent staining. (f)
Fibers 2023, 11, 39 23 of 40

The cross-section of a retrieved tubular HA/collagen nanofibrous graft 6 weeks after the transplant
at 10× (a,c,f) compared with rabbit carotid artery (b,d,g). Scale bars, 20 µm. Fluorescence relative to
vascular ECs (red), SMCs (green) and nuclei (blue). Quantification of the percentage of elastin area
(e), CD31 (endothelial cell marker), (h) and α-SMA (smooth muscle actin)-positive (i) in the retrieved
explants 6 weeks after transplant and carotid artery (n = 3). * p < 0.05, ** p < 0.01. Image reproduced
with permission from [210].

Table 4. List of the recent innovations relating fiber-based scaffolds for engineering vascular tissue
based on biophysical properties of the scaffold.

Type of Electrical
Material Used Mechanical Properties Results Reference
Electrospinning Properties

• Arterial vascular grafts with

differences in wall stiffness were The content of collagen varied
fabricated by altering the based on stiffness, with increased
PGS/PCL Coaxial electrospinning core-sheath structure - smooth muscle cell and elastin [213]
• Reduced swelling of the core content in the fibers with a
and eliminated stresses in thicker core
sheath

Functionalization with
fibronectin helps in fibrogenesis
Improved mechanical properties, while with decorin repelled
Polycarbonate–
Direct electrospinning including elasticity and burst - endothelial cells Functional [214]
urethane
pressure endothelium was formed in
dynamic conditions with
fibronectin functionalization

• Addition of aorta ECM

improved hydrophilicity
Decellularized • The scaffolds made from Increased cell viability and cell
extracellular matrix Direct electrospinning decellularized ECM had a - adherence with the use of ECM [215]
from aorta higher mechanical strength than aorta
PCL scaffold

A TEVG prepared from

Increased porosity of fibers with
PCL cotton and electrospun materials when
Direct electrospinning comparable mechanical properties - [216]
membranes implanted in rats had similar
of native tissue
characteristics as native vessels
Increased anti-burst pressure and Improved cell proliferation and
PCL Direct electrospinning - [217]
suture retention strength viability of cells
Improved cell viability and cell
proliferation with formation of
Improved mechanical strength and
PLGA Direct electrospinning - tight junctions in a coculture with [218]
burst pressure
smooth muscle cells and
endothelial cells

• Increased micro-vessel
• Randomly arranged fibrous
density and fewer
structure with high mechanical
calcifications after long-term
PCL/fibrin Direct electrospinning compliance - [219]
implantation in rats
• The scaffolds had controlled
• The graft induced the
degradation rates
regeneration of arteries

Increased cell adhesion,

Gelatin/PCL with High porosity of the scaffolds with
Direct electrospinning - proliferation, and increased [220]
chondroitin sulfate anticoagulant properties
endothelial cell responses

• Increased hydrophilicity of
scaffolds Conductive
• Concentration of gelatin Increased cell adhesion and cell
scaffolds with
provided control over proliferation demonstrated
PU, gelatin and electrical
Direct electrospinning mechanical properties and in vitro, characterized by a dense [221]
CNT conductivity such
degradation rates layer of myocardial and
as native
• Carbon nanotubes increased endothelial cells after 7 days
myocardium
mechanical strength
Fibers 2023, 11, 39 24 of 40

Table 4. Cont.

Type of Electrical
Material Used Mechanical Properties Results Reference
Electrospinning Properties

• Nanotopographical cues helped

in cell infiltration
PCL, • The electrospinning process The graft when implanted in a
polydioxanone Co-electrospinning provided flexibility in - porcine model demonstrated [222]
polydopamine improving mechanical good patency rates
properties, including tensile
strength and burst pressure

• PEO ratio influenced the

mechanical strength and
PLA and diameter of fibers Increased cell infiltration and
polyethylene oxide Coaxial electrospinning • Nanoscale pores were obtained - [223]
growth were observed in vitro
(PEO) due to phase separation during
the electrospinning process

Y-shaped structure was obtained by

Direct electrospinning Increased directional growth and
PCL/collagen electrospinning with topographical - [224]
with modified collector infiltration of endothelial cells
cues along a particular direction
Aligned scaffolds interacted more
Direct electrospinning Different types of topographical with endothelial cells than
Cellulose acetate,
with different types of cues were obtained in terms of - platelets, they also helped in [225]
chitosan, and PCL
collectors alignment of fibers and diameter increasing proliferation and
promoting angiogenesis

• Small-diameter fibers with

increased hydrophilicity and
Poly(L-lactide-co- mechanical properties were The cell proliferation and
caprolactone)/tussah Coaxial electrospinning obtained - adhesion were demonstrated [226]
silk fibroin • The core-sheath structure in vitro
enhanced the axial tensile
strength

• Nitric oxide generated in the

Fibrous scaffolds with good presented of GSH and GSNO
PCL/sulfonated • The scaffolds increased
Co-electrospinning mechanical properties, favoring - [227]
keratin endothelial cell proliferation
endothelial cell growth
and reduced platelet adhesion

Some of the fiber orientations

Direct electrospinning Different fiber orientations were were better than the other, but a
PU and PLA - [228]
with different collectors fabricated multiwalled structure was better
able to mimic the rat aorta
PEGylated Narrow pore size was obtained
Increased cell growth and
CdSe-ZnS quantum Direct electrospinning with increased mechanical - [229]
proliferation
dots in PCL properties

• The addition of PCL improved

mechanical properties of the
graft Improved cell viability
PET and PCL Direct electrospinning - [230]
• Increased suture retention demonstrated in vitro
strength

• The bilayer structure of the

vascular graft provided
interconnectivity and a porous Improved patency rate, cell
Bombyx mori-BM structure
Direct electrospinning - infiltration, graft remodeling, [231]
silk • The degradation of scaffold also neo-tissue formation
provided in vivo remodeling
ability

PCL and heparin

Vascular grafts with a pore
conjugated 50:50
Scaffolds with different pore sizes diameter smaller than 4 um had a
poly (l-lactide-co-ε- Coaxial electrospinning - [232]
were fabricated higher patency and survival rate
caprolactone)
in vivo
copolymer
Abbreviations: PU, polyurethane; PGS, poly(glycerol sebacate); PCL, polycaprolactone; PLLA, poly L-lactic acid;
PLGA, poly(lactic-co-glycolic acid); PEG, polyethylene glycol; GO, graphene oxide; PLA, poly lactic acid; PEO,
polyethylene oxide; PET, polyethylene terephthalate; CNT, carbon nanotubes.
Fibers 2023, 11, 39 25 of 40

3.3. Renal
The kidney serves as an important excretory organ by homeostatic regulation and
purifying out waste and toxins from the blood. Apart from this key function, the kidney
is also responsible for producing renin, erythropoietin, and prostaglandins. The kidney
has more than a million nephrons composed of several glomerulus and tubular structures
which aid in the filtration process. The glomerulus is covered by a Bowman’s capsule
with capillaries. These capillaries are surrounded by a negatively charged glomerular
basement membrane and podocytes. The tubular structures are composed of proximal
tubules, loop of Henle, distal tubules, and collecting ducts. The first stage of filtration
occurs in the glomerulus, followed by secondary filtration in the tubular structures. There
has been significant progress in understanding the morphogenesis of the kidney and
recovery of the kidney after an injury to understand the intricate mesenchymal–epithelial
transitions. Understanding the recovery process and functioning of the cells involved
helps in designing efficient scaffolds for renal tissue engineering. Understanding the ECM
composition of the kidney helps in better design of the scaffolds in accordance with the
mechanical properties. The basement membrane of the glomerulus is controlled by the
podocytes and the endothelium, and is mainly composed of structural and regulatory
proteins such as collagen (I, IV and VI), laminins, and heparan sulfate proteoglycans. The
mesangial cells in the glomerulus also produce ECM composed of tenascin, fibronectin
and proteoglycans, which play a role in regulating the electrical charge in the glomerulus.
The tubular structures have a different composition as compared to the glomerulus. The
ubiquitous and heterogenous ECM composition helps in providing a complex niche for
the proper functioning of the kidney. The following review by Vermue et al. (2021)
analyzes some of the latest design strategies for creating an artificial proximal tubule
using electrospinning [233]. A summary of recent innovations relating nanofiber scaffold
materials and respective mechanical, electrical and crystallinity properties studied for renal
differentiation, is listed in Table 5.

Table 5. List of the recent innovations relating fiber-based scaffolds for engineering renal tissue based
on biophysical properties of the scaffold.

Material Type of Electrical

Mechanical Properties Results Reference
Used Electrospinning Properties
Scaffolds with a porous
PCL and Improved cell–fiber and
Direct electrospinning structure with a fibrous - [234]
laminin cell–cell interaction.
morphology were fabricated

• L-DOPA and collagen

were coated to increase the
hydrophilicity of scaffolds
• The polymer Proximal tubules
concentration played a remained viable and
PCL Direct electrospinning major role in determining - [235]
maintained functionality
the mechanical properties for more than 3 weeks
and morphology of the
scaffold, and interacted
with the cells differently

Electrospinning with a
Polyvinylidene
rotating collector was used to Scaffolds with different
fluoride Direct electrospinning - [236]
obtain scaffold characteristics pore sizes were obtained
(PVDF)
such as native renal tissue
Abbreviations: PU, polyurethane; PGS, poly(glycerol sebacate); PCL, polycaprolactone; PLLA, poly L-lactic acid;
PLGA, poly(lactic-co-glycolic acid); PEG, polyethylene glycol; GO, graphene oxide; PLA, poly lactic acid; PEO,
polyethylene oxide; PET, polyethylene terephthalate; CNT, carbon nanotubes.
Fibers 2023, 11, 39 26 of 40

3.4. Nerve
The restorative function in humans after injury is limited to the central nervous sys-
tems, where functional unions are formed between severed ends, while peripheral nerve
tissue regeneration occurs after simple lesions. The process of peripheral nerve regener-
ation is complex, involving a myriad of factors. Nerve injuries can broadly be classified
into neurapraxia, axonotmesis, and neurotmesis, in increasing order of injury severity.
Following trauma, the neuron undergoes Wallerian degeneration, followed by several mor-
phological and chemical changes, which can broadly be called chromatolysis. The Schwann
cells are activated and macrophage recruitment to the injury site, helping in the cleanup
and serving as a guide for axonal regeneration (Bands of Bunger). They also produce
the required cytokines, which recruit macrophages to help with neuronal regeneration.
The neuron undergoes a change from transmission mode to a growth mode, whereby the
cytoskeletal proteins (slow-component b-tubulin and actin) are rapidly produced. This
neuronal regeneration is different in the case of the central nervous systems (CNS), where
the oligodendrocytes inhibit repair unlike the Schwann cells. Tissue engineering strategies
for peripheral nerve regeneration include the use of artificial nerve guides or conduits to
reconstruct the nerves when the gaps are larger than 3 cm. The Wallerian generation results
in growth factors that promote regeneration. The conduits are generally placed between
the two ends of the nerve to be repaired and the conduit provides guidance for the cells to
bridge the gap. Some of the essential mechanical features of the tissue include electrical
conductivity, protection against compression and appropriate tensile strength, which has
been well reviewed [237–239]. A summary of recent innovations relating nanofiber scaffold
materials and respective mechanical, electrical and crystallinity properties studied for
vascular differentiation, is listed in Table 6.

Table 6. List of the recent innovations relating fiber-based scaffolds for engineering neural tissue
based on biophysical properties of the scaffold.

Type of Elec- Electrical

Material Used Mechanical Properties Results Reference
trospinning Properties
Cellulose modified with
conductive polymers derivates Electrically
Increased cell adhesion and
(poly (N-(methacryl ethyl) The composite fibers had conductive
growth with clear-cell
pyrrole), poly Direct electro- high hydrophilicity, scaffolds that
morphology were observed [240]
(N-(2-hydroxyethyl) pyrrole), spinning surface roughness, and benefitted nerve
when PC12 cells were
poly (3-(Ethoxycarbonyl) porosity growth were
seeded on scaffolds
thiophene), and poly obtained
(3-thiophenethanol)
The use of melatonin
The fibers had higher
Direct electro- helped in the growth and
PCL/gelatin with melatonin hydrophilicity and - [241]
spinning proliferation of nerve cells
surface properties
in vitro
A 3D scaffold with higher
porosity, mechanical The prevascularization of
Silk fibroin/poly(l-lactic Direct electro-
strength with adequate the scaffolds aided in nerve [242]
acid-co-ε-caprolactone) spinning
strength for suture functional recovery
implantation
A shape memory
polymer was used to
fabricate conduits with The structure provides
Poly(lactide-co-trimethylene high porosity and uniform loading of cells
Direct electro-
carbonate) based on lactic acid mechanical strength by - and topographical cues for [243]
spinning
and trimethylene carbonate combining aligned fiber axon elongation and nerve
mat and random fiber regeneration
mat to form a
multichannel
Fibers 2023, 11, 39 27 of 40

Table 6. Cont.

