Bioactive Nanostructured Scaffold-Based Approach for Tendon and Ligament Tissue Engineering

Abstract

An effective therapeutic strategy to treat tendon or ligament injury continues to be a clinical challenge due to the limited natural healing capacity of these tissues. Furthermore, the repaired tendons or ligaments usually possess inferior mechanical properties and impaired functions. Tissue engineering can restore the physiological functions of tissues using biomaterials, cells, and suitable biochemical signals. It has produced encouraging clinical outcomes, forming tendon or ligament-like tissues with similar compositional, structural, and functional attributes to the native tissues. This paper starts by reviewing tendon/ligament structure and healing mechanisms, followed by describing the bioactive nanostructured scaffolds used in tendon and ligament tissue engineering, with emphasis on electrospun fibrous scaffolds. The natural and synthetic polymers for scaffold preparation, as well as the biological and physical cues offered by incorporating growth factors in the scaffolds or by dynamic cyclic stretching of the scaffolds, are also covered. It is expected to present a comprehensive clinical, biological, and biomaterial insight into advanced tissue engineering-based therapeutics for tendon and ligament repair.

Full text

Citation: Govindaraju, D.T.; Chen,
C.-H.; Shalumon, K.T.; Kao, H.-H.;
Chen, J.-P. Bioactive Nanostructured
Scaffold-Based Approach for Tendon
and Ligament Tissue Engineering.
Nanomaterials 2023,13, 1847. https://
doi.org/10.3390/nano13121847
Academic Editors: Luca Valentini
and Antonino Morabito
Received: 5 May 2023
Revised: 5 June 2023
Accepted: 9 June 2023
Published: 12 June 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
nanomaterials
Review
Bioactive Nanostructured Scaffold-Based Approach for Tendon
and Ligament Tissue Engineering
Darshan Tagadur Govindaraju 1, Chih-Hao Chen 2,3 , K. T. Shalumon 4, Hao-Hsi Kao 5
and Jyh-Ping Chen 1,3,6,7,8,*
1Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San,
Taoyuan City 33302, Taiwan; darshu.tg@gmail.com
2Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital at Keelung,
Chang Gung University College of Medicine, Anle, Keelung 20401, Taiwan; chchen5027@gmail.com
3Craniofacial Research Center, Chang Gung Memorial Hospital at Linkou, Kwei-San,
Taoyuan City 33305, Taiwan
4Department of Chemistry, Sacred Heart College, Mahatma Gandhi University, Kochi 682013, India;
shaumon@gmail.com
5Division of Nephrology, Chang Gung Memorial Hospital at Keelung, Chang Gung University College of
Medicine, Anle, Keelung 20401, Taiwan; kao95812@yahoo.com.tw
6Department of Neurosurgery, Chang Gung Memorial Hospital at Linkou, Kwei-San,
Taoyuan City 33305, Taiwan
7
Research Center for Food and Cosmetic Safety, College of Human Ecology, Chang Gung University of Science
and Technology, Kwei-San, Taoyuan City 33305, Taiwan
8Department of Materials Engineering, Ming Chi University of Technology, Tai-Shan,
New Taipei City 24301, Taiwan
*Correspondence: jpchen@mail.cgu.edu.tw; Tel.: +886-3-2118800
Abstract:
An effective therapeutic strategy to treat tendon or ligament injury continues to be a clinical
challenge due to the limited natural healing capacity of these tissues. Furthermore, the repaired
tendons or ligaments usually possess inferior mechanical properties and impaired functions. Tissue
engineering can restore the physiological functions of tissues using biomaterials, cells, and suitable
biochemical signals. It has produced encouraging clinical outcomes, forming tendon or ligament-
like tissues with similar compositional, structural, and functional attributes to the native tissues.
This paper starts by reviewing tendon/ligament structure and healing mechanisms, followed by
describing the bioactive nanostructured scaffolds used in tendon and ligament tissue engineering,
with emphasis on electrospun fibrous scaffolds. The natural and synthetic polymers for scaffold
preparation, as well as the biological and physical cues offered by incorporating growth factors in the
scaffolds or by dynamic cyclic stretching of the scaffolds, are also covered. It is expected to present a
comprehensive clinical, biological, and biomaterial insight into advanced tissue engineering-based
therapeutics for tendon and ligament repair.
Keywords:
tissue engineering; scaffold; electrospinning; tendon; ligament; growth factors; dynamic culture
1. Tissue Engineering of Tendon and Ligament
Tissue engineering is an evolving multidisciplinary field for regenerating damaged
tissue/organs by transplanting a cell-seeded functional scaffold. Using converged knowl-
edges from biology, materials science, chemistry, and engineering, tissue engineering has
combined the use of cells, growth factors, and scaffolds to make remarkable progress
over the last decade (Figure 1) [
1
]. Within the three components, the scaffold is the most
important one; it interacts with cells and growth factors and provides a structural sup-
port for cell attachment and tissue development [
2
]. To facilitate cell attachment, growth,
and differentiation, the scaffold should be endowed with unique properties, such as bio-
compatibility, biodegradability, and a degradation rate that can match the rate of tissue
Nanomaterials 2023,13, 1847. https://doi.org/10.3390/nano13121847 https://www.mdpi.com/journal/nanomaterials

Nanomaterials 2023,13, 1847 2 of 35
regeneration [
3
]. The extracellular matrix (ECM) is secreted by cells to provide a structural
support for cells. It also contains many highly regulated biologically active molecules
that can determine the action and fate of cells [
4
]. The proteins and glycosaminoglycans
(GAGs) are types of biomolecules found in the ECM, and they include fibrous proteins,
adhesive glycoproteins, and proteoglycans. The proteoglycans consist of a core protein
and covalently attached GAG chains [
5
]. The fibrous proteins can provide tensile strength
while the proteoglycans can resist compressive forces [
6
]. Although the composition of
ECM may be different, cell adhesion, cell-to-cell communication, and cell differentiation
are the common functions of the ECM. The ECM can bind, release, and activate signaling
molecules, and also modify cellular response to soluble factors [7]. Therefore, the scaffold
should mimic the morphological structure and chemical composition of the ECM, allowing
cells to attach to the scaffold, proliferate, and differentiate into new tissues [8].
Figure 1.
The three important components to be considered for the application of tissue engineering
in damaged tissues repair. A coordination of cells cultured in a biomaterial-based scaffold, which
is endowed with suitable biochemical signals provided by growth factors, can regenerate tissues
in vitro and repair damaged tissues in vivo.
1.1. Structure of Tendons and Ligaments
The tendons and ligaments are fibrous, dense, regular connective tissue, allowing
for movement and maintenance of body posture. The main functions of tendons and
ligaments are to connect and transmit forces from muscles to bones and from one bone
to another, respectively. They share a comparable composition and hierarchical structure.
The ECM of tendons is composed predominantly of collagen, which accounts for up to
85% of the dry weight of a tendon tissue. Within different types of collagen in tendon
ECM, collagen type I represents ~95% of the dry weight, with the rest being collagen
type III, V, XI, XII, and XIV. The tendon is composed of two primary cellular components:
tenocytes that are fully differentiated cells and account for 90–95% of cellular content in
a healthy mature tendons, and tendon stem/progenitor cells that are located within the
ECM of the tendon. Additional cellular components include synovial cells located on
the surface and vascular cells present in the endotenon and epitenon regions. In normal

