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vi Editorial Board

Prof. Kwang-Sup Lee Prof. E.M. Terentjev

Department of Advanced Materials Cavendish Laboratory
Hannam University Madingley Road
561-6 Jeonmin-Dong Cambridge CB 3 OHE, UK
Yuseong-Gu 305-811 [email protected]
Daejeon, South Korea
[email protected]
Prof. Maria Jesus Vicent
Prof. L. Leibler Centro de Investigacion Principe Felipe
Matière Molle et Chimie Medicinal Chemistry Unit
Ecole Supérieure de Physique Polymer Therapeutics Laboratory
et Chimie Industrielles (ESPCI) Av. Autopista del Saler, 16
10 rue Vauquelin 46012 Valencia, Spain
75231 Paris Cedex 05, France [email protected]
[email protected]
Prof. Brigitte Voit
Prof. Timothy E. Long
Leibniz-Institut für Polymerforschung
Department of Chemistry
Dresden
and Research Institute
Hohe Straße 6
Virginia Tech
01069 Dresden, Germany
2110 Hahn Hall (0344)
[email protected]
Blacksburg, VA 24061, USA
[email protected]
Prof. Gerhard Wegner
Prof. Ian Manners
Max-Planck-Institut
School of Chemistry für Polymerforschung
University of Bristol Ackermannweg 10
Cantock’s Close 55128 Mainz, Germany
BS8 1TS Bristol, UK [email protected]
[email protected]

Prof. Martin Möller Prof. Ulrich Wiesner

Deutsches Wollforschungsinstitut Materials Science & Engineering
an der RWTH Aachen e.V. Cornell University
Pauwelsstraße 8 329 Bard Hall
52056 Aachen, Germany Ithaca, NY 14853, USA
[email protected] [email protected]
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viii Advances in Polymer Sciences Also Available Electronically

Aims and Scope

The series presents critical reviews of the present and future trends in polymer and
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Preface

Electrospinning is a very attractive method used for the preparation of polymeric or

composite fibers. During electrospinning, a high voltage is applied to a polymer solution
to produce a polymer jet. With the fast evaporation of solvent combined with high
extension ratios of the polymer solution, uniform diameter nanofibers can be produced.
There has been an explosion of interest wherein these nanofibrous polymer mats are
used for a variety of biomedical applications such as water treatment, biosensors,
superhydrophobic surfaces, tissue engineering, wound healing and drug delivery.
The present volume entitled, “Biomedical Applications of Polymeric Nanofi-
bers” attempts to provide a broad overview on the preparation techniques, struc-
tures and biomedical applications of different biopolymeric nanofibers. The book
consists of 9 chapters:
Chapter 1 deals with the current designs of multiscale scaffolds and discusses
their physico-chemical characteristics, as well as their potential applications in
regenerative medicine. Chapter 2 focuses on the current state-of-the-art and future
perspectives of stem cells and their differentiation on nanoengineered substrates for
advanced tissue regeneration. Chapter 3 describes the methods utilized to create
biomimetic structures for bone tissue engineering, as well as highlighting the
advancements made in this field using these methods. Chapter 4 gives overviews
of several tissue engineering approaches that have exploited composite design
features and discusses new promising avenues for study. In addition, the drug-
and growth-factor delivery capabilities of these systems will similarly be reviewed.
Chapter 5 focuses on the development of artificial conduits for nerve regeneration
using nanofibers as alternatives to the autograft. Chapter 6 deals with the fabrication
of highly aligned nanofiber structures and their challenges and applications in the
biomedical field. Chapter 7 summarizes the research and development related to the
electrospinning of some common biocompatible polymers as well as an overview
of their potential in many biomedical applications such as tissue engineering,
wound dressing, carriers for drug delivery or controlled release, and enzyme

ix
x Preface

immobilization. Chapter 8 reviews the recent advances of electrospun nanofibrous

scaffolds based on biodegradable and biocompatible polymers for controlled drug and
biomolecule delivery applications. Chapter 9 summarizes the preparation and
biomedical applications of silver nanoparticles incorporated into polymeric
nanofibers.