Type of Elec- Electrical

Material Used Mechanical Properties Results Reference
trospinning Properties
Studies performed on
The use of Conduction
Pure porcine decellularized rabbits demonstrated the
Direct electro- proanthocyanidins velocities of
nerve matrix and elongation of axon and [244]
spinning increased the mechanical 8.86 ± 3.57 m/s
proanthocyanidins myelination using the
properties of the fibers. were obtained
scaffolds

• The presence of HA
helped in controlling
the degradation rates The scaffolds promoted
PCL conduit filled with Direct electro-
• The scaffolds were - Schwann cell regeneration [245]
collagen–hyaluronic acid spinning
porous and had good and axon growth
mechanical strength

• Different ratios of
polylactic and
polyglycolic acid were
used to obtain The rat Schwann cells
Direct electro- scaffolds with layers
PLGA - exhibited favorable growth [246]
spinning • The layers had on the scaffolds in vitro
different degradation
rates and mechanical
properties

Abbreviations: PCL, polycaprolactone; HA, hyaluronic acid; PLGA, poly(lactic-co-glycolic acid).

3.5. Cardiac Tissue

The heart is a marvel of engineering and is structurally and functionally a complex
pump that circulates blood within the body and is hence vital to survival. The heart is com-
posed of cardiomyocytes and fibroblasts with a collagen ECM. The muscles in the heart use
a lot of oxygen and are supported by vasculature. The heart tissue is electrically conductive
and uses this to control the contraction of muscles to pump blood. Failure of the heart is
beyond repair and is one of the leading causes of death in the USA. Myocardial infarction
of the heart muscles leads to the formation of scar tissues due to the action of macrophages,
endothelial cells, and fibroblasts that are recruited to the site through inflammatory signals.
The presence of scar tissue in turn reduces the mechanical capability of the heart to pump
blood, which ultimately leads to total heart failure. The reconstruction of heart tissue using
cardiac patches is currently being researched to aid in cardiac regeneration and prevention
of scar tissue formation. Apart from challenges in the choice of cells and type of grafts,
the mechanical and electrical properties are an important factor. The graft or patch needs
to work synchronously with the existing tissue in propagating the signal. This includes
mimicking the electrical resistance of the tissue or having similar mechanical properties
(Figure 11).
Fibers 2023,
Fibers 11, x FOR
2023, 11, 39PEER REVIEW 32 of 43
28 of 40

Figure 11. Comparison of rat cardiomyoblast (H9c2) cells on PCL, PCL-PANI, and PCL-PANI-PVDF
Figure 11. Comparison of rat cardiomyoblast (H9c2) cells on PCL, PCL-PANI, and PCL-PANI-PVDF
scaffolds on different days. Blue: DAPI nuclei stain; green: Alexa fluor®
® 488 Phalloidin for F-actin
scaffolds on different days. Blue: DAPI nuclei stain; green: Alexa fluor 488 Phalloidin for F-actin
filaments. Image
filaments. Imageadapted
adapted from [247].
from [247].

Roshanbinfar et
Roshanbinfar et al.
al. (2020)
(2020)reported thethe
reported useuse
of electrospun nanofiber
of electrospun scaffolds,
nanofiber using di-using
scaffolds,
rect electrospinning for cardiac tissue engineering. They used collagen (9.89%), hyaluronic
direct electrospinning for cardiac tissue engineering. They used collagen (9.89%), hyalu-
acid (1.1%), and polyaniline (PANi, 1.34%). The cardiomyocytes seeded on scaffolds ex-
ronic acid (1.1%), and polyaniline (PANi, 1.34%). The cardiomyocytes seeded on scaffolds
hibited longer contraction times, improved electrical coupling with lower cytotoxicity,
exhibited longercell
and enhanced contraction
attachmenttimes, improved
and cardiac electrical
function. coupling
The polymer with lower
composition cytotoxicity,
used gave
and enhanced
rise celland
to electrical attachment
mechanical and cardiac similar
properties function. Thenative
to the polymer composition
myocardium. used gave
The same
rise to electrical
study comparedand mechanical
outcomes properties similar
with combinations to the
of different native myocardium.
polymers, The same
including collagen,
study compared
hyaluronic outcomes
acid and with combinations
four different concentrationsofof different polymers, including collagen,
PANi [248].
hyaluronic acid and four different concentrations of PANi [248].
3.6. Retinal Pigment Epithelium
The retinal
3.6. Retinal Pigmentpigment epithelium (RPE) is a layer of cells in the eye that supports
Epithelium
photoreceptors that are responsible for sensing light. In vision disorders such as age-related
The retinal
macular pigmentthe
degeneration, epithelium (RPE) isand
RPE can degrade a layer
causeofa cells inphotoreceptor
loss of the eye that supports
function pho-
toreceptors that are responsible for sensing light. In vision disorders
and vision. A number of tissue engineering approaches are being advanced to repair such as age-related
the
macular degeneration,
RPE [249,250]. Some ofthe RPE
these can degrade
strategies and cause
entail growing the aRPE
lossinof photoreceptor
a lab on a fiber-basedfunction
and vision.and
scaffold A then
number of tissue
implanting theengineering
cell–scaffold approaches
construct intoare thebeing advanced
eye [251]. Several to repair the
studies
RPEhave demonstrated
[249,250]. Some that the electrospun
of these strategies fibers
entailmimic
growingthe structure
the RPE of in the native
a lab on aBruch’s
fiber-based
scaffold and then implanting the cell–scaffold construct into the eye [259].electrospun
membrane upon which the RPE resides in vivo, and that the RPE cultured on Several studies
fibers retained many of the properties of native the RPE [252–258]. Liu et al. (2014) found
have demonstrated that the electrospun fibers mimic the structure of the native Bruch’s
that the in vitro culture of RPE on fiber-based scaffolds led to a deeper pigmentation and
membrane upon which the RPE resides in vivo, and that the RPE cultured on electrospun
more uniform hexagonal tight junctions, and that the native RPE migrated onto the scaffolds
fibers retained
following many of the
implantation properties
[259]. In a studyofofnative
rodent the
andRPE [252–258].
pig models Liu laser-induced
that had et al. (2014) found
that the in vitro culture of RPE on fiber-based scaffolds led to a
RPE injuries, the induced pluripotent stem-cell-derived RPE that was generateddeeper pigmentation
on an and
more uniformfiber
electrospun hexagonal
patch was tight junctions,
implanted and to
and able that the native
improve RPE migrated
RPE functionality onto the scaf-
as compared
folds following implantation
to scaffold-free cell suspensions [259].
[260].In a study of rodent and pig models that had laser-
induced RPE injuries, the induced pluripotent stem-cell-derived RPE that was generated
on an electrospun fiber patch was implanted and able to improve RPE functionality as
compared to scaffold-free cell suspensions [260].

4. Challenges for Fiber-Based Scaffolds

Fibers 2023, 11, 39 29 of 40

4. Challenges for Fiber-Based Scaffolds

Several challenges exist for advancing fiber-based scaffolds for tissue engineering
applications. Herein, the focus has been on cell responses to fibers. However, in vitro cell
culture responses and responses following human implantation do not have a 1:1 correla-
tion. Cell differentiation on a fiber scaffold in vitro does not mean that an implanted fiber
scaffold will induce tissue regeneration in humans. It is important to improve the quality of
in vitro models to better mimic human physiological response. Next, environmental control
is important for fiber processing and manufacturing, but it is hard to achieve. Temperature,
humidity, particulates, and solvent build up in the atmosphere during electrospinning,
and can have a large impact on fiber properties. It is much more expensive and difficult
to establish environmental control than it is to set up an electrospinning system. There
are no clear mechanisms to establish reliable environmental controls to enable consistent
fiber manufacturing, and each lab and organization must develop their own mechanisms
to suit their budget and capability, largely through trial and error. Fully understanding
the environmental impact on nanofiber properties will be essential for the reproducibility
and scale-up of nanofiber technologies for tissue engineering. Another issue is profitability.
Often, the required implant for each patient may be tiny, with dimensions such as 1 mm
× 2 mm. A large-scale manufacturer may be able to make 1-m-wide rolls of fiber scaf-
folds, providing enough scaffold material for tens of thousands of patients in a day. In
addition, the amount of raw polymer material required to treat one patient may be a few
milligrams, with a value of only a few dollars. Nevertheless, the raw material provider
becomes exposed to significant medical liability by allowing their materials to be used
to treat humans. Thus, supplying a scaffold for humans use may have a high risk with a
small profit margin, which presents a challenge to investors that want to commercialize
fiber-scaffold-based therapies.

5. Conclusions
The vast body of research available on nanofiber scaffolds reiterates the wide range
of tunability, both in terms of materials that can be used and structures that can be cre-
ated. Different tissues rely on different cues: physical, chemical, and biological, that guide
cell growth, migration, and differentiation. Nanofiber scaffolds have several key advan-
tages, and have carved out a niche in the field of engineered tissues and regenerative
medicine. Several key aspects, such as mechanical properties, pore size, porosity, crys-
tallinity, alignment, and fiber diameter, support different requirements of the cells. Despite
the widespread use of nanofiber scaffolds for 3D cultures and tissue engineering, there
remains a lack of industry standard for the manufacturing, quality control, and guidelines
for the application of specific scaffolds for specific tissues or cells. Thus, there is a huge
unmet need in the standardization of 3D tissue culture approaches using nanofiber scaffolds
that may lead to conflicting outcomes. While the majority of the work has been focused on
identifying materials and scaffold properties, efforts to better understand the biomechanical
interactions will establish scaffold parameters needed for specific tissues. For example,
further investigation into how nanofiber alignment affects porosity, and how this in turn
affects tissue regeneration, is needed. Complex tissue systems with multilayered structures
and vasculature will benefit from further exploring polymer blends, controlling morphol-
ogy and nanotopography, and mimicking the physiological, biological, and mechanical
properties of the different layers of the tissue. Consideration should be given to improving
cost and time, and achieving the scale-up of manufacturing. In addition, investigating
a combination of electrospun scaffolds with other techniques, such as bioprinting or hy-
brid systems combining hydrogels and scaffolds, should be further explored. These have
the potential to improve in vitro outcomes and may prove successful in vivo. Nanofiber
scaffolds have several advantages that make them a viable option for tissue regeneration
applications. Combining this technology with others may prove to be the final step in
realizing tissue regeneration through tissue engineering.
Fibers 2023, 11, 39 30 of 40