Nanomaterials 2023,13, 1847 3 of 35
circumstances, the maintenance and repair of tendons are carried out by tenocytes, but
tendon stem/progenitor cells also perform a crucial function in this process by engaging
themselves in self-renewal and differentiation into tenocytes [
9
]. Structurally, a tendon has
a well-organized structure with three layers of fibrous bundles: the primary fiber bundle
(sub-fascicle) formed from a bunch of collagen fibers, the secondary fiber bundle (fascicle)
formed from a group of primary bundles, and the tertiary bundles formed from a bundle of
secondary fiber bundles (Figure 2) [
10
,
11
]. The diameters of the sub-fascicles and fascicles
are dependent on the overall length of the tendon, but small tendons (such as flexor and
extensor tendons in the fingers and toes) have smaller sub-fascicles and fascicles, while
large tendons (such as the Achilles tendons) have larger sub-fascicles and fascicles. In
humans, the fascicles have diameters ranging from 150 to 1000
µ
m, while the tertiary fiber
bundles have diameters ranging from 1 to 3 mm [12].
Figure 2. The anatomical structure (a) and the histological image (b) of a tendon.
The structure and composition of a tendon leads to its unique ability to withstand a
high tensile strength, and this distinct mechanical characteristic distinguishes it from other
tissues in the body. Indeed, in order to play a key function in joint stability during move-
ment and physical activity, the tendon and ligament need to display superior mechanical
strength and flexibility [
13
–
15
]. This can be revealed from the four distinct regions in a
typical force vs. displacement curve of a tendon (Figure 3) [
16
]. However, it should be noted
that although the typical force-displacement curves of tendons and ligaments show similar
profiles, these tissues show different displacement values at the end of the toe region as
well as with different failure forces. The collagen fibers in a tendon or ligament are crimped
while at rest and start to align with each other when a load is applied, and this leads to the
loss of the crimped structure found in the toe region. The second region is referred to as
the elastic/linear region as all recruited collagen fibers are straightened when a tendon is
stretched in this region. In this region, the tissue provides excellent elastic recovery when
the load is removed, and the fibers gradually begin to slide in relation to one another. The
slope of the curve changes when the force is raised beyond the linear region, at the end
of which the plastic region begins. Continued straining causes microscopic tears in the
tissue, which can result in tendinopathy. As the fibers are subjected to further stress/load,
the macroscopic failure finally appears and eventually tendon rupture occurs [
17
]. An
inflection point in the stress–strain curve represents the start of the damage to the tendon,
while an inflection point in the stress–strain curve marks the point from strain-stiffening
to strain-softening, and may indicate a damage threshold [
18
]. The mechanical proper-
ties of tendons and ligaments are closely associated with their cross-sectional areas and
physiological roles, as well as the rate of deformation during loading.

Nanomaterials 2023,13, 1847 4 of 35
Figure 3.
A typical force vs. displacement curve of a tendon and its collagen fiber structure during stretching.
The musculoskeletal tissues contain both soft and hard tissues, which provide a gradi-
ent of mechanical properties with progressive transition from one end to the other. To meet
this need, material gradients existed at the junctions for reducing stress concentrations
found at these sites. At the interface of tendon/ligament to muscle or bone, the junctions
are called entheses and myotendinous junctions, which enable a physiological transmission
of load over the junction over a wide area [
19
,
20
]. The entheses connect tendons/ligaments
to bones and vary widely based on the anatomical sites and the structures involved, but
in general they can be divided into two main categories: the fibrous entheses and the
fibrocartilaginous entheses. In a fibrous enthesis, tendons/ligaments are connected to
bones through acute angles with Sharpey’s fibers and collagen fibers extending directly
from the periosteum [
21
]. In contrast, the fibrocartilaginous enthesis contains a progres-
sive mineralization gradient, which can be separated into four zones: tendon/ligament,
unmineralized fibrocartilage, mineralized fibrocartilage, and bone. The complex and het-
erogeneous structure of the enthesis is essential to ensure smooth mechanical stress transfer
between bones and tendons/ligaments. As the interface is not regenerated following
injury and usually results in high rupture recurrence rates, the tissue engineering approach
represents a promising strategy for the regeneration of a functional enthesis [
22
,
23
]. The
interface between the tendon and the muscle tissue is a myotendinous junction, which
offers a gradual transition between stiff tendons to soft muscles. Different to entheses,
a myotendinous junction connects a high cellularity tissue (muscle) to a low cellularity
ECM-rich tissue (tendon). The microscopic structure of a myotendinous junction shows
myofibers that can generate conical finger-like projections interlocking the tendon ECM [
24
].
At the macroscale, it creates a network overlapping the muscle and tendon tissues and
increases their interface area. Sensini et al. have provided a comprehensive review of
notable works using electrospun scaffolds for tendon and ligament tissue engineering at
the enthesis and myotendinous junction [25].
1.2. Healing of Injured Tendon
As tendons and ligaments have a unique structure and function, their repair after
injury remains a challenging task for clinicians. The low cellularity and hypovascularity in
these tissues usually leads to ineffective treatment and reduced healing ability [
26
]. Indeed,
tendon and ligament injuries are among the most prevalent types of injuries sustained by
the body, especially in the young and physically active population. Apart from common
injuries, the elderly are often affected by chronic deterioration of these tissues, which

Nanomaterials 2023,13, 1847 5 of 35
occurs as a result of aging. Annually, around 300,000 surgical repairs of tendons in the
hand, foot, and ankle, and roughly 350,000 anterior cruciate ligament (ACL) reconstructive
procedures are performed in the United States, costing approximately USD 30 billion
dollars [
27
]. Tendon injuries may occur from acute or chronic changes, or a combination
of both. Chronic injuries are often related to intrinsic factors such as genetics, sex, age,
nutrition, and general health, while acute injuries are commonly associated with extrinsic
factors such as excessive and inadequate mechanical loadings [
28
,
29
]. As mentioned
before, the low cellularity, hypovascularity, and low metabolic activity of a tendon make its
ability to heal after injury particularly poor, and in most cases the repaired tendon cannot
fully restore its mechanical properties as before, and re-rupture is likely to occur. This
problem could be linked to insufficient tissue regeneration, where substantial differences in
molecular and histological structure between neo-tendon and native tendon are commonly
found [
30
]. This situation may arise from several reasons, such as change in tenocyte
phenotype as well as change in ECM composition and arrangement in the neo-tissue [
31
].
All these problems eventually lead to weakness of regenerated tissue, with post-operative
discomfort and fibrous adhesion, and eventually results in tearing or complete rupturing
of tendons [32].
There are three main stages during tendon healing post-injury, including inflammation,
proliferation, and remodeling (Figure 4) [
33
]. After tendon injury, the next 3 to 7 days
are the inflammation phase. During this period, various chemotactic agents are released,
modulating the inflammatory responses, stimulating the proliferation of fibroblasts and
tenocytes, activating the angiogenesis process, and synthesizing mostly type III collagen.
The proliferation phase, which is the second phase, usually lasts until the third or fourth
week post-injury and is characterized by a rapid increase in cellularity by fibroblasts, as well
as by random ECM structures. The last remodeling phase can be further subdivided into
two stages: consolidation and maturation. During the consolidation stage, the production
of collagen and GAGs is reduced. At the same time, the tenocytes and collagen fibers begin
to align themselves along the longitudinal axis of the tendon, and finally become capable
of bearing load. At this time, the cellularity of the tissue decreases, while production rate
of type I collagen increases. The maturation stage is a lengthy process that may take up
to a year to complete. During this period of tendon healing, the scar tissue gradually
starts to show and a histological appearance that is more resemblance of a healthy tendon
may appear as the healing process progresses and tendon tissue matures. However, the
biomechanical properties of repaired tendons are usually weaker than those of the native
tendons, and collagens in the repaired tendons usually have less crosslinking and have
smaller diameters than those in undamaged tendon, rendering the tendon more vulnerable
to re-injury [34].
Figure 4.
Overview of the tendon repair process in humans. The healing of ruptured tendons passes
through three phases with distinctive cellular and molecular cascades. Their duration depends
on the location and severity of the injury. During the first two stages (indicated by white arrows),
growth factors, stem cells or biomaterials are implemented. Reproduced with permission from [
35
].
Copyright 2015. Elsevier.