Prof. R. Jayakumar
Prof. Shantikumar V. Nair
Contents

Multiscale Fibrous Scaffolds in Regenerative Medicine . . . . . . . . . . . . . . . . . . . . 1

Sowmya Srinivasan, R. Jayakumar, K.P. Chennazhi, Erica J. Levorson,
Antonios G. Mikos, and Shantikumar V. Nair

Stem Cells and Nanostructures for Advanced Tissue Regeneration . . . . . . 21

Molamma P. Prabhakaran, J. Venugopal, Laleh Ghasemi-Mobarakeh,
Dan Kai, Guorui Jin, and Seeram Ramakrishna

Creating Electrospun Nanofiber-Based Biomimetic Scaffolds

for Bone Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Eleni Katsanevakis, Xuejun Wen, and Ning Zhang

Synthetic/Biopolymer Nanofibrous Composites as Dynamic Tissue

Engineering Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
J.A. Kluge and R.L. Mauck

Electrospun Fibers as Substrates for Peripheral Nerve Regeneration . . . 131

Jörg Mey, Gary Brook, Dorothée Hodde, and Andreas Kriebel

Highly Aligned Polymer Nanofiber Structures: Fabrication

and Applications in Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Vince Beachley, Eleni Katsanevakis, Ning Zhang, and Xuejun Wen

Electrospinning of Biocompatible Polymers and Their Potentials

in Biomedical Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
Pitt Supaphol, Orawan Suwantong, Pakakrong Sangsanoh, Sowmya Srinivasan,
Rangasamy Jayakumar, and Shantikumar V. Nair

xi
xii Contents

Electrospun Nanofibrous Scaffolds-Current Status and Prospects

in Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
M. Prabaharan, R. Jayakumar, and S.V. Nair

Biomedical Applications of Polymer/Silver Composite Nanofibers . . . . . . 263

R. Jayakumar, M. Prabaharan, K.T. Shalumon,
K.P. Chennazhi, and S.V. Nair

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Adv Polym Sci (2012) 246: 1–20
DOI: 10.1007/12_2011_163
# Springer-Verlag Berlin Heidelberg 2011
Published online: 3 November 2011

Multiscale Fibrous Scaffolds in Regenerative

Medicine

Sowmya Srinivasan, R. Jayakumar, K.P. Chennazhi, Erica J. Levorson,

Antonios G. Mikos, and Shantikumar V. Nair

Abstract In recent years, multiscale fibrous scaffolds containing a combination of

micro- and nanoscale fibers have attracted a lot of attention in the tissue engineering
field. The multiscale concept is inspired by the hierarchical structure of many
tissues, such as bone. Fibrous scaffolds have been traditionally microscale; how-
ever, it has been determined that many physicochemical and biological properties
are influenced by fiber scale. For this reason, in an effort to optimize tissue
regeneration the use of multiple scales has been investigated for obtaining innova-
tive property combinations not otherwise attainable with a single fiber scale.
Multiscale architectures have been found to be favorable not only in bone regener-
ation but also in the regeneration of soft tissues including cardiovascular tissue,
neural tissue, cartilage, and skin. The unique properties of multiscale scaffolds have
been pivotal in better mimicking the extracellular matrix and promoting vasculari-
zation, a key step towards the development of engineered tissue. In this review, we
present current designs of multiscale scaffolds and discuss their physicochemical
characteristics, as well as their potential applications in regenerative medicine.

S. Srinivasan, R. Jayakumar (*), K.P. Chennazhi, and S.V. Nair (*)

Amrita Center for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences
and Research Centre, Cochin 682 041, India
e-mail: [email protected]; [email protected]
E.J. Levorson
Department of Bioengineering, Rice University, MS-142, P.O. Box 1892, Houston, TX
77251-1892, USA
A.G. Mikos
Department of Bioengineering, Rice University, MS-142, P.O. Box 1892, Houston, TX
77251-1892, USA
Department of Chemical and Biomolecular Engineering, Rice University, MS-362,
P.O. Box 1892, Houston, TX 77251-1892, USA
2 S. Srinivasan et al.

Keywords Bone
Cardiovascular tissue engineering
Electrospinning

Microfibers
Multiscale fibrous scaffolds
Nanofibers
Neural

Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Multiscale Scaffolds: Principles and Fundamentals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
3 Mechanical, Physicochemical, and Biological Properties of Multiscale Scaffolds . . . . . . . . 5
4 Applications of Multiscale Scaffolds in Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.1 Bone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.2 Cartilage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.3 Skin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.4 Neural . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.5 Cardiovascular . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5 Logistics and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6 Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1 Introduction