Author Contributions: Conceptualization, J.D., S.N.H., S.F. and S.R.; Methodology, J.D., S.N.H., S.F.
and S.R.; Validation, S.N.H., S.F., C.G.S.J. and S.R.; Writing-original draft preparation, J.D., S.N.H.
and S.R.; Writing-review and editing, J.D., S.N.H., S.F., S.R. and C.G.S.J.; Project Administration, J.D.,
S.N.H. and S.R.; Supervision, S.N.H., S.F., S.R. and C.G.S.J. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: No new data were created or analyzed in this study. Data sharing is
not applicable to this article.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Na, S.; Collin, O.; Chowdhury, F.; Tay, B.; Ouyang, M.; Wang, Y.; Wang, N. Rapid signal transduction in living cells is a unique
feature of mechanotransduction. Proc. Natl. Acad. Sci. USA 2008, 105, 6626–6631. [CrossRef]
2. Dessauge, F.; Schleder, C.; Perruchot, M.-H.; Rouger, K. 3D in vitro models of skeletal muscle: Myopshere, myobundle and
bioprinted muscle construct. Vet. Res. 2021, 52, 72. [CrossRef] [PubMed]
3. Cha, S.H.; Lee, H.J.; Koh, W.G. Study of myoblast differentiation using multi-dimensional scaffolds consisting of nano and
micropatterns. Biomater. Res. 2017, 21, 1. [CrossRef]
4. Nguyen-Truong, M.; Li, Y.V.; Wang, Z. Mechanical Considerations of Electrospun Scaffolds for Myocardial Tissue and Regenerative
Engineering. Bioengineering 2020, 7, 122. [CrossRef] [PubMed]
5. Ding, S.; Kingshott, P.; Thissen, H.; Pera, M.; Wang, P.-Y. Modulation of human mesenchymal and pluripotent stem cell behavior
using biophysical and biochemical cues: A review. Biotechnol. Bioeng. 2017, 114, 260–280. [CrossRef]
6. Liu, H.; Paul, C.; Xu, M. Optimal Environmental Stiffness for Stem Cell Mediated Ischemic Myocardium Repair. In Adult Stem
Cells; Springer: New York, NY, USA, 2017; pp. 293–304.
7. Saxena, N.; Mogha, P.; Dash, S.; Majumder, A.; Jadhav, S.; Sen, S. Matrix elasticity regulates mesenchymal stem cell chemotaxis. J.
Cell Sci. 2018, 131, jcs211391. [CrossRef]
8. Wang, M.; Cheng, B.; Yang, Y.; Liu, H.; Huang, G.; Han, L.; Li, F.; Xu, F. Microchannel Stiffness and Confinement Jointly Induce
the Mesenchymal-Amoeboid Transition of Cancer Cell Migration. Nano Lett. 2019, 19, 5949–5958. [CrossRef] [PubMed]
9. Pauly, H.M.; Kelly, D.J.; Popat, K.C.; Trujillo, N.A.; Dunne, N.J.; McCarthy, H.O.; Donahue, T.L.H. Mechanical properties and
cellular response of novel electrospun nanofibers for ligament tissue engineering: Effects of orientation and geometry. J. Mech.
Behav. Biomed Mater. 2016, 61, 258–270. [CrossRef]
10. Choi, Y.S.; Vincent, L.G.; Lee, A.R.; Kretchmer, K.C.; Chirasatitsin, S.; Dobke, M.K.; Engler, A.J. The alignment and fusion assembly
of adipose-derived stem cells on mechanically patterned matrices. Biomaterials 2012, 33, 6943–6951. [CrossRef] [PubMed]
11. Jagiełło, A.; Hu, Q.; Castillo, U.; Botvinick, E. Patterned photocrosslinking to establish stiffness anisotropies in fibrous 3D
hydrogels. Acta Biomater. 2022, 141, 39–47. [CrossRef]
12. Yang, C.-Y.; Huang, W.-Y.; Chen, L.-H.; Liang, N.-W.; Wang, H.-C.; Lu, J.; Wang, X.; Wang, T.-W. Neural tissue engineering:
The influence of scaffold surface topography and extracellular matrix microenvironment. J. Mater. Chem. B 2021, 9, 567–584.
[CrossRef] [PubMed]
13. Lee, C.H.; Shin, H.J.; Cho, I.H.; Kang, Y.-M.; Kim, I.A.; Park, K.-D.; Shin, J.-W. Nanofiber alignment and direction of mechanical
strain affect the ECM production of human ACL fibroblast. Biomaterials 2005, 26, 1261–1270. [CrossRef] [PubMed]
14. Forte, G.; Pagliari, S.; Ebara, M.; Uto, K.; Van Tam, J.K.; Romanazzo, S.; Escobedo-Lucea, C.; Romano, E.; Di Nardo, P.; Traversa,
E.; et al. Substrate stiffness modulates gene expression and phenotype in neonatal cardiomyocytes in vitro. Tissue Eng. Part A
2012, 18, 1837–1848. [CrossRef] [PubMed]
15. ASTM F3510-21; Standard Guide for Characterizing Fiber-Based Constructs for TissueEngineered Medical Products. ASTM
International: West Conshohocken, PA, USA, 2021.
16. Roldán, G.J.C.; Martínez, Y.Q.; Gómez, L.M.A.; Vinasco, L.F.R.; Palacio, L.M.H. Influence of the molecular weight of polymer,
solvents and operational condition in the electrospinning of polycaprolactone. Rev. Fac. Ing. Univ. Antioq. 2017, 2017, 35–45.
[CrossRef]
17. Ameer, J.M.; Pr, A.K.; Kasoju, N. Strategies to Tune Electrospun Scaffold Porosity for Effective Cell Response in Tissue Engineering.
J. Funct. Biomater. 2019, 10, 30. [CrossRef]
18. Harley, B.A.; Kim, H.-D.; Zaman, M.H.; Yannas, I.V.; Lauffenburger, D.A.; Gibson, L.J. Microarchitecture of three-dimensional
scaffolds influences cell migration behavior via junction interactions. Biophys. J. 2008, 95, 4013–4024. [CrossRef]
19. Vagaská, B.; Bačáková, L.; Filová, E.; Balík, K. Osteogenic Cells on Bio-Inspired Materials for Bone Tissue Engineering. Physiol.
Res. 2010, 59, 309–322. [CrossRef]
20. Filippi, M.; Born, G.; Chaaban, M.; Scherberich, A. Natural Polymeric Scaffolds in Bone Regeneration. Front. Bioeng. Biotechnol.
2020, 8, 474. [CrossRef]
21. Smith, I.O.; Liu, X.H.; Smith, L.A.; Ma, P.X. Nanostructured polymer scaffolds for tissue engineering and regenerative medicine.
Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2009, 1, 226–236. [CrossRef]
Fibers 2023, 11, 39 31 of 40

22. Murphy, C.M.; Haugh, M.G.; O’Brien, F.J. The effect of mean pore size on cell attachment, proliferation and migration in
collagen-glycosaminoglycan scaffolds for bone tissue engineering. Biomaterials 2010, 31, 461–466. [CrossRef]
23. Chen, X.; Fu, X.; Shi, J.-G.; Wang, H. Regulation of the osteogenesis of pre-osteoblasts by spatial arrangement of electrospun
nanofibers in two- and three-dimensional environments. Nanomed. Nanotechnol. Biol. Med. 2013, 9, 1283–1292. [CrossRef]
24. Bružauskaitė, I.; Bironaitė, D.; Bagdonas, E.; Bernotienė, E. Scaffolds and cells for tissue regeneration: Different scaffold pore
sizes-different cell effects. Cytotechnology 2016, 68, 355–369. [CrossRef] [PubMed]
25. Nitti, P.; Gallo, N.; Natta, L.; Scalera, F.; Palazzo, B.; Sannino, A.; Gervaso, F. Influence of Nanofiber Orientation on Morphological
and Mechanical Properties of Electrospun Chitosan Mats. J. Healthc. Eng. 2018, 2018, 3651480. [CrossRef] [PubMed]
26. Li, W.-J.; Mauck, R.L.; Cooper, J.A.; Yuan, X.; Tuan, R.S. Engineering controllable anisotropy in electrospun biodegradable
nanofibrous scaffolds for musculoskeletal tissue engineering. J. Biomech. 2007, 40, 1686–1693. [CrossRef]
27. Patel, K.H.; Dunn, A.J.; Talovic, M.; Haas, G.J.; Marcinczyk, M.; Elmashhady, H.; Kalaf, E.G.; Sell, S.A.; Garg, K. Aligned nanofibers
of decellularized muscle ECM support myogenic activity in primary satellite cells in vitro. Biomed. Mater. 2019, 14, 035010.
[CrossRef] [PubMed]
28. Patel, K.H.; Talovic, M.; Dunn, A.J.; Patel, A.; Vendrell, S.; Schwartz, M.; Garg, K. Aligned nanofibers of decellularized muscle
extracellular matrix for volumetric muscle loss. J. Biomed. Mater. Res. Part B Appl. Biomater. 2020, 108, 2528–2537. [CrossRef]
[PubMed]
29. Kawano, T.; Sato, M.; Yabu, H.; Shimomura, M. Honeycomb-shaped surface topography induces differentiation of human
mesenchymal stem cells (hMSCs): Uniform porous polymer scaffolds prepared by the breath figure technique. Biomater. Sci. 2014,
2, 52–56. [CrossRef]
30. Narayanan, N.; Jiang, C.; Wang, C.; Uzunalli, G.; Whittern, N.; Chen, D.; Jones, O.G.; Kuang, S.; Deng, M. Harnessing Fiber
Diameter-Dependent Effects of Myoblasts Toward Biomimetic Scaffold-Based Skeletal Muscle Regeneration. Front. Bioeng.
Biotechnol. 2020, 8, 203. [CrossRef]
31. Kroehne, V.; Heschel, I.; Schügner, F.; Lasrich, D.; Bartsch, J.W.; Jockusch, H. Use of a novel collagen matrix with oriented pore
structure for muscle cell differentiation in cell culture and in grafts. J. Cell. Mol. Med. 2008, 12, 1640–1648. [CrossRef]
32. Lowery, J.L.; Datta, N.; Rutledge, G.C. Effect of fiber diameter, pore size and seeding method on growth of human dermal
fibroblasts in electrospun poly(ε-caprolactone) fibrous mats. Biomaterials 2010, 31, 491–504. [CrossRef]
33. Nguyen, J.H.; Chung, J.D.; Lynch, G.S.; Ryall, J.G. The Microenvironment Is a Critical Regulator of Muscle Stem Cell Activation
and Proliferation. Front. Cell Dev. Biol. 2019, 7, 254. [CrossRef] [PubMed]
34. Perez-Puyana, V.; Wieringa, P.; Yuste, Y.; de la Portilla, F.; Guererro, A.; Romero, A.; Moroni, L. Fabrication of hybrid scaffolds
obtained from combinations of PCL with gelatin or collagen via electrospinning for skeletal muscle tissue engineering. J. Biomed
Mater. Res. A 2021, 109, 1600–1612. [CrossRef] [PubMed]
35. Cooper, A.; Jana, S.; Bhattarai, N.; Zhang, M. Aligned chitosan-based nanofibers for enhanced myogenesis. J. Mater. Chem. 2010,
20, 8904. [CrossRef]
36. Badami, A.S.; Kreke, M.R.; Thompson, M.S.; Riffle, J.S.; Goldstein, A.S. Effect of fiber diameter on spreading, proliferation, and
differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates. Biomaterials 2006, 27, 596–606. [CrossRef]
37. Ma, B.; Xie, J.; Jiang, J.; Shuler, F.D.; E Bartlett, D. Rational design of nanofiber scaffolds for orthopedic tissue repair and
regeneration. Nanomedicine 2013, 8, 1459–1481. [CrossRef]
38. Bloise, N.; Berardi, E.; Gualandi, C.; Zaghi, E.; Gigli, M.; Duelen, R.; Ceccarelli, G.; Cortesi, E.E.; Costamagna, D.; Bruni,
G.; et al. Ether-Oxygen Containing Electrospun Microfibrous and Sub-Microfibrous Scaffolds Based on Poly(butylene 1,4-
cyclohexanedicarboxylate) for Skeletal Muscle Tissue Engineering. Int. J. Mol. Sci. 2018, 19, 3212. [CrossRef]
39. Jo, S.B.; Erdenebileg, U.; Dashnyam, K.; Jin, G.-Z.; Cha, J.-R.; El-Fiqi, A.; Knowles, J.C.; Patel, K.D.; Lee, H.-H.; Lee, J.-H.; et al.
Nano-graphene oxide/polyurethane nanofibers: Mechanically flexible and myogenic stimulating matrix for skeletal tissue
engineering. J. Tissue Eng. 2020, 11, 2041731419900424. [CrossRef]
40. Saburi, E.; Islami, M.; Hosseinzadeh, S.; Moghadam, A.S.; Mansour, R.N.; Azadian, E.; Joneidi, Z.; Nikpoor, A.R.; Ghadiani, M.H.;
Khodaii, Z.; et al. In vitro osteogenic differentiation potential of the human induced pluripotent stem cells augments when grown
on Graphene oxide-modified nanofibers. Gene 2019, 696, 72–79. [CrossRef]
41. Gaharwar, A.K.; Nikkhah, M.; Sant, S.; Khademhosseini, A. Anisotropic poly (glycerol sebacate)-poly (-caprolactone) electrospun
fibers promote endothelial cell guidance. Biofabrication 2014, 7, 015001. [CrossRef]
42. Shi, D.; Mi, G.; Wang, M.; Webster, T.J. In vitro and ex vivo systems at the forefront of infection modeling and drug discovery.
Biomaterials 2019, 198, 228–249. [CrossRef]
43. Sarkar, K.; Gomez, C.; Zambrano, S.; Ramirez, M.; de Hoyos, E.; Vasquez, H.; Lozano, K. Electrospinning to Forcespinning (TM).
Mater. Today 2010, 13, 12–14. [CrossRef]
44. Gajjar, C.R.; Stallrich, J.W.; Pasquinelli, M.A.; King, M.W. Process–Property Relationships for Melt-Spun Poly(lactic acid) Yarn.
ACS Omega 2021, 6, 15920–15928. [CrossRef] [PubMed]
45. Dorthé, E.W.; Williams, A.B.; Grogan, S.P.; D’lima, D.D. Pneumatospinning Biomimetic Scaffolds for Meniscus Tissue Engineering.
Front. Bioeng. Biotechnol. 2022, 10, 810705. [CrossRef] [PubMed]
46. Song, J.; Li, Z.; Wu, H. Blowspinning: A New Choice for Nanofibers. ACS Appl. Mater. Interfaces 2020, 12, 33447–33464. [CrossRef]
47. Brown, T.D.; Slotosch, A.; Thibaudeau, L.; Taubenberger, A.; Loessner, D.; Vaquette, C.; Dalton, P.D.; Hutmacher, D.W. Design and
Fabrication of Tubular Scaffolds via Direct Writing in a Melt Electrospinning Mode. Biointerphases 2012, 7, 13. [CrossRef]
Fibers 2023, 11, 39 32 of 40