Nanomaterials 2023,13, 1847 6 of 35
1.3. Treatment of Tendon and Ligament Injuries
Tendon and ligament injuries can be treated with extracorporeal shockwave therapy,
eccentric exercise therapy, ultrasound, and low-intensity laser therapy, all of which are
considered conservative and unsatisfactory [
36
]. Additionally, the use of a non-steroidal
anti-inflammatory drugs for pain management is controversial because there is a high risk
of spontaneous tendon and ligament rupture after taking these medicines [
37
]. Tendon
pathology can be mainly divided into tendon laceration or tendinopathy [
38
]. Tendon lacer-
ations are commonly treated with advanced suturing techniques; however, initial problems
such as repair site rupture and postoperative adhesions may persist despite improved
surgical methods and rehabilitation approaches [
39
]. In cases where substantial tissue
defects occur, tendon and ligament grafting may be necessary by employing autografts or
allografts [
40
,
41
]. Allografts have several limitations, including the high risk of infectious
disease transmission, potential immune response from the recipient, and the difficulty of
preservation during transportation [
42
]. Autografts are used widely in clinical practice for
tendon repair with excellent long-term results when compared to allografts, but problems
such as donor site morbidity and lack of autograft material still persist [
43
]. Furthermore,
when graft efficiency is low during the tendon/ligament restoration process, the graft
cannot meet the mechanical strength requirements [
44
]. A significant amount of emphasis
is therefore being paid to the fabrication of scaffolds made from degradable natural or
synthetic polymers for the treatment of tendon/ligament injuries by tissue engineering.
After implantation of the cell/scaffold construct, the tissue engineering approach for ten-
don/ligament repair could be accomplished pending successful regeneration of functional
neo-tendon/ligament tissues to solve the problems faced by tissue grafting, which usually
fail to restore the lost functionality.
2. Preparation of Scaffolds for Tendon and Ligament Tissue Engineering
2.1. Scaffold Materials
Various polymers have been employed as scaffold materials for tendon and liga-
ment tissue engineering [
45
–
52
]. Scaffolds produced from biodegradable natural poly-
mers or synthetic polymers such as gelatin [
45
], collagen [
53
], chitosan, cellulose [
51
],
poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), poly(glycolic acid) (PGA) [
54
],
and poly(caprolactone) (PCL) [
46
] have been studied before with promising results. Syn-
thetic and natural polymers have their own abilities and inabilities. Synthetic polymers
have controllable properties and good mechanical strength but poor biocompatibility; on
the other hand, natural polymers can promote cell proliferation and attachment and can
also mimic the native ECM components. The main drawbacks of natural polymers are their
low mechanical strength and fast degradation rates [
55
]. Overcoming these drawbacks is
possible by combining two or more polymers to create a scaffold with desirable proper-
ties [
47
,
48
,
52
,
54
,
56
]. However, it should be noted that we do not differentiate biomaterials
that can be used as augmentation strategies or as tendon substitutes for tendon/ligament
repair in the following review.
2.1.1. Synthetic Polymers
Synthetic polymers have been widely used to produce tendon/ligament scaffolds due
to their availability, ease of processing, and reproducibility [
10
,
14
]. In addition, synthetic
polymers exhibit low immunogenicity and are more versatile with controllable chemical
and physical properties to meet different requirements by the scaffolds [
57
,
58
]. Because of
their mechanical strength and biodegradability, synthetic polymers such as PLA, PCL, PGA,
PLGA, and poly(L-lactide-co-caprolactone) (PLCL) are widely used in tissue engineering
applications [
57
,
59
]. Moreover, these polymers have been approved by the US Food and
Drug Administration (FDA) for certain human uses. However, they still lack proper
biological cues for cell adhesion and proliferation, which must be overcome by blending or
coating with natural polymers [60,61].

Nanomaterials 2023,13, 1847 7 of 35
Poly(caprolactone) (PCL) is a linear hydrophobic polymer with good mechanical
properties, a low melting point, and good processability [
62
,
63
]. It is used for a variety of
biomedical applications in humans, including as a suitable candidate to fabricate fibrous
scaffolds [
56
,
64
]. However, the PCL is hydrophobic in nature, which may reduce its ability
to induce cell adhesion, proliferation, and differentiation. Pouly et al. produced random
and aligned PCL nanofibers in flat sheets and rolled them into nanofiber bundles that
could mimic the size scale of fascicle units in soft musculoskeletal tissues and bear tensile
loads [
65
]. The geometry and orientation of aligned nanofiber can significantly increase the
strength of the scaffold, with aligned fiber sheets and bundles showing modulus 125% and
45% higher than random nanofiber sheets and bundles, respectively. In addition, the yield
stress and yield strain of aligned nanofiber bundles were 107% and 140% higher than those
of aligned nanofiber sheets. The adipose derived stem cells (ADSCs) were cultured in these
nanofiber scaffolds over a period of 7 days, with cells attaching preferentially along the
fiber axis in aligned nanofiber bundles. Overall, the PCL scaffold has potential for ligament
and tendon tissue engineering and can provide a milieu for cell adhesion, proliferation, and
elongation, as well as providing mechanical strength from fiber bundles [
65
]. Bosworth
et al. fabricated yarn scaffolds based on PCL fibers and seed human mesenchymal stem
cells (MSCs) in the scaffolds for cell culture in both dynamic and static modes [
66
]. The
yarn fiber porosity was ~56%, and the scaffold provided a suitable macroporous structure
to support cell infiltration. The dynamically loaded PCL fiber yarn scaffold showed a
thicker cell layer around the scaffold’s exterior than the statistically cultured yarn, with cell
proliferation and ECM deposition occurring predominantly along the axial cell orientation
direction after 21-day
in vitro
culture. The dynamic cultured fibrous yarn had the highest
tensile strength, as well as up-regulated specific gene marker expression; endorsing the
electrospun yarn scaffold can promote seeded MSCs to differentiate towards a tendon
lineage [
66
]. In a different study, aligned and nonaligned multilayered PCL fibrous scaffolds
were seeded with human ADSCs. The scaffolds showed cell infiltration and collagen
deposition throughout the thickness of the scaffold, as depicted from scanning electron
microscopy (SEM) images after 28-day
in vitro
culture [
67
]. Most importantly, the aligned
multilayered PCL scaffold was found to enhance collagen alignment and tendon-related
gene expression and construct mechanical properties when compared with nonaligned
multilayered scaffolds. To introduce tendon-specific biochemical cues into electrospun
PCL scaffolds, three PCL single yarns composed of aligned PCL nanofibers were braided
to form a multi-yarn scaffold. The electrospun multi-yarn fibrous scaffold was surface
modified with oxygen plasma, conjugated with heparin and immobilized with fibrous
growth factor-2 (FGF2) for the culture of tendon-derived fibroblasts. The biochemical cues
from FGF2 as well as the physical cues from aligned fibrous structure can accelerate cell
proliferation, increase ECM synthesis, and promote tendon maturation [
68
]. Dynamic
culture in a bioreactor under uniaxial cyclic tensile loading with 5% strain further increased
the rates of cell proliferation and tenogenic differentiation, and the cell/scaffold construct
demonstrated superior mechanical properties and tendon regeneration capabilities 6-week
post-implantation to repair extensor digitorum tendon defects. However, it should be
noted that a scaffold fabricated from PCL may have limited tensile strength and a slow
degradation rate when compared to other polymers used in tendon and ligament tissue
engineering [59,69].
Poly(lactic acid) (PLA) is a linear aliphatic polymer, with lactide being the monomer
component [
70
,
71
]. It undergoes hydrolytic degradation in aqueous solutions by randomly
cleaved ester bonds into lactic acid. The degradation rate is controlled by the amount
of absorbed water, the solubility of degradation products, and the diffusion coefficients
of chain fragments within the polymer [
72
]. The lactic acid could be eliminated from
the body by incorporation into the tricarboxylic acid cycle, which is mainly eliminated
through respiration by the lungs as CO
2
. However, it undergoes slow degradation within a
period of 10 months to 4 years [
73
]. PLA has been mainly used as a scaffold material for
tendon/ligament engineering based on its molecular weight, crystallinity, shape, and site