Replacement of damaged tissues or organs by natural tissue instead of using

synthetic implants is now seen as a realistic goal [1]. The emerging field of tissue
engineering essentially involves engineering a suitably shaped biocompatible scaf-
fold that has the potential to regenerate the host tissue when infused with the right
mix of cells and other growth factors [2–7]. The regeneration can be conducted
in vitro giving the opportunity to partially develop the host tissue extracorporeally,
followed by implantation. An ideal scaffold must exhibit a porous, interconnected,
and permeable structure to permit the infiltration of cells and nutrients and also
should exhibit the appropriate surface structure and chemistry for cell adhesion and
proliferation [8–12]. The ideal scaffold must also have mechanical properties
comparable with the tissue being engineered and have just the right degree of
biodegradability, such that it degrades to form soluble nontoxic products when
the tissue is fully formed [13, 14].
The question of scale in this review is uniquely concerned with the scaffold
structure. Early studies focused on scaffolds containing micrometer-sized inter-
connected pores to allow for infiltration of cells into the scaffold [15–19]. Since
2000, with advances in new nanomaterials and processing techniques a large
number of studies [20–24] have focused on the role of the nanostructure of scaffolds
in tissue regeneration. This was also prompted by the recognition that the environ-
ment of the cells in actual tissues (the extracellular matrix; ECM) is fundamentally
nanostructured. On the other hand, microscale fibers allow the fabrication of
scaffolds with controlled cell–cell interactions. Microfibers have the potential to
be utilized as tissue engineering scaffolds in different forms for numerous
applications such as wound dressings and bone regeneration [25, 26]. The appeal
of nanofibers in tissue engineering is their structural similarity to native ECM;
Multiscale Fibrous Scaffolds in Regenerative Medicine 3

however, ECM not only has a structural role but also a functional one. This network
creates a dynamic, three-dimensional (3D) microenvironment in which cells are
maintained. Signals are transmitted from cell surfaces in contact with ECM to the
cell nucleus, enabling communication influencing cell adhesion, migration, growth,
differentiation, programmed cell death, modulation of cytokine and growth factor
activity, and activation of intracellular signaling [27]. Spun nanofibers also offer
several advantages such as an extremely high surface-to-volume ratio, tunable
porosity, flexibility with respect to scaffold size and shape, as well as the ability
to control the nanofiber composition to achieve the desired results from its
properties and functionality [27]. Though there are many studies that have proposed
nanofibrous polymeric mats for tissue engineering, they have a limitation for 3D
applications due to their pore size, which is smaller than a cellular diameter and
restricts cell migration within the structure [28–30].
The use of toxic organic solvents for the fabrication of micro- and nanofibers is a
major limitation of fibers obtained from synthetic polymers, which would require
thorough washing or solvent evaporation treatment prior to use with cells. Natural
polymers act as alternatives to synthetic polymers and offer a few advantages such
as solubility in aqueous media due to their hydrophilic nature in addition to limited
or nonexistent immunogenicity and cytotoxicity [31]. A variety of natural and
synthetic polymers have been used to develop scaffolds; however, these scaffolds
do not interact with cells in the desired manner. The growth of these cells may be
affected by their interaction with the substrate [32–34]. In vivo, the complex
structure of vasculature ensures a maximum nutrient diffusion distance of nearly
200 mm for the supply of nutrients to tissue. However, this distance exceeds 200 mm
within in vitro constructs, resulting in reduced nutrient supply that compromises
cell growth and differentiation [35–37]. Very few studies have looked at the
vascularization of scaffolds, which is also of major concern [38]. Most of the
current scaffolds lack an interconnected microcapillary network that would help
cells to survive within these scaffolds. Also, the cells seeded onto larger and thicker
scaffolds have limited success as the cells in the deeper layers are devoid of
nutrients and oxygen. Hence, there are more cells at the surfaces of thick
3D scaffolds than in the deeper layers [39]. Therefore these scaffolds should
be provided with an inbuilt nutrient distribution network to support the uniform
growth of cells. Some fabrication techniques fail to form interconnected pores,
which are an important requirement of tissue engineering scaffolds [40–43]. In
addition, fabrication techniques in general fail to develop 3D constructs that mimic
the complex natural tissues, specifically with regard to mechanical properties
[38, 44, 45].
To overcome the current limitations, one approach developed mainly in the last
5 years has been to study multiscale scaffolds in an attempt to mimic hierarchical
tissue structures. This review essentially focuses on studies involving multiscale
fibrous scaffolds, their development, use of different fabrication methods, their
properties, and specific applications in tissue engineering.
4 S. Srinivasan et al.

2 Multiscale Scaffolds: Principles and Fundamentals

ECM is a hierarchical structure comprised of both micro- and nanoscale structures.