48. Breitenbach, J. Melt extrusion: From process to drug delivery technology. Eur. J. Pharm. Biopharm. 2002, 54, 107–117. [CrossRef]
49. Lavin, D.M.; Harrison, M.W.; Tee, L.Y.; Wei, K.A.; Mathiowitz, E. A novel wet extrusion technique to fabricate self-assembled
microfiber scaffolds for controlled drug delivery. J. Biomed. Mater. Res. A 2012, 100, 2793–2802. [CrossRef]
50. Mohd Pu’Ad, N.A.S.; Haq, R.H.A.; Noh, H.M.; Abdullah, H.Z.; Idris, M.I.; Lee, T.C. Review on the fabrication of fused deposition
modelling (FDM) composite filament for biomedical applications. Mater. Today Proc. 2020, 29, 228–232. [CrossRef]
51. Martella, D.; Parmeggiani, C. Advances in Cell Scaffolds for Tissue Engineering: The Value of Liquid Crystalline Elastomers.
Chem. A Eur. J. 2018, 24, 12206–12220. [CrossRef]
52. Cheng, X.; Gurkan, U.A.; Dehen, C.J.; Tate, M.P.; Hillhouse, H.W.; Simpson, G.J.; Akkus, O. An electrochemical fabrication process
for the assembly of anisotropically oriented collagen bundles. Biomaterials 2008, 29, 3278–3288. [CrossRef]
53. Yadavalli, N.S.; Asheghali, D.; Tokarev, A.; Zhang, W.; Xie, J.; Minko, S. Gravity Drawing of Micro- and Nanofibers for Additive
Manufacturing of Well-Organized 3D-Nanostructured Scaffolds. Small 2020, 16, 1907422. [CrossRef]
54. Wu, S.; Wang, Y.; Streubel, P.N.; Duan, B. Living nanofiber yarn-based woven biotextiles for tendon tissue engineering using cell
tri-culture and mechanical stimulation. Acta Biomater. 2017, 62, 102–115. [CrossRef]
55. Wu, S.; Duan, B.; Liu, P.; Zhang, C.; Qin, X.; Butcher, J.T. Fabrication of Aligned Nanofiber Polymer Yarn Networks for Anisotropic
Soft Tissue Scaffolds. Acs Appl. Mater. Interfaces 2016, 8, 16950–16960. [CrossRef]
56. Wu, T.; Zhang, J.; Wang, Y.; Li, D.; Sun, B.; El-Hamshary, H.; Yin, M.; Mo, X. Fabrication and preliminary study of a biomimetic
tri-layer tubular graft based on fibers and fiber yarns for vascular tissue engineering. Mater. Sci. Eng. C-Mater. Biol. Appl. 2018, 82,
121–129. [CrossRef] [PubMed]
57. Chartrain, N.A.; Williams, C.B.; Whittington, A.R. A review on fabricating tissue scaffolds using vat photopolymerization. Acta
Biomater. 2018, 74, 90–111. [CrossRef] [PubMed]
58. Ahn, J.-H.; Kim, J.; Han, G.; Kim, D.; Cheon, K.-H.; Lee, H.; Kim, H.-E.; Kim, Y.-J.; Jang, T.-S.; Jung, H.-D. 3D-printed biodegradable
composite scaffolds with significantly enhanced mechanical properties via the combination of binder jetting and capillary rise
infiltration process. Addit. Manuf. 2021, 41, 101988. [CrossRef]
59. Kim, C.K.; Jeong, J.I.; Choi, S.G.; Kim, J.H.; Cho, Y.T. High-throughput directed energy deposition process with an optimized
scanning nozzle. J. Mater. Process. Technol. 2021, 295, 117165. [CrossRef]
60. Zhu, J.L.; Kaufman, L.J. Collagen I Self-Assembly: Revealing the Developing Structures that Generate Turbidity. Biophys. J. 2014,
106, 1822–1831. [CrossRef]
61. FDA. Guidance for the Preparation of a Premarket Notification Application for a Surgical Mesh. In Guidance for Industry and/or for
FDA Reviewers/Staff and/or Compliance; Food and Drug Administration, U.S. Department of Health and Human Services, Center
for Devices and Radiological Health, Eds.; FDA: Silver Spring, MD, USA, 1999.
62. Mao, N.; Russell, S.J.; Pourdeyhimi, B. Characterisation, testing, and modelling of nonwoven fabrics. In Handbook of Nonwovens;
Elsevier: Amsterdam, The Netherlands, 2022; pp. 509–626.
63. Tucker, N.; Stanger, J.J.; Staiger, M.P.; Razzaq, H.; Hofman, K. The history of the science and technology of electrospinning from
1600 to 1995. J. Eng. Fibers Fabr. 2012, 7 (Suppl. S2), 155892501200702S10. [CrossRef]
64. Bhardwaj, N.; Kundu, S.C. Electrospinning: A fascinating fiber fabrication technique. Biotechnol. Adv. 2010, 28, 325–347. [CrossRef]
65. Teo, W.E.; Ramakrishna, S. A review on electrospinning design and nanofibre assemblies. Nanotechnology 2006, 17, R89. [CrossRef]
66. Subbiah, T.; Bhat, G.S.; Tock, R.W.; Parameswaran, S.; Ramkumar, S.S. Electrospinning of nanofibers. J. Appl. Polym. Sci. 2005, 96,
557–569. [CrossRef]
67. Bognitzki, M.; Czado, W.; Frese, T.; Schaper, A.; Hellwig, M.; Steinhart, M.; Greiner, A.; Wendorff, J.H. Nanostructured fibers via
electrospinning. Adv. Mater. 2001, 13, 70–72. [CrossRef]
68. Li, D.; Xia, Y. Electrospinning of nanofibers: Reinventing the wheel? Adv. Mater. 2004, 16, 1151–1170. [CrossRef]
69. Tong, H.-W.; Wang, M. Electrospinning of fibrous polymer scaffolds using positive voltage or negative voltage: A comparative
study. Biomed. Mater. 2010, 5, 054110. [CrossRef]
70. Doshi, J.; Reneker, D.H. Electrospinning process and applications of electrospun fibers. J. Electrost. 1995, 35, 151–160. [CrossRef]
71. Mailley, D.; Hebraud, A.; Schlatter, G. A review on the impact of humidity during electrospinning: From the nanofiber structure
engineering to the applications. Macromol. Mater. Eng. 2021, 306, 2100115. [CrossRef]
72. Pelipenko, J.; Kristl, J.; Janković, B.; Baumgartner, S.; Kocbek, P. The impact of relative humidity during electrospinning on the
morphology and mechanical properties of nanofibers. Int. J. Pharm. 2013, 456, 125–134. [CrossRef] [PubMed]
73. Cai, Y.; Gevelber, M. The effect of relative humidity and evaporation rate on electrospinning: Fiber diameter and measurement
for control implications. J. Mater. Sci. 2013, 48, 7812–7826. [CrossRef]
74. Kumar, P. Effect of Colletor on Electrospinning to Fabricate Aligned Nano Fiber. Bachelor’s Thesis, National Institute of
Technology, Rourkela, India, 2012.
75. Hohman, M.M.; Shin, M.; Rutledge, G.; Brenner, M.P. Electrospinning and electrically forced jets. II. Applications. Phys. Fluids
2001, 13, 2221–2236. [CrossRef]
76. Rosell-Llompart, J.; Grifoll, J.; Loscertales, I.G. Electrosprays in the cone-jet mode: From Taylor cone formation to spray
development. J. Aerosol Sci. 2018, 125, 2–31. [CrossRef]
77. Yarin, A.L.; Koombhongse, S.; Reneker, D.H. Taylor cone and jetting from liquid droplets in electrospinning of nanofibers. J. Appl.
Phys. 2001, 90, 4836–4846. [CrossRef]
Fibers 2023, 11, 39 33 of 40

78. Stanger, J.; Tucker, N.; Kirwan, K.; Staiger, M. Effect of charge density on the Taylor cone in electrospinning. Int. J. Mod. Phys. B
2009, 23, 1956–1961. [CrossRef]
79. Vaseashta, A. Controlled formation of multiple Taylor cones in electrospinning process. Appl. Phys. Lett. 2007, 90, 093115.
[CrossRef]
80. Li, Z.; Wang, C. Effects of working parameters on electrospinning. In One-Dimensional Nanostructures; Springer: Berlin/Heidelberg,
Germany, 2013; pp. 15–28.
81. Li, Y.; Zhu, J.; Cheng, H.; Li, G.; Cho, H.; Jiang, M.; Gao, Q.; Zhang, X. Developments of advanced electrospinning techniques: A
critical review. Adv. Mater. Technol. 2021, 6, 2100410. [CrossRef]
82. Nagam Hanumantharao, S.; Que, C.; Rao, S. Self-assembly of 3D nanostructures in electrospun polycaprolactone-polyaniline
fibers and their application as scaffolds for tissue engineering. Materialia 2019, 6, 100296. [CrossRef]
83. Park, S.M.; Eom, S.; Kim, W.; Kim, D.S. Role of grounded liquid collectors in precise patterning of electrospun nanofiber mats.
Langmuir 2018, 34, 284–290. [CrossRef]
84. Li, S.; Lee, B.K. Electrospinning of circumferentially aligned polymer nanofibers floating on rotating water collector. J. Appl.
Polym. Sci. 2020, 137, 48759. [CrossRef]
85. Zhang, D.; Chang, J. Electrospinning of three-dimensional nanofibrous tubes with controllable architectures. Nano Lett. 2008, 8,
3283–3287. [CrossRef] [PubMed]
86. Zhang, D.; Chang, J. Patterning of electrospun fibers using electroconductive templates. Adv. Mater. 2007, 19, 3664–3667.
[CrossRef]
87. Lei, T.; Xu, Z.; Cai, X.; Xu, L.; Sun, D. New insight into gap electrospinning: Toward meter-long aligned nanofibers. Langmuir
2018, 34, 13788–13793. [CrossRef]
88. Liu, Y.; Zhang, X.; Xia, Y.; Yang, H. Magnetic-field-assisted electrospinning of aligned straight and wavy polymeric nanofibers.
Adv. Mater. 2010, 22, 2454–2457. [CrossRef]
89. Dalton, P.D.; Klee, D.; Möller, M. Electrospinning with dual collection rings. Polymer 2005, 46, 611–614. [CrossRef]
90. Yoon, J.; Yang, H.-S.; Lee, B.-S.; Yu, W.-R. Recent progress in coaxial electrospinning: New parameters, various structures, and
wide applications. Adv. Mater. 2018, 30, 1704765. [CrossRef]
91. Khalf, A.; Madihally, S.V. Recent advances in multiaxial electrospinning for drug delivery. Eur. J. Pharm. Biopharm. 2017, 112, 1–17.
[CrossRef]
92. Bazilevsky, A.V.; Yarin, A.L.; Megaridis, C.M. Co-electrospinning of core- shell fibers using a single-nozzle technique. Langmuir
2007, 23, 2311–2314. [CrossRef] [PubMed]
93. Wannatong, L.; Sirivat, A.; Supaphol, P. Effects of solvents on electrospun polymeric fibers: Preliminary study on polystyrene.
Polym. Int. 2004, 53, 1851–1859. [CrossRef]
94. Luo, C.; Nangrejo, M.; Edirisinghe, M. A novel method of selecting solvents for polymer electrospinning. Polymer 2010, 51,
1654–1662. [CrossRef]
95. Hodge, J.; Quint, C. The improvement of cell infiltration in an electrospun scaffold with multiple synthetic biodegradable
polymers using sacrificial PEO microparticles. J. Biomed. Mater. Res. Part A 2019, 107, 1954–1964. [CrossRef]
96. Dang, J.M.; Leong, K.W. Natural polymers for gene delivery and tissue engineering. Adv. Drug Deliv. Rev. 2006, 58, 487–499.
[CrossRef] [PubMed]
97. Sell, S.A.; Wolfe, P.S.; Garg, K.; McCool, J.M.; Rodriguez, I.A.; Bowlin, G.L. The use of natural polymers in tissue engineering: A
focus on electrospun extracellular matrix analogues. Polymers 2010, 2, 522–553. [CrossRef]
98. Keshvardoostchokami, M.; Majidi, S.S.; Huo, P.; Ramachandran, R.; Chen, M.; Liu, B. Electrospun nanofibers of natural and
synthetic polymers as artificial extracellular matrix for tissue engineering. Nanomaterials 2020, 11, 21. [CrossRef] [PubMed]
99. Tong, X.; Pan, W.; Su, T.; Zhang, M.; Dong, W.; Qi, X. Recent advances in natural polymer-based drug delivery systems. React.
Funct. Polym. 2020, 148, 104501. [CrossRef]
100. Janoušková, O. Synthetic polymer scaffolds for soft tissue engineering. Physiol. Res. 2018, 67, S335–S348. [CrossRef]
101. Ghasemi-Mobarakeh, L.; Kolahreez, D.; Ramakrishna, S.; Williams, D. Key terminology in biomaterials and biocompatibility.
Curr. Opin. Biomed. Eng. 2019, 10, 45–50. [CrossRef]
102. Nagam Hanumantharao, S. A 3D Biomimetic Scaffold Using Electrospinning for Tissue Engineering Applications; Michigan Technologi-
cal University: Houghton, MI, USA, 2017; p. 75.
103. Ricotti, L.; Polini, A.; Genchi, G.G.; Ciofani, G.; Iandolo, D.; Vazão, H.; Mattoli, V.; Ferreira, L.; Menciassi, A.; Pisignano, D.
Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun
nanofibrous PHB scaffolds. Biomed. Mater. 2012, 7, 035010. [CrossRef] [PubMed]
104. Fujie, T.; Shi, X.; Ostrovidov, S.; Liang, X.; Nakajima, K.; Chen, Y.; Wu, H.; Khademhosseini, A. Spatial coordination of cell
orientation directed by nanoribbon sheets. Biomaterials 2015, 53, 86–94. [CrossRef] [PubMed]
105. Jana, S.; Leung, M.; Chang, J.; Zhang, M. Effect of nano- and micro-scale topological features on alignment of muscle cells and
commitment of myogenic differentiation. Biofabrication 2014, 6, 035012. [CrossRef]
106. Das, S.; Browne, K.D.; Laimo, F.A.; Maggiore, J.C.; Hilman, M.C.; Kaisaier, H.; Aguilar, C.A.; Ali, Z.S.; Mourkioti, F.; Cullen,
D.K. Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss.
Commun. Biol. 2020, 3, 330. [CrossRef]
Fibers 2023, 11, 39 34 of 40