Nanomaterials 2023,13, 1847 8 of 35
of impact [
73
–
78
]. Sensini et al. cultured human fibroblasts cells on structured bundles
of fibrous PLA/collagen scaffolds and found the electrospun scaffolds showed weaker
mechanical properties, such as stiffness, strength, and toughness, after ageing in phosphate
buffer saline (PBS) [
79
]. However, increasing the mass ratio of PLA in the scaffold improves
mechanical properties such as stiffness and ductility. Cooper et al. fabricated fibrous
scaffolds by braiding PLA fibers using a 3D braiding machine and seeded the scaffolds
with four different types of connective tissue cells from anterior cruciate ligament (ACL),
medical collateral ligament (MCL), Achilles tendon (AT), and patellar tendon (PT) [
74
].
Their study revealed that ACL cells showed the highest cell proliferation rates and gene
expression of fibronectin, collagen type I (COL I), and collagen type III (COL III), followed
by PT and AT cells during
in vitro
culture. Their study suggested that the ACL is the
most suitable cell source for tissue-engineered ligament [
74
]. Jenkins et al. proposed
biodegradable scaffolds from aligned melt-blown PLA fabrics as scaffolds for rotator cuff
tendon tissue engineering [
80
]. The PLA scaffold demonstrated mechanical anisotropy for
the attachment, proliferation, and spreading of human ADSCs. The ADSC-seeded scaffolds
showed a significant decrease in Young’s modulus and loss of integrity as found in cell-free
scaffolds over 28 days. However, the DNA, sulfated glycosaminoglycans (GAGs), and
collagen content significantly increased during this period, and histology and polarized
light microscopy demonstrated deposition of aligned collagen fibers in the scaffolds [
80
].
Considering PLA is a hard and brittle polymer, which may not be suited for soft tissue
applications as in tendons, Vuornos et al. braided filaments fabricated from a PLA/PCL
copolymer PLCL into fibrous scaffolds for the efficient production of a tendon-like matrix
in vitro
using human ADSCs [
81
]. However, they reported that the braided PLA scaffold
had higher cell adhesion, proliferation, and tenogenic differentiation ability compared to
a PLCL scaffold by significantly enhancing tendon-like matrix production in a tenogenic
medium. Furthermore, total collagen content and tendon-specific gene markers were also
significantly higher using a PLA scaffold, which provided an elastic modulus as high as
that of a native Achilles tendon [81].
Poly(lactic-co-glycolic acid) (PLGA) is a linear aliphatic polymer formed from both
lactide and glycolide monomers and has been approved by the FDA for a wide range
of clinical applications [
70
,
82
]. For tendon and ligament tissue engineering, the PLGA
scaffold has attracted particular attention for its design flexibility, good mechanical strength,
and complete bioresorption ability
in vivo
[
70
,
83
–
86
]. Sahoo et al. fabricated a nano-
micro fibrous PLGA scaffold by deposition of electrospun PLGA nanofibers onto the
surfaces of a knitted PLGA scaffold for tendon/ligament tissue engineering [
84
]. The bone
marrow stromal cells from pigs were seeded by directly pipetting a cell suspension or by
immobilizing in a fibrin gel matrix coated to the knitted PLGA scaffold. Although the cell
attachment was comparable, faster cell proliferation and up-regulation of specific gene
markers such as COL I, decorin, and biglycan was found by direct pipetting. Although
many studies on 3D braided scaffolds have been carried out for anterior cruciate ligament
(ACL) replacement, an optimized material selection based on cellular response remains
a challenging issue to be solved [
87
,
88
]. Towards this, Lu et al. used
in vitro
study to
optimize a braided fibrous scaffold for ACL regeneration by focusing on the influence
of composition of three synthetic poly-
α
-hydroxyester fibers, poly(glycolic acid) (PGA),
poly(L-lactic acid) (PLLA), and poly(lactic-co-glycolic acid) (PLGA), on scaffold mechanical
properties, degradation, and cellular response [
85
]. The scaffolds were pre-coated with
fibronectin, an important protein that is upregulated during ligament healing, prior to
in vitro
culture with rabbit ACL cells. Within the three scaffolds, the PGA scaffold showed
the highest tensile strength, and the PLGA scaffold showed the lowest, but the PGA scaffold
showed rapid degradation and resulted in matrix disruption and cell death over time.
Overall, the fibronectin-coated PLLA scaffold was found to be the most suitable substrate
for ACL tissue engineering based on overall cellular response, mechanical properties, and
degradation
in vitro
[
85
]. Ouyang et al. examined a knitted PLGA scaffold for applications
in tendon regeneration in a rabbit model with 10-mm Achilles tendon gap defects. They

Nanomaterials 2023,13, 1847 9 of 35
observed regenerated tendon tissues containing COL I and Col III as early as 2 weeks post-
implantation [
89
]. Additionally, at 12 weeks post-implantation, both the tensile stiffness
and the modulus of the regenerated tendon reached ~50% that of a normal tendon, with
even better results achieved for scaffolds seeded with bone marrow stromal cells. However,
as knitted scaffolds require a coated gel layer from fibrin or collagen for cell seeding, it may
not be used for ligament reconstruction in the knee joint, because the cell-loaded gel will be
dissociated from the scaffold during motion.
2.1.2. Natural Polymers
Natural polymers are gaining attention in biomedical applications due to their unique
properties, such as biocompatibility, and as components in the ECM [71]. These polymers
are recognizable in a biological environment because they have a similar composition to
macromolecules found
in vivo
, which provides them a physiologically related degradation
rate [
90
,
91
]. In addition, natural polymers contain many functional groups for conjuga-
tion with growth factors, which is beneficial for application in tendon/ligament tissue
engineering. Even with these benefits, natural polymers typically show poor mechanical
properties compared with synthetic polymers, as well as low processing ability and poor
reproducibility due to their inconsistency in purity and molecular weight shown from
different samples [92].
The collagen-based biomaterials are the most obvious and common choice for mus-
culoskeletal tissue engineering. As a major component in ECM of the connective tissue,
collagens represent 25% total protein in the body, with low immunogenicity, easy modifica-
tion, and high biocompatibility characteristics [
93
,
94
]. However, as collagens are purified
from animal tissues, the extracted collagens require removal of foreign antigens to avoid
disease transmission. A purified collagen sample usually requires crosslinking to improve
its mechanical strength, which can also slow down the degradation rate. However, the
mechanical strength of a crosslinked collagenous scaffold still fails to match those of native
tendon/ligament tissues with either physical or chemical crosslinking methods, and the
scaffold is still associated with a relatively fast
in vivo
degradation rate [
10
,
73
]. Bellincampi
et al. fabricated collagen fibers and cross-linked them to produce collagen fibrous scaffolds
for seeding rabbit ACL or skin fibroblasts for autogenous reconstruction of the ACL in the
knee [
95
]. They crosslinked ovine dermal collagen fibers extruded through a polyethylene
tubing and formed in a fiber-formation buffer by ultraviolet light to increase the tensile
strength of the fibers. This was followed by aligning two hundred crosslinked fibers in
parallel to form a scaffold with 3-mm diameter. The cells seeded in the scaffold can survive
for at least 6 weeks post-implantation with high cell viability, and the scaffold can be com-
pletely reabsorbed within 6 weeks [
95
]. Similarly, rabbit ACL or PT fibroblasts were seeded
in a crosslinked collagen fiber scaffold to investigate cell adhesion and viability [
96
]. The
PT fibroblasts can proliferate faster than the ACL fibroblasts, but both fibroblasts showed a
10-fold higher collagen synthesis rate in the scaffold than on a tissue culture plate, and the
cells remained viable after autogenous transplantation into a knee joint [
96
]. For guided tis-
sue regeneration of ruptured ACL, the cells isolated from an ACL must retain the ability to
migrate into an implanted scaffold for repair of ligament rupture. For this purpose, Murray
et al. confirmed that ACL fibroblasts isolated from ruptured human ACLs can retain their
ability to migrate into collagen-glycosaminoglycan scaffolds
in vitro
, with cellular growth
and expression of a contractile actin isoform [
97
]. Numerous cross-linking methods for
collagens have been investigated to enhance the mechanical properties of collagen-based
scaffolds [
98
]. Although the mechanical properties of crosslinked collagen scaffolds can be
improved, they are still incapable of mimicking the strength of a natural ACL [
99
,
100
]. To-
ward this, Uquillas et al. optimized the cross-linking parameters to improve the mechanical
properties of collagen scaffolds fabricated from electronically aligned collagen threads [
101
].
The mechanical properties of the collagen thread scaffold after crosslinking with 2% genipin
were significantly improved to mimic the mechanical strength of a native tendon tissue.
The cell adhesion and proliferation rate of human mesenchymal stem cells seeded in the