The micrometer scale offers a porous structure large enough for the migration of
cells through the construct, whereas the nanoscale allows for enhanced protein
adsorption, thus improving protein-mediated interactions with the cell surface and
so favoring cell adhesion and improving cell viability [46]. The nanoscale fiber
meshes exhibit high porosity and reduced pore sizes. Fiber diameter plays an
important role in adhesion of cells to the fibers. Reports have suggested that
nanofibers may serve as suitable biomaterials for tissue engineering since they
can be used to enhance cell differentiation, adhesion, and proliferation [47]. Cells
attach at multiple focal points, extend filopodia along the length of the fibers, and
spread throughout the nanofiber matrix [48]. However, the effect of fiber diameter
on tissue regeneration still remains unknown. Scaffold architecture and design are
key factors in determining vascularization and nutrient supply, which are crucial for
the development of regenerating tissue [49]. Studies have shown that pores of
100–300 mm favor vascularization and nutrient supply [50]. However, the relatively
small pore size of nanoscale fibrous scaffolds compared to cell diameter (5–20 mm)
limits cell migration and infiltration within the scaffold, leading to the formation of
a monolayer of cells on the scaffold surface rather than a 3D cellular construct. For
this reason, nanoscale fibrous scaffolds cannot mimic the natural thickness of
certain tissues such as human articular cartilage, which ranges from 0.5 to
7.1 mm thick [51].
Microfiber scaffolds could be potentially advantageous because they are
comprised of larger pores than nanofiber scaffolds. These larger pores could facili-
tate cellular infiltration and/or diffusion of nutrients during in vitro culture. To
study the mechanism of interaction between cells and nanofibers, it is important to
control the architecture of nanofibers to minimize the interaction of a single cell
with neighboring fibers [47]. Controlling the nanofiber to microfiber ratio can easily
vary the porosity of multiscale scaffolds. Pham et al., who developed a bimodal
scaffold consisting of a top nanofiber layer and a bottom microfiber layer,
demonstrated this [52]. These scaffolds were developed by a sequential
electrospinning technique wherein the multiscale layers were electrospun using
the same polymer with different morphologies. These scaffolds were characterized
to evaluate the extent of nanofiber deposition as a function of duration of
electrospinning time. Electrospinning for longer periods increased the amount
and thickness of the nanofibrous layer. Hence, thickness and coverage of
nanofibrous layer could be controlled by modulating the electrospinning time.
Cross-sectional images helped in distinguishing the alternating layers, even in the
presence of a thin nanofiber layer. Also, scaffolds with different densities of
nanofiber layers were considered to investigate the effect on cellular infiltration
into these bimodal scaffolds. Cell attachment at different intervals of time was
found to be similar; however, cell spreading was affected by the presence of
nanofibers. The cells initially appearing to exhibit rounded morphologies became
Multiscale Fibrous Scaffolds in Regenerative Medicine 5

more spread with time, with the greatest amount of spreading being observed on
scaffolds with nanofibers. Complete infiltration of cells was only observed in the
presence of a flow perfusion bioreactor. Thus the presence of nanofibers was found
to hinder cell migration due to the presence of smaller pores, but at the same time
influenced cell spreading, proliferation, and differentiation [52]. However, these 3D
scaffolds combining nano- and microscale fibers (nano/microfiber scaffolds) have a
layered nanofibrous structure instead of a randomly mixed structure composed of
nanofibers and microfibers. Therefore, the nanofibrous layers, which were formed
in sheet-like structures over microfibers, can prevent the cells from infiltrating
adequately into the scaffolds.

3 Mechanical, Physicochemical, and Biological Properties

of Multiscale Scaffolds

The success of a tissue engineering scaffold ultimately depends on the interplay of