107. Ma, P.X.; Zhang, R. Microtubular architecture of biodegradable polymer scaffolds. J. Biomed. Mater. Res. 2001, 56, 469–477.
[CrossRef]
108. Rowland, D.C.; Aquilina, T.; Klein, A.; Hakimi, O.; Alexis-Mouthuy, P.; Carr, A.J.; Snelling, S.J. A comparative evaluation of the
effect of polymer chemistry and fiber orientation on mesenchymal stem cell differentiation. J. Biomed. Mater. Res. A 2016, 104,
2843–2853. [CrossRef] [PubMed]
109. Hu, L.-Y.; Mileti, C.J.; Loomis, T.; Brashear, S.E.; Ahmad, S.; Chellakudam, R.R.; Wohlgemuth, R.P.; Gionet-Gonzales, M.A.; Leach,
J.K.; Smith, L.R. Skeletal muscle progenitors are sensitive to collagen architectural features of fibril size and cross linking. Am. J.
Physiol. Cell Physiol. 2021, 321, C330–C342. [CrossRef] [PubMed]
110. Suresh, S.; Becker, A.; Glasmacher, B. Impact of apparatus orientation and gravity in electrospinning—A review of empirical
evidence. Polymers 2020, 12, 2448. [CrossRef]
111. Suresh, S.; Gryshkov, O.; Glasmacher, B. Impact of setup orientation on blend electrospinning of poly-ε-caprolactone-gelatin
scaffolds for vascular tissue engineering. Int. J. Artif. Organs 2018, 41, 801–810. [CrossRef] [PubMed]
112. Nagam Hanumantharao, S.; Rao, S. Multi-functional electrospun nanofibers from polymer blends for scaffold tissue engineering.
Fibers 2019, 7, 66. [CrossRef]
113. Simon, C.G., Jr.; Kumar, G.; Tison, C.K. Nanofiber Scaffolds Induce Morphological Changes in hBMSCs Critical for Osteogenic
Differentiation; NIST: Gaithersburg, MD, USA, 2011.
114. Kumar, G.; Tison, C.K.; Chatterjee, K.; Pine, P.S.; McDaniel, J.H.; Salit, M.L.; Young, M.F.; Simon, C.G. The determination of stem
cell fate by 3D scaffold structures through the control of cell shape. Biomaterials 2011, 32, 9188–9196. [CrossRef] [PubMed]
115. Farooque, T.M.; Camp, C.H., Jr.; Tison, C.K.; Kumar, G.; Parekh, S.H.; Simon, C.G., Jr. Measuring stem cell dimensionality in
tissue scaffolds. Biomaterials 2014, 35, 2558–2567. [CrossRef]
116. Tutak, W.; Jyotsnendu, G.; Bajcsy, P.; Simon, C.G. Nanofiber scaffolds influence organelle structure and function in bone marrow
stromal cells. J. Biomed. Mater. Res. B Appl. Biomater. 2017, 105, 989–1001. [CrossRef]
117. Thomas, C.H.; Collier, J.H.; Sfeir, C.S.; Healy, K.E. Engineering gene expression and protein synthesis by modulation of nuclear
shape. Proc. Natl. Acad. Sci. USA 2002, 99, 1972–1977. [CrossRef]
118. Tsimbouri, P.M.; Murawski, K.; Hamilton, G.; Herzyk, P.; Oreffo, R.O.; Gadegaard, N.; Dalby, M.J. A genomics approach in
determining nanotopographical effects on MSC phenotype. Biomaterials 2013, 34, 2177–2184. [CrossRef]
119. Chen, D.; Sarkar, S.; Candia, J.; Florczyk, S.J.; Bodhak, S.; Driscoll, M.K.; Simon, C.G.; Dunkers, J.P.; Losert, W. Machine learning
based methodology to identify cell shape phenotypes associated with microenvironmental cues. Biomaterials 2016, 104, 104–118.
[CrossRef]
120. Chen, D.; Dunkers, J.P.; Losert, W.; Sarkar, S. Early time-point cell morphology classifiers successfully predict human bone
marrow stromal cell differentiation modulated by fiber density in nanofiber scaffolds. Biomaterials 2021, 274, 120812. [CrossRef]
121. Xie, J.; Shen, H.; Yuan, G.; Lin, K.; Su, J. The effects of alignment and diameter of electrospun fibers on the cellular behaviors and
osteogenesis of BMSCs. Mater. Sci. Eng. C Mater. Biol. Appl. 2021, 120, 111787. [CrossRef] [PubMed]
122. Ma, J.; He, X.; Jabbari, E. Osteogenic differentiation of marrow stromal cells on random and aligned electrospun poly(L-lactide)
nanofibers. Ann. Biomed. Eng. 2011, 39, 14–25. [CrossRef] [PubMed]
123. Pandey, S.; Rathore, K.; Johnson, J.; Cekanova, M. Aligned nanofiber material supports cell growth and increases osteogenesis in
canine adipose-derived mesenchymal stem cells in vitro. J. Biomed. Mater. Res. A 2018, 106, 1780–1788. [CrossRef]
124. Newman, P.; Galenano-Niño, J.L.; Graney, P.; Razal, J.M.; Minett, A.I.; Ribas, J.; Ovalle-Robles, R.; Biro, M.; Zreiqat, H. Relationship
between nanotopographical alignment and stem cell fate with live imaging and shape analysis. Sci. Rep. 2016, 6, 37909. [CrossRef]
125. Kolambkar, Y.M.; Bajin, M.; Wojtowicz, A.; Hutmacher, D.W.; García, A.J.; Guldberg, R.E. Nanofiber orientation and surface
functionalization modulate human mesenchymal stem cell behavior in vitro. Tissue Eng. Part A 2014, 20, 398–409. [CrossRef]
126. Lanfer, B.; Seib, F.P.; Freudenberg, U.; Stamov, D.; Bley, T.; Bornhäuser, M.; Werner, C. The growth and differentiation of
mesenchymal stem and progenitor cells cultured on aligned collagen matrices. Biomaterials 2009, 30, 5950–5958. [CrossRef]
127. Kim, M.-J.; Lee, B.; Yang, K.; Park, J.; Jeon, S.; Um, S.H.; Kim, D.-I.; Im, S.G.; Cho, S.-W. BMP-2 peptide-functionalized
nanopatterned substrates for enhanced osteogenic differentiation of human mesenchymal stem cells. Biomaterials 2013, 34,
7236–7246. [CrossRef]
128. Vo, T.N.; Tabata, Y.; Mikos, A.G. Effects of cellular parameters on the in vitro osteogenic potential of dual-gelling mesenchymal
stem cell-laden hydrogels. J. Biomater. Sci. Polym. Ed. 2016, 27, 1277–1290. [CrossRef] [PubMed]
129. Hu, X.; Park, S.-H.; Gil, E.S.; Xia, X.-X.; Weiss, A.; Kaplan, D.L. The influence of elasticity and surface roughness on myogenic and
osteogenic-differentiation of cells on silk-elastin biomaterials. Biomaterials 2011, 32, 8979–8989. [CrossRef] [PubMed]
130. Shin, Y.C.; Lee, J.H.; Jin, L.; Kim, M.J.; Kim, Y.-J.; Hyun, J.K.; Jung, T.-G.; Hong, S.W.; Han, D.-W. Stimulated myoblast
differentiation on graphene oxide-impregnated PLGA-collagen hybrid fibre matrices. J. Nanobiotechnol. 2015, 13, 21. [CrossRef]
131. Shimizu, K.; Fujita, H.; Nagamori, E. Alignment of skeletal muscle myoblasts and myotubes using linear micropatterned surfaces
ground with abrasives. Biotechnol. Bioeng. 2009, 103, 631–638. [CrossRef] [PubMed]
132. Patel, A.; Mukundan, S.; Wang, W.; Karumuri, A.; Sant, V.; Mukhopadhyay, S.M.; Sant, S. Carbon-based hierarchical scaffolds for
myoblast differentiation: Synergy between nano-functionalization and alignment. Acta Biomater. 2016, 32, 77–88. [CrossRef]
133. Yeo, M.; Kim, G. Nano/microscale topographically designed alginate/PCL scaffolds for inducing myoblast alignment and
myogenic differentiation. Carbohydr. Polym. 2019, 223, 115041. [CrossRef] [PubMed]
Fibers 2023, 11, 39 35 of 40