Nanomaterials 2023,13, 1847 10 of 35
scaffold also increased after scaffold cross-linking. Similarly, Younesi et al. formed a
3D textile scaffold from collagen-based threads to mimic the mechanical properties and
load-displacement behavior of native tendons [
102
]. Topographically, the scaffolds made
from collagen threads showed a densely packed texture and aligned substrate structure.
The scaffolds can stimulate tenogenesis, with seeded mesenchymal stem cells showing
up-regulated expression of tenogenic markers such as tenomodulin, COL I, and cartilage
oligomeric matrix proteins.
The silk fibers from the cocoons of the mulberry silkworm Bombyx mori were used in
most of the research works using silk as a raw material for scaffold fabrication, which is
particularly promising in the musculoskeletal field [
103
,
104
]. The exceptional high strength
and toughness provided by silk, when compared to other natural and synthetic polymers,
make it an interesting choice for regeneration of tendons and ligaments [
105
–
107
]. The silk
fibers will lose their tensile strength with time
in vivo
and complete degradation by prote-
olysis is expected within two years. This degradation rate matches the mechanical strength
requirement of a scaffold for tendon and ligament regeneration. The strength requirement
met by a silk scaffold can also be gradually transferred to a neo-tissue during scaffold
degradation [
108
]. Furthermore, silk scaffolds can enable the attachment and proliferation
of a variety of primary cells and cell lines, including bone marrow stem cells (BMSCs) and
fibroblasts [
108
]. Liu et al. fabricated a hybrid scaffold by impregnating microporous silk
sponges to a knitted silk fiber scaffold [
109
]. They studied the cellular responses of rabbit
ACL fibroblasts (ACLFs) and BMSCs in the scaffold for ACL tissue engineering. The BMSCs
proliferated faster than the ACLFs, and the expression of ligament phenotypic marker
genes was significantly upregulated for BMSCs in comparison with ACLFs. The BMSCs
also produced more ligament-related ECM than ACLFs, leading to the conclusion that
BMSCs are a preferred cell source for the regeneration of ACL using silk-based scaffolds.
Similarly, a scaffold made from bundles of silk fibers was found to be suitable for the
tissue engineering of ACL, which can also match the mechanical requirements of a native
human ACL [
110
]. Besides unique mechanical properties, the biocompatibility and slow
degradability of this silk-based scaffold also provide a suitable milieu for differentiation
of BMSCs toward the ligament lineage. Tissue engineering scaffolds based on silk can be
fabricated using a variety of textile techniques, such as twisting, braiding, and knitting. The
braiding of silk fibers into a wire rope-like structure can form a scaffold. This was reported
by Teuschl et al., who used this method to prepare a silk ACL graft for seeding autologous
stem cells [
111
]. The cell/scaffold construct was effective in stimulating ACL regeneration
in a sheep model when tested
in vivo
, and the elastic modulus of the implanted construct
was equivalent to that of a native ovine ACL [
111
]. Although silk fibroin is a good substrate
for adhesion, proliferation, and differentiation into a ligament lineage of BMSCs, Chen et al.
further modified a silk film with short polypeptides, and the modified silk film permitted
higher rates of ECM production, adhesion, and proliferation of BMSCs compared with pris-
tine silk matrices [
112
]. A few studies investigating silk-based ACL grafts in large animal
models have generated encouraging results [
107
,
113
]. Fan et al. prepared a scaffold by
rolling a knitted microporous silk mesh around a braided silk cord [
107
]. The mesenchymal
stem cells seeded in these scaffolds proliferated and differentiated into fibroblast-like cells
with expression of COL I, COL III, and tenascin C genes. The cell/scaffold construct can
be implanted into a larger animal pig model to regenerate the ACL. The regenerated ACL
neo-tissue can compensate for the tensile strength loss from degradation of the scaffold
24-week postoperatively, and robust cell proliferation and fibroblastic differentiation of
stem cells were found [
107
]. Liu et al. developed a combined scaffold for ACL tissue
engineering by incorporating microporous materials into a knitted silk scaffold [
109
]. After
seeding with BMSCs or ACL fibroblasts, the scaffold was implanted into rabbits and the
BMSCs demonstrated better cell proliferation and glycosaminoglycan synthesis, as well as
ligament-related ECM marker gene expression and protein synthesis, when compared to
ACL fibroblasts 4-week post-implantation [109].

Nanomaterials 2023,13, 1847 11 of 35
Using the collagen derivative gelatin, Yang et al. [
114
] fabricated injectable gelatin
microcryogels (GMs), which showed a microporous structure and good pore connectivity
as well as excellent biocompatibility toward ADSCs. The microporous gelatin microcryogel
scaffold could promote cell attachment and proliferation. They repaired Achilles tendon
defects with ADSCs + GMs, ADSCs, GMs, or a blank control and found both the ADSCs
+ GMs and ADSCs groups enhanced the macroscopic appearance and the biomechanical
properties of repaired tissue without inducing an unfavorable immune response. Other
than suggesting that ADSCs have stimulatory effects on Achilles tendon healing, the
injectable microgel scaffold can provide a less traumatic injectable cell/scaffold delivery
method for the repair of ruptured Achilles tendons [114].
Hyaluronic acid (HA) or hyaluronan is a non-sulfated, linear GAG made up of repeat-
ing disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. With a backbone
containing 5000–30,000 sugar molecules, this biopolymer occurs naturally and provides a
good starting materials for fabricating scaffolds for ACL
replacement [115–117]
. As the nat-
ural form of HA is in a gel from with rapid degradation rates, chemical modification of HA
to enhance its processability and adjust its biodegradation rate have been suggested [
117
].
Using esterified sodium hyaluronate, Cristino et al. fabricated a HA-based scaffold with a
multilayered knitted cylindrical array of fibers, which were disposed transversally to the
longitudinal axis externally and disposed parallel to the central axis of the scaffold in the
center [
117
]. After short-term cell culture with mesenchymal stem cells, the seeded cells
completely wrapped the fibers in the scaffold and expressed CD44, which is an important
receptor for interaction with HA in the scaffold. The cells also secreted proteins responsible
for the functional characteristics of ligaments, including COL I, COL III, laminin, fibronectin,
and actin, but did not secrete collagen type II and bone sialoprotein [
117
]. Alternatively,
coating scaffolds with HA is another venue that can be explored for the tissue engineering
of tendons and ligaments, where the controlled release of growth factors from the HA
gel could also be accomplished. Funakoshi et al. coated chitosan fibers prepared by wet
spinning with HA gel [
118
]. The HA coating could significantly increase the mechanical
properties of the fibrous scaffolds as well as enhance the
in vitro
biological response of
cultured rabbit fibroblasts in terms of cell attachment, proliferation, and production of
COL I. With HA hybridization with the chitosan fiber sheet stacked scaffold, ECM pro-
duction, cell adhesion, and proliferation rates increased when compared to controls using
chitosan fiber scaffold by seeding with rabbit fibroblasts. On day 1, more cells were found
in hybrid HA/chitosan scaffolds from DNA content analysis over chitosan scaffolds, and
immunostaining signal and mRNA level of COL I, as well as mechanical properties, were
clearly predominant in the hybrid scaffolds using HA coating [
118
]. Recently, Chen et al.
developed a HA/PCL core–shell fibrous membrane to act as an anti-adhesive barrier by
preventing fibroblast attachment, penetration, and focal adhesion while providing lubrica-
tion for smooth tendon gliding during post-surgical tendon repair [
119
]. This membrane
scaffold could promote tenocyte migration and fasten tendon healing, indicating that HA
hybridization with biodegradable synthetic polymers can improve the biological properties
of cultured tenocytes.
2.1.3. Natural Polymer Blend
It is challenging to find a single natural or synthetic polymer that can meet all the
requirements as a scaffold material [
120
]. A composite material can combine the beneficial
properties from its constituents, with the possibility to show synergistic features not found
in pristine single materials. Therefore, a composite scaffold that can mimic the complex
structures of tendons or ligaments is expected to show improved biological, biophysical,
and mechanical properties for tendon and ligament tissue engineering [
14
,
90
,
121
,
122
].
Polymer composite materials, which are also known as polymer-based nanocomposite
materials, have emerged as promising options for load-bearing applications in various
fields [
123
,
124
]. The use of composite scaffolds has been reported to completely restore
the functions of a tendon or ligament tissue to a state before injury, using simple surgical