various biological, mechanical and physicochemical properties. For every success-
ful application, these tissue engineered constructs must satisfy the essential
requirements of biological, mechanical, and physical properties. The scaffold
architecture should support and withstand compressive forces and contraction
in vivo. The scaffold should provide adequate support for cell attachment and
growth in vitro and facilitate mass transfer of nutrients and oxygen when implanted
in vivo. For biodegradable scaffolds, the rate of degradation should balance the rate
of neo-tissue formation and remodeling, wherein the degree of remodeling depends
on the type of tissue. Shalumon et al. prepared multiscale poly(e-caprolactone)
(PCL) fibrous scaffolds [46]. Various processing parameters such as viscosity,
applied voltage, and flow rate influence the fiber diameter and result in the forma-
tion of a simultaneous micro/nanofiber matrix. The presence of nanofibers embed-
ded in the microfiber matrix was found to enhance the mechanical strength in
comparison to microfibers alone. Also, the strain values were increased, which
may be attributed to the cumulative elongation effect in the presence of the nano/
microfiber combination. The incorporation of nanocrystalline hydroxyapatite
(nHA) into these multiscale fibers did not alter the fiber morphology; however,
the fiber diameter was slightly larger. This was probably due to the increase in
polymer viscosity. The cellular activity on multiscale scaffolds, regardless of the
incorporation of nHA, was higher than on microfibers or nanofibers alone. Cells
attached to the multiscale fibers were flattened, laterally stretched, and extended
(Fig. 1) [46].
Similarly Shalumon et al. also fabricated electrospun porous poly(lactic acid)
(PLA) multiscale scaffolds and evaluated their physicochemical and biological
properties in detail [53]. These multiscale scaffolds were developed through a
bimodal fiber fabrication system. The three main solution properties, solution
viscosity, conductivity, and surface tension, were determined to be the governing
6 S. Srinivasan et al.

Fig. 1 SEM images of cell attachment after 12 h of incubation. (a) Cell attachment on multiscale
scaffold. (b) Cell growth into the multiscale scaffold; arrows indicate spherical morphology of
cells attached to microfibers. (c) Cell access to the interior of the multiscale scaffold through the
pores in the microfibers (Adapted from [46])

Fig. 2 3D projection images of cell infiltration behavior into (a) micro/nanofibrous scaffolds and
(b) micro/nano/nHAp fibrous scaffolds (Adapted from [53])

factors in the formation of multiscale fibers. Cells seeded on the multiscale scaffold
displayed a well-flattened and laterally stretched morphology over the scaffold
surface. Cell penetration and proliferation on multiscale scaffolds was enhanced
in comparison to nano- or microscale scaffolds (Fig. 2). This was due to the
combined effect of porosity and low contact angle offered by the microfibers and
the capacity for spreading offered by nanofibers. Increased cell penetration
also resulted in homogenous distribution of the cells throughout the multiscale
scaffold [53].
Santos et al. elucidated the importance of endothelial cell (EC) colonization and
angiogenic potential of nano/microfibrous combined scaffolds [54]. The ECs
spanned between microfibers with the support of nanobridges formed by the
nanofibers, yielding a higher density of adherent ECs. This spanning of cells
between the fibers was absent in the microfiber control. This could be due to the
presence of a nanonetwork that induced a different cytoskeletal arrangement, which
was reflected in the stretched shape with numerous cellular extensions. Besides
improving the interconnectivity, these nano/micro scaffolds provided a means of
physical support for the enhanced growth of ECs, thus showing close resemblance
to morphology of a capillary-like network [54]. Edwards et al. also described
similar work wherein a multiwalled carbon nanotube–PLGA nanofiber composite
presented ideal pore spanning, allowing uniform distribution of fibroblast-like cells
throughout the scaffold surface [55]. With an increase in cell spanning and
stretching, the receptors also get stretched and activated. Tuzlakoglu et al.
Multiscale Fibrous Scaffolds in Regenerative Medicine 7

developed polymeric micro/nanofiber scaffolds from a blend of starch and PCL.