134. Zhou, J.; Yang, X.; Liu, W.; Wang, C.; Shen, Y.; Zhang, F.; Zhu, H.; Sun, H.; Chen, J.; Lam, J.; et al. Injectable OPF/graphene
oxide hydrogels provide mechanical support and enhance cell electrical signaling after implantation into myocardial infarct.
Theranostics 2018, 8, 3317–3330. [CrossRef] [PubMed]
135. Malikmammadov, E.; Tanir, T.E.; Kiziltay, A.; Hasirci, V.; Hasirci, N. PCL and PCL-based materials in biomedical applications. J.
Biomater. Sci. Polym. Ed. 2018, 29, 863–893. [CrossRef]
136. Homaeigohar, S.; Boccaccini, A.R. Nature-Derived and Synthetic Additives to poly(ε-Caprolactone) Nanofibrous Systems for
Biomedicine; an Updated Overview. Front. Chem. 2022, 9, 809676. [CrossRef] [PubMed]
137. Gao, J.; Chen, S.; Tang, D.; Jiang, L.; Shi, J.; Wang, S. Mechanical Properties and Degradability of Electrospun PCL/PLGA Blended
Scaffolds as Vascular Grafts. Trans. Tianjin Univ. 2018, 25, 152–160. [CrossRef]
138. Leung, M.; Cooper, A.; Jana, S.; Tsao, C.-T.; Petrie, T.A.; Zhang, M. Nanofiber-based in vitro system for high myogenic differentia-
tion of human embryonic stem cells. Biomacromolecules 2013, 14, 4207–4216. [CrossRef]
139. Jana, S.; Cooper, A.; Zhang, M. Chitosan scaffolds with unidirectional microtubular pores for large skeletal myotube generation.
Adv. Healthc. Mater. 2013, 2, 557–561. [CrossRef]
140. Stern, M.M.; Myers, R.L.; Hammam, N.; Stern, K.A.; Eberli, D.; Kritchevsky, S.B.; Soker, S.; Van Dyke, M. The influence of
extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex
vivo. Biomaterials 2009, 30, 2393–2399. [CrossRef] [PubMed]
141. Wingate, K.; Bonani, W.; Tan, Y.; Bryant, S.; Tan, W. Compressive elasticity of three-dimensional nanofiber matrix directs
mesenchymal stem cell differentiation to vascular cells with endothelial or smooth muscle cell markers. Acta Biomater. 2012, 8,
1440–1449. [CrossRef] [PubMed]
142. Somaiah, C.; Kumar, A.; Mawrie, D.; Sharma, A.; Patil, S.D.; Bhattacharyya, J.; Swaminathan, R.; Jaganathan, B.G. Collagen
Promotes Higher Adhesion, Survival and Proliferation of Mesenchymal Stem Cells. PLoS ONE 2015, 10, e0145068. [CrossRef]
[PubMed]
143. Bertram, U.; Steiner, D.; Poppitz, B.; Dippold, D.; Köhn, K.; Beier, J.P.; Detsch, R.; Boccaccini, A.R.; Schubert, D.W.; Horch, R.E.;
et al. Vascular Tissue Engineering: Effects of Integrating Collagen into a PCL Based Nanofiber Material. BioMed Res. Int. 2017,
2017, 9616939. [CrossRef]
144. Choi, J.S.; Lee, S.J.; Christ, G.J.; Atala, A.; Yoo, J.J. The influence of electrospun aligned poly(epsilon-caprolactone)/collagen
nanofiber meshes on the formation of self-aligned skeletal muscle myotubes. Biomaterials 2008, 29, 2899–2906. [CrossRef]
145. Brzeska, J.; Tercjak, A.; Sikorska, W.; Kowalczuk, M.; Rutkowska, M. Morphology and Physicochemical Properties of Branched
Polyurethane/Biopolymer Blends. Polymers 2019, 12, 16. [CrossRef]
146. McClure, M.J.; Garg, K.; Simpson, D.G.; Ryan, J.J.; Sell, S.A.; Bowlin, G.L.; Ericksen, J.J. The influence of platelet-rich plasma on
myogenic differentiation. J. Tissue Eng. Regen. Med. 2016, 10, E239–E249. [CrossRef]
147. Shin, Y.C.; Kim, C.; Song, S.-J.; Jun, S.; Kim, C.-S.; Hong, S.W.; Hyon, S.-H.; Han, D.-W.; Oh, J.-W. Ternary Aligned Nanofibers
of RGD Peptide-Displaying M13 Bacteriophage/PLGA/Graphene Oxide for Facilitated Myogenesis. Nanotheranostics 2018, 2,
144–156. [CrossRef]
148. Wen, F.; Wong, H.K.; Tay, C.Y.; Yu, H.; Li, H.; Yu, T.; Tijore, A.; Boey, F.Y.C.; Venkatraman, S.S.; Tan, L.P.; et al. Induction of
myogenic differentiation of human mesenchymal stem cells cultured on Notch agonist (Jagged-1) modified biodegradable scaffold
surface. ACS Appl. Mater. Interfaces 2014, 6, 1652–1661. [CrossRef] [PubMed]
149. Jahanmard, F.; Eslaminejad, M.B.; Amani-Tehran, M.; Zarei, F.; Rezaei, N.; Croes, M.; Yavari, S.A. Incorporation of F-MWCNTs
into electrospun nanofibers regulates osteogenesis through stiffness and nanotopography. Mater. Sci. Eng. C Mater. Biol. Appl.
2020, 106, 110163. [CrossRef]
150. Gaharwar, A.K.; Mukundan, S.; Karaca, E.; Dolatshahi-Pirouz, A.; Patel, A.; Rangarajan, K.; Mihaila, S.M.; Iviglia, G.; Zhang, H.;
Khademhosseini, A. Nanoclay-enriched poly(varepsilon-caprolactone) electrospun scaffolds for osteogenic differentiation of
human mesenchymal stem cells. Tissue Eng. Part A 2014, 20, 2088–2101. [CrossRef]
151. Wang, X.; Gittens, R.A.; Song, R.; Tannenbaum, R.; Olivares-Navarrete, R.; Schwartz, Z.; Chen, H.; Boyan, B.D. Effects of structural
properties of electrospun TiO2 nanofiber meshes on their osteogenic potential. Acta Biomater. 2012, 8, 878–885. [CrossRef]
152. Chen, H.; Huang, X.; Zhang, M.; Damanik, F.; Baker, M.B.; Leferink, A.; Yuan, H.; Truckenmüller, R.; van Blitterswijk, C.;
Moroni, L. Tailoring surface nanoroughness of electrospun scaffolds for skeletal tissue engineering. Acta Biomater. 2017, 59, 82–93.
[CrossRef]
153. Faia-Torres, A.B.; Charnley, M.; Goren, T.; Guimond-Lischer, S.; Rottmar, M.; Maniura-Weber, K.; Spencer, N.D.; Reis, R.L.; Textor,
M.; Neves, N.M. Osteogenic differentiation of human mesenchymal stem cells in the absence of osteogenic supplements: A
surface-roughness gradient study. Acta Biomater. 2015, 28, 64–75. [CrossRef]
154. Gittens, R.A.; McLachlan, T.; Olivares-Navarrete, R.; Cai, Y.; Berner, S.; Tannenbaum, R.; Schwartz, Z.; Sandhage, K.H.; Boyan,
B.D. The effects of combined micron-/submicron-scale surface roughness and nanoscale features on cell proliferation and
differentiation. Biomaterials 2011, 32, 3395–3403. [CrossRef]
155. McBeath, R.; Pirone, D.M.; Nelson, C.M.; Bhadriraju, K.; Chen, C.S. Cell shape, cytoskeletal tension, and RhoA regulate stem cell
lineage commitment. Dev. Cell 2004, 6, 483–495. [CrossRef] [PubMed]
156. Sarkar, S. Roles of Nanofiber Scaffold Structure and Chemistry in Directing Human Bone Marrow Stromal Cell Response. Adv.
Tissue Eng. Regen. Med. 2016, 1, 6–18. [CrossRef]
157. Nair, L.S.; Laurencin, C.T. Biodegradable polymers as biomaterials. Prog. Polym. Sci. 2007, 32, 762–798. [CrossRef]
Fibers 2023, 11, 39 36 of 40

158. Moradi, Y.; Atyabi, S.A.; Ghiassadin, A.; Bakhshi, H.; Irani, S.; Dadgar, N. Cold Atmosphere Plasma Modification on Beta-
Carotene-Loaded Nanofibers to Enhance Osteogenic Differentiation. Fibers Polym. 2021, 23, 18–27. [CrossRef]
159. Atyabi, S.M.; Sharifi, F.; Irani, S.; Zandi, M.; Mivehchi, H.; Nagheh, Z. Cell Attachment and Viability Study of PCL Nano-fiber
Modified by Cold Atmospheric Plasma. Cell Biochem. Biophys. 2016, 74, 181–190. [CrossRef]
160. Kooshki, H.; Ghollasi, M.; Halabian, R.; Kazemi, N.M. Osteogenic differentiation of preconditioned bone marrow mesenchymal
stem cells with lipopolysaccharide on modified poly-l-lactic-acid nanofibers. J. Cell. Physiol. 2019, 234, 5343–5353. [CrossRef]
[PubMed]
161. Xing, Y.; Zhang, Y.; Jia, L.; Xu, X. Lipopolysaccharide from Escherichia coli stimulates osteogenic differentiation of human
periodontal ligament stem cells through Wnt/beta-catenin-induced TAZ elevation. Mol. Oral Microbiol. 2019, 34, omi.12249.
162. Onak, G.; Şen, M.; Horzum, N.; Ercan, U.K.; Yaralı, Z.B.; Garipcan, B.; Karaman, O. Aspartic and Glutamic Acid Templated
Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A
Comparative Study. Sci. Rep. 2018, 8, 17620. [CrossRef]
163. Liu, H.; Li, W.; Wen, W.; Luo, B.; Liu, M.; Ding, S.; Zhou, C. Mechanical properties and osteogenic activity of poly(l-lactide)
fibrous membrane synergistically enhanced by chitosan nanofibers and polydopamine layer. Mater. Sci. Eng. C Mater. Biol. Appl.
2017, 81, 280–290. [CrossRef] [PubMed]
164. Sanaei-Rad, P.; Kashi, T.-S.J.; Seyedjafari, E.; Soleimani, M. Enhancement of stem cell differentiation to osteogenic lineage on
hydroxyapatite-coated hybrid PLGA/gelatin nanofiber scaffolds. Biologicals 2016, 44, 511–516. [CrossRef]
165. Parvathi, K.; Krishnan, A.G.; Anitha, A.; Jayakumar, R.; Nair, M.B. Poly(L-lactic acid) nanofibers containing Cissus quadrangularis
induced osteogenic differentiation in vitro. Int. J. Biol. Macromol. 2018, 110, 514–521. [CrossRef]
166. Zhang, K.; Wang, Y.; Sun, T.; Wang, B.; Zhang, H. Bioinspired Surface Functionalization for Improving Osteogenesis of Electrospun
Polycaprolactone Nanofibers. Langmuir 2018, 34, 15544–15550. [CrossRef]
167. Seyedjafari, E.; Soleimani, M.; Ghaemi, N.; Shabani, I. Nanohydroxyapatite-coated electrospun poly(l-lactide) nanofibers enhance
osteogenic differentiation of stem cells and induce ectopic bone formation. Biomacromolecules 2010, 11, 3118–3125. [CrossRef]
[PubMed]
168. Engler, A.J.; Sen, S.; Sweeney, H.L.; Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 2006, 126, 677–689.
[CrossRef] [PubMed]
169. ASTM F3369-19e1; Standard Guide for Assessing the Skeletal Myoblast Phenotype. ASTM International: West Conshoshocken,
PA, USA, 2019.
170. Engler, A.J.; Griffin, M.A.; Sen, S.; Bönnemann, C.G.; Sweeney, H.L.; Discher, D.E. Myotubes differentiate optimally on substrates
with tissue-like stiffness: Pathological implications for soft or stiff microenvironments. J. Cell Biol. 2004, 166, 877–887. [CrossRef]
171. Xu, J.; Xie, Y.; Zhang, H.; Ye, Z.; Zhang, W. Fabrication of PLGA/MWNTs composite electrospun fibrous scaffolds for improved
myogenic differentiation of C2C12 cells. Colloids Surf. B Biointerfaces 2014, 123, 907–915. [CrossRef] [PubMed]
172. Uribe-Gomez, J.; Posada-Murcia, A.; Shukla, A.; Alkhamis, H.; Salehi, S.; Ionov, L. Soft Elastic Fibrous Scaffolds for Muscle Tissue
Engineering by Touch Spinning. ACS Appl. Bio Mater. 2021, 4, 5585–5597. [CrossRef] [PubMed]
173. Nam, J.; Johnson, J.; Lannutti, J.J.; Agarwal, S. Modulation of embryonic mesenchymal progenitor cell differentiation via control
over pure mechanical modulus in electrospun nanofibers. Acta Biomater. 2011, 7, 1516–1524. [CrossRef] [PubMed]
174. Fu, C.; Bai, H.; Zhu, J.; Niu, Z.; Wang, Y.; Li, J.; Yang, X.; Bai, Y. Enhanced cell proliferation and osteogenic differentiation
in electrospun PLGA/hydroxyapatite nanofibre scaffolds incorporated with graphene oxide. PLoS ONE 2017, 12, e0188352.
[CrossRef] [PubMed]
175. Rho, J.Y.; Ashman, R.B.; Turner, C.H. Young’s modulus of trabecular and cortical bone material: Ultrasonic and microtensile
measurements. J. Biomech. 1993, 26, 111–119. [CrossRef]
176. Wang, Z.; Liang, R.; Jiang, X.; Xie, J.; Cai, P.; Chen, H.; Zhan, X.; Lei, D.; Zhao, J.; Zheng, L. Electrospun PLGA/PCL/OCP
nanofiber membranes promote osteogenic differentiation of mesenchymal stem cells (MSCs). Mater. Sci. Eng. C Mater. Biol. Appl.
2019, 104, 109796. [CrossRef]
177. Daňková, J.; Buzgo, M.; Vejpravová, J.; Kubíčková, S.; Sovková, V.; Vysloužilová, L.; Mantlíková, A.; Nečas, A.; Amler, E. Highly
efficient mesenchymal stem cell proliferation on poly-epsilon-caprolactone nanofibers with embedded magnetic nanoparticles.
Int. J. Nanomed. 2015, 10, 7307–7317. [CrossRef]
178. Bateni, F.; Motlagh, G.H. Electrospun polyamide/graphene oxide nanofibers as fillers for polyethylene: Preparation and
characterization. J. Appl. Polym. Sci. 2021, 139, 51506. [CrossRef]
179. Ramazani, S.; Karimi, M. Aligned poly(epsilon-caprolactone)/graphene oxide and reduced graphene oxide nanocomposite
nanofibers: Morphological, mechanical and structural properties. Mater. Sci. Eng. C Mater. Biol. Appl. 2015, 56, 325–334.
[CrossRef]
180. Yao, Q.; Fuglsby, K.E.; Zheng, X.; Sun, H. Nanoclay-functionalized 3D nanofibrous scaffolds promote bone regeneration. J. Mater.
Chem. B 2020, 8, 3842–3851. [CrossRef]
181. Yi, Q.; Wen, X.; Li, L.; He, B.; Wu, Y.; Nie, Y.; Gu, Z. The polymeric crystallinity effect on the responses of bone marrow stromal
cells. e-Polymers 2009, 9, 42. [CrossRef]
182. Cui, H.; Sinko, P.J. The role of crystallinity on differential attachment/proliferation of osteoblasts and fibroblasts on poly
(caprolactone-co-glycolide) polymeric surfaces. Front. Mater. Sci. 2011, 6, 47–59. [CrossRef]
Fibers 2023, 11, 39 37 of 40