Nanomaterials 2023,13, 1847 12 of 35
procedures with minimal patient morbidity [
14
,
90
,
98
,
125
]. Although scaffolds made from
collagen are suitable for cell attachment and cell growth, their inadequate mechanical
properties prevent their practical use in tendon and ligament regeneration. Similar chal-
lenges in mechanical properties have been found for various biological materials, such
as gelatin, HA, and silk, although these biological materials can support cell attachment
and cell growth [
106
,
126
,
127
]. Specifically, collagen-based scaffolds are usually in a gel or
sponge form with low mechanical strength, which restricts their use in the regeneration of
load-bearing tendons. To solve the inherent deficiency in mechanical properties of pristine
collagen scaffolds, Panas et al. fabricated different collagen/silk composite fiber scaffolds
and demonstrated that a scaffold contains 14% (v/v) silk and 86% (v/v) collagen can pro-
vide similar or even higher ultimate tensile stress in comparison with human ACL [
128
].
Walters et al. used a braid-twist design to fabricate a COL I fiber-based scaffold, which
after crosslinking and adding gelatin was used for a mechanical property evaluation and
cellular response study using rat ligament fibroblast cells [
129
]. The crosslinked scaffolds
with gelatin displayed a Young’s modulus and an ultimate tensile strength closest to those
of human ACL, in addition to increased cellular activity [
129
]. Chen et al. fabricated
knitted silk scaffolds impregnated with freeze-dried collagen sponges for the culture of
BMSCs [
130
]. In a rabbit medial collateral ligament repair model, the defect treated with a
silk/collagen scaffold improved structural and functional ligament repair, forming collagen
fibrils and collagen fibril assembly with a larger diameter than those treated with a silk
scaffold. In a follow up study, the knitted silk-collagen sponge scaffold was used for the
culture of human embryonic stem cell-derived mesenchymal stem cells (hESC-MSCs) under
mechanical stimulation
in vitro
[
131
]. The hESC-MSCs exhibited tenocyte-like morphology
and upregulated expression of tendon-specific gene markers and other mechano-sensory
molecules. After ectopic implantation in rats, the cell/scaffold construct displayed more
aligned cells and larger collagen fibers, as well as higher tendon regeneration ability from
histology and mechanical property characterization.
The physiologic benefits of HA, such as improved cellular adhesion and proliferation
and anti-inflammatory properties, may improve ligament tissue regeneration. Using
this benefit from HA, Majima et al. fabricated composite chitosan/HA fibrous scaffolds
containing different weight percent of HA. They showed that the scaffold made from
0.1% HA provided adequate biodegradability and biocompatibility [
132
].
In vivo
animal
experiments using fibroblasts from rabbit Achilles tendon in the chitosan/HA scaffold
revealed less inflammation induction
in vivo
, and the mechanical properties of regenerated
tendon/ligament tissues could stabilize the joint. A silk/collagen scaffold with collagen
microsponges formed within a knitted silk scaffold has been shown to be effective for
tendon and ligament repair [
133
–
136
]. Bi et al. used a silk/collagen scaffold for ACL
reconstruction and found good infiltration of fibroblast-like cells in the graft. The stiffness
of the graft was much higher than that of an autograft 16-week post-operation [
133
].
Zheng et al. developed an aligned collagen/silk scaffold and evaluated its biomechanical
performance after implantation in a rabbit massive rotator cuff tear model [
136
]. The
scaffold had similar 3D alignment of collagen fibers found in natural tendons and provided
superior mechanical properties. The seeded tendon stem/progenitor cells displayed well-
aligned spindle morphology in the scaffold, with intercellular contacts and ECM deposition
after 7-day
in vitro
culture. The rotator cuff tendon defects in rabbits were repaired with this
aligned collagen/silk scaffold, which could regenerate a neo-tendon tissue with a failure
force 13-fold higher than that provided by a collagen sponge 12-week post-implantation.
The regenerated tendon tissue also displayed abundant collagen fibrils, resembling the
native microstructure but with larger diameters and better alignments [136].
To evaluate the angiogenesis ability of a composite silk/collagen/HA scaffold, Seo
et al. prepared a knitted silk scaffold and fabricate it into a composite scaffold by freeze
drying a collagen-HA solution in the scaffold for ACL regeneration [
137
]. By seeding
human ACL cells in the scaffolds, the composite scaffold exhibited the highest cell at-
tachment and proliferation rates, but no difference in the immune response was found

Nanomaterials 2023,13, 1847 13 of 35
between the two scaffolds by T-lymphocyte culture
in vitro
. By implanting the scaffold
as a graft for ACL repair in the knees of dogs, inflammatory tissue response at implant
site was noted in both scaffolds from gross examination. The composite scaffold-grafted
group revealed granulation tissues consisting of fibroblasts and collagen fibers from his-
tological study, in addition to new blood vessel formation from CD31 staining. On the
other hand, no blood vessels, cells, or collagens were found in the pristine silk scaffold,
suggesting that the lyophilized collagen-HA in silk fibers can enhance cell migration and
new blood vessel formation in vivo [137]. The fibrin hydrogel scaffold formed from fibrin
protein is biodegradable, biocompatible, and has a porous fibrillar structure to treat tendon
injuries [
138
]. However, both biomechanical properties and
in vivo
stability of a fibrin
hydrogel scaffold remain a challenging task for its application in tendon tissue engineering.
To solve this problem, nanostructured fibrin/collagen and fibrin/agarose hydrogels were
demonstrated to provide enhanced healing efficacy in the surgical repair of rat Achilles
tendon ruptures with better functional, histological, and histochemical results [
139
,
140
].
A summary of composite scaffolds from natural polymer blends for tendon and ligament
tissue engineering is included in Table 1.
Table 1.
Summary of composite scaffolds from natural polymer blends for tendon and ligament
tissue engineering.
Type of Polymers Fabrication Methods Type of Study Type of Tissue References
Collagen/gelatin Chemical crosslinking In vitro Ligament [129]
Silk/collagen Freeze-drying In vitro and in vivo Ligament [130]
Silk/collagen Freeze-drying In vitro and in vivo Tendon [131]
Collagen/silk Freeze-drying In vivo Tendon [136]
Chitosan/HA Wet-spinning In vitro Ligament [118]
Chitosan/HA Wet-spinning In vitro and in vivo Ligament/Tendon [132]
Silk/collagen/HA Freeze-drying In vitro and in vivo Ligament [137]
2.1.4. Synthetic and Natural Polymer Blends
Other than blending natural polymers, a key strategy for producing hybrid scaffolds in
ligament and tendon regeneration has been the combination of natural and synthetic poly-
mers. Natural and synthetic polymers each have their own advantages and disadvantages.
Although natural polymers are biocompatible and can promote cell proliferation/adhesion
and mimic the natural ECM, they show fast degradation and low mechanical strength [
55
].
On the other hand, synthetic polymers show controllable physico–chemical characteris-
tics and good mechanical strength, but low biocompatibility. Hence, a synergetic effect
between natural and synthetic polymers may occur by combining them as scaffold mate-
rials, with the expectation that a balance between biological properties and mechanical
performance could be accomplished for tendon and ligament regeneration [
59
,
141
,
142
]. A
hybrid nanofibrous membrane scaffold with a PCL shell and a HA + Ag nanoparticle (NP)
core was produced to prevent peritendinous adhesion by Chen et al., anticipating that HA
can provide effective lubrication during tendon regeneration while Ag NPs can provide
antibacterial activity [
143
]. Rabbit fibroblasts grown on a PCL/HA + Ag NPs nanofibrous
membrane showed very high viability to discount the possible cytotoxicity from Ag NPs.
In vivo
studies with a rabbit flexor tendon repair model indicated that the PCL/HA + Ag
NPs membrane can reduce peritendinous adhesion along with an increased mechanical
strength of healed tendons [
143
]. The same group used random and aligned electrospun
PCL/silk fibroin nanofibrous scaffolds for the culture of rabbit dermal fibroblasts
in vitro
,
followed by
in vivo
study with a cell/scaffold construct to repair Achilles tendon defects in
rabbits [
63
]. Silk fibroin can promote cell proliferation and up-regulate the gene expression
of tendon marker proteins to a higher extent than that provided by fiber alignment alone.