The results indicated changes in cell morphology and cytoskeletal rearrangement.
The cell receptors were stretched and activated, leading to the expression of
different genes in comparison to the unstretched cells [48]. Mota et al. developed
dual-scale scaffolds consisting of aligned PCL microfilaments and PLGA
nanofibers [32]. They observed that in the dual-scale scaffolds, cells were able to
colonize the interfilament gap caused by the microfilaments. This was due to the
effect of the guidance in cellular adhesion exerted by the electrospun nanofibers,
which were also responsible for the morphological changes between cells adhered
on fibers with different orientations. These dual-scale scaffolds are also
characterized by fiber alignment and high spatial connectivity capable of directing
cell orientation and migration [32]. The interaction of cells with ECM is not only an
anisotropic behavior, but is also dependent on the chemical changes experienced
from the surrounding environment. The cells endure chemical stimulation by direct
contact with the surrounding ECM or the neighbors, which greatly depends on the
spatial concentration distribution of the fibers. By altering the composition of
nanofibers while electrospinning, multilayered scaffolds with specified chemistry,
thickness, and morphology can be obtained [56].
Porosity and pore size are among the key factors responsible for the success of
tissue engineered scaffolds. Optimum porosity enhances cell spreading and migra-
tion while supporting the exchange of nutrients between the scaffold and the
surrounding environment. Porosity and pore size in electrospun scaffolds are
mainly dependent on the fiber diameter and their packing density. Soliman et al.
developed a bio-inspired multiscale 3D scaffold for soft tissue engineering wherein
the fiber distributions were intermixed into a single, multimodal layer [57]. They
also stated the importance of combining nano- and microfibers in a single scaffold
wherein cell motility and adhesion was improved by the network of nanofibers. The
web of nanofibers helped in bridging the cells across the microfibers, thus
colonizing the entire scaffold, while the microfibers produced larger pores to
accommodate the cells. This is a valuable characteristic because in a single-scale
scaffold the cells are frequently confined to a single fiber due to the absence of
bridging across microfibers that are far apart. The multimodal scaffold also
exhibited superior strength and stiffness in comparison with the controls [57].
Multiscale scaffolds composed of nano- and microscale fiber meshes using
two different electrospinning techniques, multilayer electrospinning and mixing
electrospinning, were reported by Kidoaki et al. [58]. Initially, four different
polymers were individually electrospun: type I collagen, styrenated gelatin (ST-
gelatin), segmented polyurethane (SPU), and poly(ethylene oxide). Then, these
individual fiber meshes were deposited layer-by-layer to form a trilayered
electrospun mesh and mixed electrospun fiber mesh. A bilayered tubular construct
composed of a thick SPU microfiber mesh as an outer layer and a thin type I
collagen nanofiber mesh as an inner layer was fabricated as a prototype scaffold of
artificial grafts and may provide compliance matching with native arteries and
tissues. Further insights into detailed in vitro and in vivo studies are essential to
visualize the functioning of the fiber mesh after implantation [58]. Thus, the
8 S. Srinivasan et al.

synergistic combination of micro- and nanofiber hierarchical scaffolds developed

using combinations of a wide variety of biopolymers showed unique mechanical,
biological, and physicochemical properties that are suitable for diverse tissue
engineering applications and are absent in single-scale scaffolds.

4 Applications of Multiscale Scaffolds in Tissue Engineering

4.1 Bone

Bone tissue engineering is a branch of tissue engineering that aims to repair and/or
regenerate bone using scaffolds with cell-based therapies and growth supplements.
It may be used to restore skeleton function in the field of orthopedic and oral-
maxillofacial surgery [59–61]. Scaffolds for bone tissue engineering are developed
with the primary aim of closely mimicking the biophysical structure of natural
ECM. Tuzlakoglu et al. developed nano- and microfiber combined scaffolds from
starch/PCL-based biomaterials to support bone cells [48]. The nanofibers were
electrospun in a random manner between the microfibers, thereby providing
nanobridges similar to those found in ECM. In the presence of nanofibers, a well-
spread cellular morphology and cytoskeletal organization of the SaOs-2 human
osteoblast-like cell line and rat bone marrow stromal cells was observed.
Osteoblasts were directed to bridge between microfibers and this resulted in
scaffolds completely filled with cells after 2 weeks of culture. With cell stretching,
the receptors also got stretched and activated, resulting in the expression of various
genes in comparison to the unstretched cells. Cell viability and alkaline phospha-
tase (ALP) activity for both of the cell types was found to be enhanced in nano/
microfiber combined scaffolds as compared to control scaffolds based on fiber
meshes without nanofibers. Consequently, the developed structures are believed
to have a great potential for the 3D organization and guidance of cells that are
important for engineering 3D bone tissues. Their unique architecture, which
supports and guides the cells, makes them suitable candidates for bone tissue
engineering applications [48]. Martins et al. also developed bone ECM-inspired
structures by conjugating electrospun chitosan (Cht) nanofibers within biodegrad-
able polymeric microfibers [poly(butylene succinate) (PBS) and PBS/Cht], assem-
bled in a fiber mesh structure [62]. The physical properties of these scaffolds could
be enhanced by developing a highly connected porous framework. The osteogenic
differentiation of human bone marrow mesenchymal stem cells (hBMSCs) pro-
duced an increased amount of calcium phosphates on these nanofiber-reinforced
composite scaffolds, confirming ECM deposition and mineralization, mainly in the
PBS/Cht-based fiber meshes. The osteogenic genotype of the cultured hBMSCs was
confirmed by the expression of osteoblastic genes, namely those coding for ALP,
osteopontin, bone sialoprotein and osteocalcin, and the transcription factors Runx2
and Osterix, all involved in different stages of osteogenesis [62]. Osathanon et al.