183. Müller, W.E.; Ackermann, M.; Al-Nawas, B.; Righesso, L.A.; Muñoz-Espí, R.; Tolba, E.; Neufurth, M.; Schröder, H.C.; Wang, X.
Amplified morphogenetic and bone forming activity of amorphous versus crystalline calcium phosphate/polyphosphate. Acta
Biomater. 2020, 118, 233–247. [CrossRef] [PubMed]
184. Seetharam, A.; Abad, J.; Baessler, A.; Badman, B.L. Use of a Nanofiber Resorbable Scaffold During Rotator Cuff Repair: Surgical
Technique and Results After Repair of Small- to Medium-Sized Tears. Orthop. J. Sport. Med. 2022, 10, 23259671221094848.
[CrossRef] [PubMed]
185. Chaudhari, A.A.; Vig, K.; Baganizi, D.R.; Sahu, R.; Dixit, S.; Dennis, V.; Singh, S.R.; Pillai, S.R. Future Prospects for Scaffolding
Methods and Biomaterials in Skin Tissue Engineering: A Review. Int. J. Mol. Sci. 2016, 17, 1974. [CrossRef]
186. Phillips, H. RenovoDerm Receives FDA Clearance for Proprietary Product, in Newswire. 2018. Available online: https:
//www.newswire.com/news/renovoderm-receives-fda-clearance-for-proprietary-product-20370988 (accessed on 3 March 2022).
187. U.S. Food and Drug Administration. 510(k) Premarket Notification—Phoenix Wound Matrix; U.S. Food and Drug Administration:
Silver Spring, MD, USA, 2018.
188. Schulz, A.; Fuchs, P.; Heitzmann, W.; Kanho, C.; Schiefer, J. Our Initial Experience In The Customized Treatment Of Donor Site
And Burn Wounds With A New Nanofibrous Temporary Epidermal Layer. Ann. Burn. Fire Disasters 2021, 34, 58–66.
189. Sadeghianmaryan, A.; Karimi, Y.; Naghieh, S.; Sardroud, H.A.; Gorji, M.; Chen, X. Electrospinning of Scaffolds from the
Polycaprolactone/Polyurethane Composite with Graphene Oxide for Skin Tissue Engineering. Appl. Biochem. Biotechnol. 2020,
191, 567–578. [CrossRef]
190. Keirouz, A.; Zakharova, M.; Kwon, J.; Robert, C.; Koutsos, V.; Callanan, A.; Chen, X.; Fortunato, G.; Radacsi, N. High-throughput
production of silk fibroin-based electrospun fibers as biomaterial for skin tissue engineering applications. Mater. Sci. Eng. C 2020,
112, 110939. [CrossRef]
191. Movahedi, M.; Asefnejad, A.; Rafienia, M.; Khorasani, M.T. Potential of novel electrospun core-shell structured polyurethane/starch
(hyaluronic acid) nanofibers for skin tissue engineering: In vitro and in vivo evaluation. Int. J. Biol. Macromol. 2020, 146, 627–637.
[CrossRef]
192. Narayanan, K.B.; Park, G.T.; Han, S.S. Electrospun poly(vinyl alcohol)/reduced graphene oxide nanofibrous scaffolds for skin
tissue engineering. Colloids Surf. B Biointerfaces 2020, 191, 110994. [CrossRef] [PubMed]
193. Rathinavel, S.; Ekambaram, S.; Korrapati, P.S.; Sangeetha, D. Design and fabrication of electrospun SBA-15-incorporated PVA
with curcumin: A biomimetic nanoscaffold for skin tissue engineering. Biomed. Mater. 2020, 15, 035009. [CrossRef] [PubMed]
194. Lopresti, F.; Campora, S.; Tirri, G.; Capuana, E.; Pavia, F.C.; Brucato, V.; Ghersi, G.; La Carrubba, V. Core-shell PLA/Kef hybrid
scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via
air-plasma treatment. Mater. Sci. Eng. C 2021, 127, 112248. [CrossRef] [PubMed]
195. Ruggeri, M.; Bianchi, E.; Rossi, S.; Boselli, C.; Cornaglia, A.I.; Malavasi, L.; Carzino, R.; Suarato, G.; Sánchez-Espejo, R.;
Athanassiou, A.; et al. Maltodextrin-amino acids electrospun scaffolds cross-linked with Maillard-type reaction for skin tissue
engineering. Mater. Sci. Eng. C 2021, 133, 112593. [CrossRef]
196. Zarei, M.; Samimi, A.; Khorram, M.; Abdi, M.M.; Golestaneh, S.I. Fabrication and characterization of conductive polypyr-
role/chitosan/collagen electrospun nanofiber scaffold for tissue engineering application. Int. J. Biol. Macromol. 2021, 168, 175–186.
[CrossRef]
197. Ababzadeh, S.; Farzin, A.; Goodarzi, A.; Karimi, R.; Farahani, M.S.; Farsani, M.E.; Gharibzad, K.; Zahiri, M.; Ai, J. High porous
electrospun poly (ε-caprolactone)/gelatin/MgO scaffolds preseeded with endometrial stem cells promote tissue regeneration
in full-thickness skin wounds: An in vivo study. J. Biomed. Mater. Res. Part B Appl. Biomater. 2020, 108, 2961–2970. [CrossRef]
[PubMed]
198. Fathi, A.; Khanmohammadi, M.; Goodarzi, A.; Foroutani, L.; Mobarakeh, Z.T.; Saremi, J.; Arabpour, Z.; Ai, J. Fabrication of
chitosan-polyvinyl alcohol and silk electrospun fiber seeded with differentiated keratinocyte for skin tissue regeneration in
animal wound model. J. Biol. Eng. 2020, 14, 27. [CrossRef]
199. Ghorbani, M.; Nezhad-Mokhtari, P.; Sohrabi, H.; Roshangar, L. Electrospun chitosan/nanocrystalline cellulose-graft-poly(N-
vinylcaprolactam) nanofibers as the reinforced scaffold for tissue engineering. J. Mater. Sci. 2020, 55, 2176–2185. [CrossRef]
200. Chen, J.; Zhang, G.; Zhao, Y.; Zhou, M.; Zhong, A.; Sun, J. Promotion of skin regeneration through co-axial electrospun fibers
loaded with basic fibroblast growth factor. Adv. Compos. Hybrid Mater. 2022, 5, 1111–1125. [CrossRef]
201. Goudarzi, Z.M.; Behzad, T.; Ghasemi-Mobarakeh, L.; Kharaziha, M. An investigation into influence of acetylated cellulose
nanofibers on properties of PCL/Gelatin electrospun nanofibrous scaffold for soft tissue engineering. Polymer 2021, 213, 123313.
[CrossRef]
202. Ramos-Rodriguez, D.H.; MacNeil, S.; Claeyssens, F.; Ortega Asencio, I. Fabrication of Topographically Controlled Electrospun
Scaffolds to Mimic the Stem Cell Microenvironment in the Dermal-Epidermal Junction. ACS Biomater. Sci. Eng. 2021, 7, 2803–2813.
[CrossRef] [PubMed]
203. Ji, X.; Li, R.; Liu, G.; Jia, W.; Sun, M.; Liu, Y.; Luo, Y.; Cheng, Z. Phase separation-based electrospun Janus nanofibers loaded with
Rana chensinensis skin peptides/silver nanoparticles for wound healing. Mater. Des. 2021, 207, 109864. [CrossRef]
204. Ji, X.; Li, R.; Jia, W.; Liu, G.; Luo, Y.-G.; Cheng, Z. Co-Axial Fibers with Janus-Structured Sheaths by Electrospinning Release Corn
Peptides for Wound Healing. ACS Appl. Bio Mater. 2020, 3, 6430–6438. [CrossRef] [PubMed]
Fibers 2023, 11, 39 38 of 40

205. Chandika, P.; Oh, G.-W.; Heo, S.-Y.; Kim, S.-C.; Kim, T.-H.; Kim, M.-S.; Jung, W.-K. Electrospun porous bilayer nano-fibrous
fish collagen/PCL bio-composite scaffolds with covalently cross-linked chitooligosaccharides for full-thickness wound-healing
applications. Mater. Sci. Eng. C 2021, 121, 111871. [CrossRef]
206. Li, P.; Ruan, L.; Wang, R.; Liu, T.; Song, G.; Gao, X.; Jiang, G.; Liu, X. Electrospun Scaffold of Collagen and Polycaprolactone
Containing ZnO Quantum Dots for Skin Wound Regeneration. J. Bionic Eng. 2021, 18, 1378–1390. [CrossRef]
207. Madub, K.; Goonoo, N.; Gimié, F.; Arsa, I.A.; Schönherr, H.; Bhaw-Luximon, A. Green seaweeds ulvan-cellulose scaffolds enhance
in vitro cell growth and in vivo angiogenesis for skin tissue engineering. Carbohydr. Polym. 2021, 251, 117025. [CrossRef]
208. Atila, D.; Hasirci, V.; Tezcaner, A. Coaxial electrospinning of composite mats comprised of core/shell poly(methyl methacry-
late)/silk fibroin fibers for tissue engineering applications. J. Mech. Behav. Biomed. Mater. 2022, 128, 105105. [CrossRef]
[PubMed]
209. Tourlomousis, F.; Jia, C.; Karydis, T.; Mershin, A.; Wang, H.; Kalyon, D.M.; Chang, R.C. Machine learning metrology of cell
confinement in melt electrowritten three-dimensional biomaterial substrates. Microsyst. Nanoeng. 2019, 5, 15. [CrossRef]
210. Niu, Y.; Galluzzi, M.; Fu, M.; Hu, J.; Xia, H. In vivo performance of electrospun tubular hyaluronic acid/collagen nanofibrous
scaffolds for vascular reconstruction in the rabbit model. J. Nanobiotechnol. 2021, 19, 349. [CrossRef]
211. Kandzari, D.E.; Birkemeyer, R. PK Papyrus covered stent: Device description and early experience for the treatment of coronary
artery perforations. Catheter. Cardiovasc. Interv. 2019, 94, 564–568. [CrossRef]
212. Hernández-Enríquez, M.; Belle, L.; Madiot, H.; Pansieri, M.; Souteyrand, G.; de Poli, F.; Piot, C.; Boueri, Z.; Gerbaud, E.; Boiffard,
E.; et al. Use and outcomes of the PK Papyrus covered stent in France: SOS PK Papyrus Registry. Catheter. Cardiovasc. Interv. 2021,
98, 874–881. [CrossRef]
213. Wu, Y.-L.; Szafron, J.M.; Blum, K.M.; Zbinden, J.C.; Khosravi, M.R.; Best, C.A.; Reinhardt, J.W.; Zheng, Q.; Yi, T.; Shinoka, T.; et al.
Electrospun Tissue-Engineered Arterial Graft Thickness Affects Long-Term Composition and Mechanics. Tissue Eng. Part A 2020,
27, 593–603. [CrossRef] [PubMed]
214. Daum, R.; Visser, D.; Wild, C.; Kutuzova, L.; Schneider, M.; Lorenz, G.; Weiss, M.; Hinderer, S.; Stock, U.A.; Seifert, M.;
et al. Fibronectin Adsorption on Electrospun Synthetic Vascular Grafts Attracts Endothelial Progenitor Cells and Promotes
Endothelialization in Dynamic In Vitro Culture. Cells 2020, 9, 778. [CrossRef] [PubMed]
215. Reid, J.A.; Callanan, A. Hybrid cardiovascular sourced extracellular matrix scaffolds as possible platforms for vascular tissue
engineering. J. Biomed. Mater. Res. Part B Appl. Biomater. 2020, 108, 910–924. [CrossRef]
216. Chen, D.; Zhang, L.; Zhang, W.; Tang, Z.; Fu, W.; Hu, R.; Feng, B.; Hong, H.; Zhang, H. Shapeable large-pore electrospun
polycaprolactam cotton facilitates the rapid formation of a functional tissue engineered vascular graft. Mater. Des. 2020, 191,
108631. [CrossRef]
217. Jin, Q.; Fu, Y.; Zhang, G.; Xu, L.; Jin, G.; Tang, L.; Ju, J.; Zhao, W.; Hou, R. Nanofiber electrospinning combined with rotary
bioprinting for fabricating small-diameter vessels with endothelium and smooth muscle. Compos. Part B Eng. 2022, 234, 109691.
[CrossRef]
218. Chew, C.H.; Sheu, B.-L.; Chen, A.; Huang, W.-T.; Cheng, T.-M.; Shih, C.-M.; Chang, A.; Chen, C.-C. Tissue-Engineered Vascular
Graft with Co-Culture of Smooth Muscle Cells and Human Endothelial Vein Cells on an Electrospun Poly(lactic-co-glycolic acid)
Microtube Array Membrane. Membranes 2021, 11, 732. [CrossRef]
219. Zhao, L.; Li, X.; Yang, L.; Sun, L.; Mu, S.; Zong, H.; Li, Q.; Wang, F.; Song, S.; Yang, C.; et al. Evaluation of remodeling and
regeneration of electrospun PCL/fibrin vascular grafts in vivo. Mater. Sci. Eng. C 2021, 118, 111441. [CrossRef]
220. Kong, X.; He, Y.; Zhou, H.; Gao, P.; Xu, L.; Han, Z.; Le Yang, L.; Wang, M. Chondroitin Sulfate/Polycaprolactone/Gelatin
Electrospun Nanofibers with Antithrombogenicity and Enhanced Endothelial Cell Affinity as a Potential Scaffold for Blood Vessel
Tissue Engineering. Nanoscale Res. Lett. 2021, 16, 62. [CrossRef]
221. Tondnevis, F.; Keshvari, H.; Mohandesi, J.A. Fabrication, characterization, and in vitro evaluation of electrospun polyurethane-
gelatin-carbon nanotube scaffolds for cardiovascular tissue engineering applications. J. Biomed. Mater. Res. Part B Appl. Biomater.
2020, 108, 2276–2293. [CrossRef]
222. A Emechebe, G.; O Obiweluozor, F.; Jeong, I.S.; Park, J.-K.; Park, C.H.; Kim, C.S. Merging 3D printing with electrospun
biodegradable small-caliber vascular grafts immobilized with VEGF. Nanomed. Nanotechnol. Biol. Med. 2020, 30, 102306.
[CrossRef] [PubMed]
223. Zhou, Y.; Sooriyaarachchi, D.; Tan, G.Z. Fabrication of Nanopores Polylactic Acid Microtubes by Core-Sheath Electrospinning for
Capillary Vascularization. Biomimetics 2021, 6, 15. [CrossRef] [PubMed]
224. Jang, S.R.; Kim, J.I.; Park, C.H.; Kim, C.S. Development of Y-shaped small diameter artificial blood vessel with controlled
topography via a modified electrospinning method. Mater. Lett. 2020, 264, 127113. [CrossRef]
225. Rubenstein, D.A.; Greene, V.K.; Yin, W. Electrospun scaffold fiber orientation regulates endothelial cell and platelet properties
associated with angiogenesis and hemocompatibility. Materialia 2020, 14, 100942. [CrossRef]
226. Liu, F.; Liao, X.; Liu, C.; Li, M.; Chen, Y.; Shao, W.; Weng, K.; Li, F.; Ou, K.; He, J. Poly(l-lactide-co-caprolactone)/tussah silk
fibroin nanofiber vascular scaffolds with small diameter fabricated by core-spun electrospinning technology. J. Mater. Sci. 2020,
55, 7106–7119. [CrossRef]
227. Dou, J.; Wang, Y.; Jin, X.; Li, P.; Wang, L.; Yuan, J.; Shen, J. PCL/sulfonated keratin mats for vascular tissue engineering scaffold
with potential of catalytic nitric oxide generation. Mater. Sci. Eng. C 2020, 107, 110246. [CrossRef]
Fibers 2023, 11, 39 39 of 40