Nanomaterials 2023,13, 1847 14 of 35
The aligned PCL/silk construct can generate neo-tendon tissues with 60.2% tensile stiffness
and 81.3% ultimate load of those found in native tendons with increased deposition of COL
I and tenascin C.
In another study, Saatcioglu et al. fabricated electrospun PCL/chitosan fibrous scaf-
folds by blending 1, 3, or 5% (w/w) chitosan with 10% (w/w) PCL for ligament regeneration
and evaluated their mechanical properties and cellular response [
144
]. The addition of
chitosan into PCL fibers can increase the fiber diameter in the scaffold in addition to its
swelling and degradation behaviors. The attached mesenchymal stem cells showed a
network-like structure in the scaffolds, and adding 3% (w/w) chitosan to PCL fibers pro-
vided the best scaffold for cellular response as well as the highest tensile strength from
tensile testing [
144
]. Similarly, Leung et al. investigated aligned chitosan/PCL nanofibers
for tendon regeneration using differentiated human BMSCs after conjugating transforming
growth factor-
β
3 (TGF-
β
3) [
145
]. The BMSCs in the aligned nanofiber scaffold proliferated
and showed an elongated morphology along the fiber orientation, showing upregulated
expression of tenogenic maker genes and collagen production compared with tissue cul-
ture plates, chitosan/PCL films, and random chitosan/PCL nanofibers. They concluded
that physical cues from the alignment of chitosan/PCL nanofibers and biological cues
from TGF-
β
3 can work synergistically for effective differentiation of BMSCs into tenogenic
progenitor cells to manage tendon defects [
145
]. Domingues et al. reported the use of
cellulose nanocrystals (CNCs) as reinforcing agents in aligned and random electrospun
PCL/chitosan fibrous scaffolds [
141
]. The incorporation of small amounts of CNCs into the
nanofibrous bundles could remarkably elevate their biomechanical parameters to the range
shown by native tendon or ligament tissues. Other than providing mechanical support,
the aligned PCL/chitosan/CNCs nanofibrous scaffold could provide tendon structure-
mimicking topographic cues, a key feature for maintaining tendon cell phenotype [
141
]. A
braided multiscale fibrous scaffold consisting of aligned PCL/collagen/bFGF nanofibers
was also fabricated by Jayashree et al. to mimic a native tendon structure [
56
]. Rabbit
primary tenocytes cultured in the multiscale braided scaffold showed higher cell prolifera-
tion rates and enhanced expression of tenogenic markers in contrast to controls without
bFGF. When subjected to dynamic stimulation
in vitro
, enhanced cellular proliferation and
tenogenic marker expression were found when compared with the static control.
Xu et al. cultured tendon-derived stem cells (TDSCs) in an electrospun poly(L-lactide-
co-
ε
-caprolactone)/collagen nanoyarn scaffold under mechanical stimulation for tendon
tissue engineering [
146
]. They reported well proliferated TDSCs associated with good
tendon ECM gene expression and protein synthesis rates when cells were cultured in
the scaffold
in vitro
under mechanical stimulation. Interestingly, after implantation into
nude mice for mechanical stimulation
in vivo
, the TDSCs showed long-term survival
and neo-tendon formation. Furthermore, the TDSCs/scaffold construct after dynamic
culture
in vitro
was used successfully to repair injured tendons in a rabbit patellar tendon
defect model, where neo-tendon tissues with enhanced production of tendon-related
proteins and good mechanical properties were produced
in vivo
[
146
]. Darshan et al.
recently fabricated suture-embedded spiral wound aligned gelatin/PCL/heparin nanofiber
scaffolds [
38
]. After immobilizing bFGF by bio-affinity to heparin, the scaffold was used
for the culture of rabbit tenocytes and the cell/scaffold construct after
in vitro
culture
was used to repair rabbit Achilles tendon defects. The seeded tenocytes showed good
cell proliferation, upregulated tenogenic gene expression, and enhanced tendon ECM
protein production
in vitro
, and successful tendon repair was demonstrated
in vivo
with
the cell/scaffold construct.
To replicate a hierarchical multi-tissue transition with change from a mineralized
to a non-mineralized tissue at the tendon-to-bone interface, Calejo et al. fabricated hy-
brid scaffolds for tendon repair [
147
]. They prepared a wet-spun fibrous scaffold con-
sisting of two parts: PCL/gelatin aligned microfibers to mimic the tendon tissue and
PCL/gelatin/hydroxyapatite random microfibers to mimic the bone tissue. The human
ADSCs seeded to the PCL/gelatin aligned microfibers showed highly aligned morphology

Nanomaterials 2023,13, 1847 15 of 35
resembling native tenogenic organization and produced tendon ECM molecules. In con-
trast, cells in the PCL/gelatin/hydroxyapatite part presented a more random cytoskeleton
orientation and only produced a osteogenic-like matrix. By assembling the PCL/gelatin
and PCL/gelatin/hydroxyapatite microfibers, this fibrous scaffold formed with a con-
tinuous topographical and compositional gradient is expected to mimic the structural
characteristics at the tendon-to-bone interface [147].
A modified PLGA/silk hybrid scaffold using knitted silk scaffolds and electrospun
PLGA nanofibers was developed by Sahoo et al., who assessed its feasibility in liga-
ment/tendon tissue engineering
in vitro
[
148
]. The mechanically robust nano-micro scaf-
folds were assembled from degummed knitted silk scaffolds coated with an intervening
adhesive layer of silk solution, followed by direct electrospinning PLGA nanofibers onto the
silk scaffold. Taking advantage of the slow degradation rate of silk, which can compensate
for the early degradation behavior of PLGA, the hybrid scaffold showed improved me-
chanical properties and provided continued support to an injured ligament/tendon tissue
during tissue regeneration, with the seeded BMSCs cells showing high cell viability and
proliferation rates. A follow-up study from this group created a similar PLGA/silk hybrid
scaffold but coated the knitted silk microfibers with bFGF-releasing electrospun PLGA
fibers [
149
]. The feasibility to use it for ligament/tendon repair was evaluated
in vitro
using rabbit bone marrow stem cells, which demonstrated good cell viability in the scaffold.
Most importantly, the released bFGF from nanofibers in the hybrid scaffold could promote
cell growth and induce gene expression of ligament/tendon-associated ECM proteins, as
well as increasing collagen production and scaffold mechanical properties [149].
Full et al. prepared PLGA/COL I/polyurethane (PU) scaffolds by electrospinning for
ligament tissue engineering with two PLGA polymers (50:50 and 85:15) and determined
its effects on scaffold mechanical properties and cell adhesion [
150
]. The 50:50 PLGA
scaffold showed similar tensile properties to those of knee ligaments, in contrast to weaker
tensile properties shown by 85:15 PLGA scaffolds. The aligned fiber scaffold also showed
improved tensile properties compared with the random fiber scaffold. By introducing COL
I into the fibers, human fibroblasts could attach firmly to the fiber surface and proliferate
within the scaffold, as is usually necessary for fibrous scaffolds prepared only with synthetic
polymers. Manning et al. fabricated a hybrid scaffold by loading ADSCs and platelet-
derived growth factor BB (PDGF-BB) in heparin/fibrin hydrogel and layered it with an
electrospun PLGA nanofibrous membrane [
151
]. The natural polymer hydrogel part of the
scaffold allowed for delivery of the growth factor and cells, while the electrospun PLGA
backbone provided structural integrity for implantation. From
in vitro
study, good cell
viability and sustained PDGF-BB release was observed.
In vivo
studies with a flexor tendon
defect model created in large animals found only mild immune response from histology
during tendon repair using ADSCs. A summary of composite scaffolds from synthetic and
natural polymer blend for tendon and ligament tissue engineering is included in Table 2.
Table 2.
Summary of composite scaffolds from natural polymer blend for tendon and ligament tissue
engineering.
Type of Polymers Fabrication Methods Type of Study Type of Tissue Reference
PCL/HA Co-axial electrospinning In vitro and in vivo Tendon [143]
PCL/silk Electrospinning In vitro ad in vivo Tendon [63]
PCL/chitosan Electrospinning In vitro Ligament [144]
Chitosan/PCL Electrospinning In vitro Tendon [145]
PCL/chitosan/cellulose Electrospinning In vitro Tendon [141]
PCL/collagen Mixed electrospinning In vitro Tendon [56]
Gelatin/PCL Mixed electrospinning In vitro and in vivo Tendon [38]
PLA/PCL/collagen Electrospinning In vitro and in vivo Tendon [146]