228. Grasl, C.; Stoiber, M.; Röhrich, M.; Moscato, F.; Bergmeister, H.; Schima, H. Electrospinning of small diameter vascular grafts with
preferential fiber directions and comparison of their mechanical behavior with native rat aortas. Mater. Sci. Eng. C 2021, 124,
112085. [CrossRef]
229. Columbus, S.; Painuly, D.; Nair, R.P.; Krishnan, V.K. Role of PEGylated CdSe-ZnS quantum dots on structural and functional
properties of electrospun polycaprolactone scaffolds for blood vessel tissue engineering. Eur. Polym. J. 2021, 151, 110430.
[CrossRef]
230. Rahmati Nejad, M.; Yousefzadeh, M.; and Solouk, A. Electrospun PET/PCL small diameter nanofibrous conduit for biomedical
application. Mater. Sci. Eng. C 2020, 110, 110692. [CrossRef]
231. Gupta, P.; Lorentz, K.L.; Haskett, D.G.; Cunnane, E.M.; Ramaswamy, A.K.; Weinbaum, J.S.; Vorp, D.A.; Mandal, B.B. Bioresorbable
silk grafts for small diameter vascular tissue engineering applications: In vitro and in vivo functional analysis. Acta Biomater.
2020, 105, 146–158. [CrossRef]
232. Matsuzaki, Y.; Iwaki, R.; Reinhardt, J.W.; Chang, Y.-C.; Miyamoto, S.; Kelly, J.; Zbinden, J.; Blum, K.; Mirhaidari, G.; Ulziibayar, A.;
et al. The effect of pore diameter on neo-tissue formation in electrospun biodegradable tissue-engineered arterial grafts in a large
animal model. Acta Biomater. 2020, 115, 176–184. [CrossRef]
233. Vermue, I.M.; Begum, R.; Castilho, M.; Rookmaaker, M.B.; Masereeuw, R.; Bouten, C.V.C.; Verhaar, M.C.; Cheng, C. Renal Biology
Driven Macro- and Microscale Design Strategies for Creating an Artificial Proximal Tubule Using Fiber-Based Technologies. ACS
Biomater. Sci. Eng. 2021, 7, 4679–4693. [CrossRef]
234. Baskapan, B.; Callanan, A. Electrospinning Fabrication Methods to Incorporate Laminin in Polycaprolactone for Kidney Tissue
Engineering. Tissue Eng. Regen. Med. 2022, 19, 73–82. [CrossRef] [PubMed]
235. Jansen, K.; Castilho, M.; Aarts, S.; Kaminski, M.M.; Lienkamp, S.S.; Pichler, R.; Malda, J.; Vermonden, T.; Jansen, J.; Masereeuw, R.
Fabrication of Kidney Proximal Tubule Grafts Using Biofunctionalized Electrospun Polymer Scaffolds. Macromol. Biosci. 2019, 19,
1800412. [CrossRef]
236. Agueda, J.R.S.; Madrid, J.; Mondragon, J.M.; Lim, J.; Tan, A.; Wang, I.; Duguran, N.; Bondoc, A. Synthesis and Characterization of
Electrospun Polyvinylidene Fluoride-based (PVDF) Scaffolds for Renal Bioengineering. J. Phys. Conf. Ser. 2021, 2071, 012005.
[CrossRef]
237. Behtaj, S.; John, J.A.S.; Ekberg, J.A.K.; Rybachuk, M. Neuron-fibrous scaffold interfaces in the peripheral nervous system: A
perspective on the structural requirements. Neural Regen. Res. 2022, 17, 1893–1897. [CrossRef]
238. Castro, V.O.; Merlini, C. Aligned electrospun nerve conduits with electrical activity as a strategy for peripheral nerve regeneration.
Artif. Organs 2021, 45, 813–818. [CrossRef] [PubMed]
239. Behtaj, S.; Ekberg, J.A.K.; John, J.A.S. Advances in Electrospun Nerve Guidance Conduits for Engineering Neural Regeneration.
Pharmaceutics 2022, 14, 219. [CrossRef] [PubMed]
240. Zha, F.; Chen, W.; Lv, G.; Wu, C.; Hao, L.; Meng, L.; Zhang, L.; Yu, D. Effects of surface condition of conductive electrospun
nanofiber mats on cell behavior for nerve tissue engineering. Mater. Sci. Eng. C 2021, 120, 111795. [CrossRef]
241. Chen, T.; Jiang, H.; Li, X.; Zhang, D.; Zhu, Y.; Chen, X.; Yang, H.; Shen, F.; Xia, H.; Zheng, J.; et al. Proliferation and differentiation
study of melatonin functionalized polycaprolactone/gelatin electrospun fibrous scaffolds for nerve tissue engineering. Int. J. Biol.
Macromol. 2022, 197, 103–110. [CrossRef]
242. Shen, J.; Wang, J.; Liu, X.; Sun, Y.; Yin, A.; Chai, Y.; Zhang, K.; Wang, C.; Zheng, X. In Situ Prevascularization Strategy with
Three-Dimensional Porous Conduits for Neural Tissue Engineering. ACS Appl. Mater. Interfaces 2021, 13, 50785–50801. [CrossRef]
[PubMed]
243. Wang, J.; Xiong, H.; Zhu, T.; Liu, Y.; Pan, H.; Fan, C.; Zhao, X.; Lu, W.W. Bioinspired Multichannel Nerve Guidance Conduit Based
on Shape Memory Nanofibers for Potential Application in Peripheral Nerve Repair. ACS Nano 2020, 14, 12579–12595. [CrossRef]
[PubMed]
244. Kong, Y.; Xu, J.; Han, Q.; Zheng, T.; Wu, L.; Li, G.; Yang, Y. Electrospinning porcine decellularized nerve matrix scaffold for
peripheral nerve regeneration. Int. J. Biol. Macromol. 2022, 209, 1867–1881. [CrossRef] [PubMed]
245. Entekhabi, E.; Nazarpak, M.H.; Shafieian, M.; Mohammadi, H.; Firouzi, M.; Hassannejad, Z. Fabrication and in vitro evaluation
of 3D composite scaffold based on collagen/hyaluronic acid sponge and electrospun polycaprolactone nanofibers for peripheral
nerve regeneration. J. Biomed. Mater. Res. Part A 2021, 109, 300–312. [CrossRef] [PubMed]
246. Yu, L.; Zhang, W.; Jiang, Y.; Guo, C. Gradient degradable nerve guidance conduit with multilayer structure prepared by
electrospinning. Mater. Lett. 2020, 276, 128238. [CrossRef]
247. Hanumantharao, S.N.; Alinezhadbalalami, N.; Kannan, S.; Friske, M.; Rao, S. Electrospun acellular scaffolds for mimicking the
natural anisotropy of the extracellular matrix. RSC Adv. 2019, 9, 40190–40195. [CrossRef]
248. Roshanbinfar, K.; Vogt, L.; Ruther, F.; Roether, J.A.; Boccaccini, A.R.; Engel, F.B. Nanofibrous Composite with Tailorable Electrical
and Mechanical Properties for Cardiac Tissue Engineering. Adv. Funct. Mater. 2020, 30, 1908612. [CrossRef]
249. Roshanbinfar, K.; Vogt, L.; Ruther, F.; Roether, J.A.; Boccaccini, A.R.; Engel, F.B. Developing cellular therapies for retinal
degenerative diseases. Investig. Ophthalmol. Vis. Sci. 2014, 55, 1191–1202.
250. Van Gelder, R.N.; Chiang, M.F.; Dyer, M.A.; Greenwell, T.N.; Levin, L.A.; Wong, R.O.; Svendsen, C.N. Regenerative and restorative
medicine for eye disease. Nat. Med. 2022, 28, 1149–1156. [CrossRef]
Fibers 2023, 11, 39 40 of 40

251. Hotaling, N.A.; Khristov, V.; Wan, Q.; Sharma, R.; Jha, B.S.; Lotfi, M.; Maminishkis, A.; Simon, C.; Bharti, K. Nanofiber Scaffold-
Based Tissue-Engineered Retinal Pigment Epithelium to Treat Degenerative Eye Diseases. J. Ocul. Pharmacol. Ther. 2016, 32,
272–285. [CrossRef]
252. Treharne, A.J.; Thomson, H.A.; Grossel, M.C.; Lotery, A.J. Developing methacrylate-based copolymers as an artificial Bruch’s
membrane substitute. J. Biomed. Mater. Res. A 2012, 100, 2358–2364. [CrossRef] [PubMed]
253. Warnke, P.H.; Alamein, M.; Skabo, S.; Stephens, S.; Bourke, R.; Heiner, P.; Liu, Q. Primordium of an artificial Bruch’s membrane
made of nanofibers for engineering of retinal pigment epithelium cell monolayers. Acta Biomater. 2013, 9, 9414–9422. [CrossRef]
[PubMed]
254. Xiang, P.; Wu, K.C.; Zhu, Y.; Xiang, L.; Li, C.; Chen, D.L.; Chen, F.; Xu, G.; Wang, A.; Li, M.; et al. A novel Bruch’s membrane-
mimetic electrospun substrate scaffold for human retinal pigment epithelium cells. Biomaterials 2014, 35, 9777–9788. [CrossRef]
[PubMed]
255. Sorkio, A.; Porter, P.J.; Juuti-Uusitalo, K.; Meenan, B.J.; Skottman, H.; Burke, G.A. Surface Modified Biodegradable Electrospun
Membranes as a Carrier for Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells. Tissue Eng. Part A 2015, 21,
2301–2314. [CrossRef]
256. Komez, A.; Baran, E.T.; Erdem, U.; Hasirci, N.; Hasirci, V. Construction of a patterned hydrogel-fibrous mat bilayer structure to
mimic choroid and Bruch’s membrane layers of retina. J. Biomed. Mater. Res. A 2016, 104, 2166–2177. [CrossRef] [PubMed]
257. Phelan, M.A.; Kruczek, K.; Wilson, J.P.; Brooks, M.J.; Drinnan, C.T.; Regent, F.; Gerstenhaber, J.A.; Swaroop, A.; Lelkes, P.I.; Li, T.
Soy Protein Nanofiber Scaffolds for Uniform Maturation of Human Induced Pluripotent Stem Cell-Derived Retinal Pigment
Epithelium. Tissue Eng. Part C Methods 2020, 26, 433–446. [CrossRef]
258. Zeng, Z.; Lam, P.T.; Robinson, M.L.; Del Rio-Tsonis, K.; Saul, J.M. Design and Characterization of Biomimetic Kerateine
Aerogel-Electrospun Polycaprolactone Scaffolds for Retinal Cell Culture. Ann. Biomed. Eng. 2021, 49, 1633–1644. [CrossRef]
259. Liu, Z.; Yu, N.; Holz, F.G.; Yang, F.; Stanzel, B.V. Enhancement of retinal pigment epithelial culture characteristics and subretinal
space tolerance of scaffolds with 200 nm fiber topography. Biomaterials 2014, 35, 2837–2850. [CrossRef]
260. Sharma, R.; Khristov, V.; Rising, A.; Jha, B.S.; Dejene, R.; Hotaling, N.; Li, Y.; Stoddard, J.; Stankewicz, C.; Wan, Q.; et al.
Clinical-grade stem cell–derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs. Sci. Transl.
Med. 2019, 11, eaat5580. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.