Nanomaterials 2023,13, 1847 16 of 35
Table 2. Cont.
Type of Polymers Fabrication Methods Type of Study Type of Tissue Reference
PCL/gelatin Wet spinning In vitro Tendon-to-bone [147]
PLGA/collagen/PU Mixed electrospinning In vitro Ligament [150]
HA/PCL Co-axial electrospinning In vitro Tendon [152]
2.2. Fabrication of Fibrous Scaffolds by Electrospinning
The electrospinning technique has been widely exploited for the fabrication of tissue
engineering scaffolds [
69
]. It can produce random or aligned fibers from natural and/or
synthetic polymers, with fiber diameters ranging from submicron to micrometer scale
(Figure 5) [153]
. Usually, electrospun nanofibers are deposited randomly on a static collec-
tor, but they can also be collected in a consistent manner using a rapidly rotating collector to
produce a membrane scaffold composed of aligned nanofibers [
154
]. A scaffold consisting
of aligned nanofibers can provide seeded cells with topographical signals to regulate direc-
tional cellular growth as well as inducing higher cell proliferation and differentiation rates
during the formation of functional ligamentous and tendinous tissue [
155
]. Furthermore,
the electrospun fibrous scaffold can be prepared to have high porosity but small pore size,
allowing for nutrient diffusion but preventing penetration of fibroblasts responsible for
post-operative adhesion formation [
156
]. Notably, electrospinning can prepare different
nano/microscale fibrous scaffolds with a complex 3D structure, resembling the natural
ECM in tissues, and these have shown great promise for making artificial functional tis-
sues [
157
,
158
]. However, electrospun fibrous scaffolds produced traditionally are made up
entirely of closed-packed fibers, which can only create a superficial porous structure. A two-
dimensional porous surface rather than a three-dimensional porous structure is formed in
this case. The small pore size on the surface also inhibits the migration of cells into the inte-
rior of the scaffolds and restricts tissue ingrowth [
159
]. Numerous scientists have examined
different nanostructured fibrous scaffolds and their roles in tissue engineering applications,
including skin, neural tissue, tendons, ligaments, bone, and cartilage [
25
,
50
,
51
,
63
,
160
]. De-
spite the fact that these newly introduced fibrous scaffolds have been shown to be effective
in simulating neo-tissue formation, more study is required to completely understand the
cellular responses to complex structures in a scaffold with different pore size distribution
and spatial arrangements. As a result, it is important to look at the complex structure of
fibrous scaffolds fabricated by electrospinning using different methods.
Figure 5.
A schematic diagram of the electrospinning process where a rotating drum (
a
) or a static
collector (b) was used for collecting aligned or random fibers.
2.2.1. Multiscale Electrospun Fibrous Scaffold
A dual electrospinning method combining two extrusion syringes can produce multi-
scale hybrid scaffolds for tendon and ligament tissue engineering. This approach allows
for a multiscale fibrous structure to be incorporated into a single scaffold by controlling
the distribution of electrospun fibers with different properties. For instance,
Jayashree et al.
prepared a braided multiscale hybrid scaffold that contained mixed nanoscale and mi-
croscale fibers by dual extrusion electrospinning, with one syringe creating nanoscale fibers

Nanomaterials 2023,13, 1847 17 of 35
and the other providing microscale fibers (Figure 6) [
56
]. They investigated the cellular
response and reported enhanced expression of tenogenic markers by seeded tenocytes
during both static and dynamic culture. Park et al. fabricated a stacked hybrid fibrous
scaffold containing alternating layers of random and aligned fibers, which provided better
mechanical support than a single-layered fibrous scaffold [
161
]. They reported that the
presence of mechano-chemical gradients in the scaffold can help to establish normal loading
properties at the tissue interface and promote scaffold integration with bones. Employing
two separate electrospinning arrangements, Sensini et al. fabricated multiscale hierar-
chical scaffolds to replicate the hierarchical arrangement present in natural tendons and
ligaments. The scaffold consisted of multiple bundles of aligned electrospun nanofibers,
which mimicked the tendon fascicles, wrapped in a sheath of nanofibrous membrane,
which replicated the tendon sheath [
162
]. The morphology of the scaffold could mimic
the hierarchical arrangement in natural tendons from X-ray tomographic images. The
scaffold also provided mechanical properties (tensile stiffness and toughness) matching
those required to replace tendons. Human fibroblasts could attach and proliferate in the
scaffold by aligning along the long-axis direction of the electrospun nanofibers for
in vivo
regeneration of tendons and ligaments. Laranjeira et al. reinforced aligned electrospun
nanofiber threads prepared from PCL/chitosan with cellulose nanocrystals and incremen-
tally assembled the nanofiber threads into hierarchical scaffolds to simultaneously mimic
the nanotopography, nano-to-macro structure and nonlinear biomechanical behavior of ten-
dons/ligaments. Using human tenocytes and ADSCs, they showed the scaffold can induce
anisotropic organization typical of tendon tissues as well as expression of tendon-related
markers, indicating the scaffold could prevent the phenotypic drift of tenocytes as well as
induce the tenogenic differentiation of ADSCs [
163
]. To improve the mechanical resilience
of the nanofibrous scaffolds, Sensini et al. developed an electrospun bundle of nanofibers
to improve the mechanical performance of the scaffolds [
164
]. By recreating the structure
and performance of tendons and ligaments, the aligned hierarchical Nylon 6,6 electrospun
assemblies showed comparable yield stress (15.6 MPa) and failure stress (235 MPa) of a
tendon or ligament tissue. In a similar study, the same group fabricated electrospun Nylon
6,6 bundles with different nanofiber alignment by controlling the collector speed [
165
]. The
electrospun fibrous scaffold could mimic the stress–strain curve of natural tendons and
showed similar transition and inflection points in the stress–strain curve, as well as similar
elastic modulus in the linear region.
Figure 6.
The fabrication and the fibrous structure of a multiscale electrospun fibrous scaffold.
Reproduced with permission from [56]. Copyright 2019. American Chemical Society.

Nanomaterials 2023,13, 1847 18 of 35
2.2.2. Co-Axial Electrospun Fibrous Scaffold
Co-axial electrospinning technology was used to create core–shell nanofibers and
was disclosed by Sun et al. in 2003, where a spinneret made up of two co-axial needles
was employed separately to deliver two immiscible spinning solutions during electrospin-
ning [
166
] (Figure 7A). Full et al. applied co-axial electrospinning to produce random and
aligned nanofibers with a PU core and a blend of PLGA/collagen core for ligament tissue
regeneration [
150
]. Mixing different mass ratios of PLGA with collagen in the core solution,
they studied the mechanical properties of the fibrous scaffolds, and attachment and prolif-
eration of seeded human foreskin fibroblasts during
in vitro
cell culture. They found higher
mechanical properties of aligned nanofiber scaffolds prepared with 50:50 PLGA/collagen
than with 85:15 PLGA/collagen. Moreover, a significant increase in cell attachment in the
aligned scaffolds of 50:50 PLGA/collagen was found compared to the other group [
150
]. Us-
ing core–shell nanofibers prepared by co-axial electrospinning, the controlled and sustained
release of drugs, growth factors, or nanoparticles could be accomplished by encapsulating
or embedding them in the core and/or the shell compartments [
167
]. Shalumon et al. fabri-
cated core–shell nanofibrous membranes with embedded silver nanoparticles (Ag NPs) in
a shell of polyethylene glycol (PEG)/PCL and a core with HA/ibuprofen. The released HA
from the core could impart a lubrication effect for smooth tendon gliding and reduce fibrob-
last attachment. Ibuprofen and Ag NPs can provide anti-inflammation and anti-infection
properties, respectively. From
in vitro
cell culture studies, initial cell attachment and focal
adhesion of fibroblasts was effectively reduced on the core–shell nanofiber membrane
surface, which also demonstrated minimum cytotoxicity. Simultaneously, the Ag NPs
released from the shell could inhibit the growth of both Gram-positive and Gram-negative
bacteria.
In vivo
studies in a rabbit flexor tendon rupture model showed the core–shell
nanofibrous membrane can reduce post-operative inflammation and tendon adhesion for-
mation to promote tendon healing [
52
]. Along this line, Chen et al. fabricated random and
aligned nanofibers with a core of HA/platelet-rich plasma (PRP) for growth factor delivery
and a shell of PCL for mechanical support (Figure 7B) [
152
]. The aligned core–sheath
nanofibers with PRP (Align
+
) provides the best mechanical properties compared with
aligned nanofibers prepared without PRP (Random) and random nanofibers prepared with
PRP (Random+). The combined effects from growth factors in PRP and fiber alignment in
the Align
+
nanofibrous membrane also led to enhanced proliferation of rabbit tenocytes
as well as up-regulated tendon-specific gene expression and increased tenogenic marker
protein synthesis. Besides biochemical cues from PRP, the cell-seeded Align
+
scaffold was
mechanically stimulated
in vitro
under cyclic tensile loading in a bioreactor, by which
a shorter tendon maturation time and a higher cell proliferation rate were found with
well-preserved tendon phenotype when compared to non-mechanically loaded cell culture
conditions. Therefore, topographical cues from nanofibers can be facilely combined with
mechanical stimulation to ameliorate the cellular response of tenocytes in a core–sheath
nanofiber membrane scaffold for tendon tissue engineering.
Figure 7.
The fabrication (
A
) and fibrous structure (
B
) of co-axial electrospun fibrous scaffolds
prepared by electrospinning. Reproduced with permission from [
152
,
166
]. Copyright 2003. John
Wiley & Sons, Inc.

Nanomaterials 2023,13, 1847 19 of 35
2.2.3. Three-Dimensional (3D) Amalgamated Fibrous Scaffold
A variety of methods to fabricate a three-dimensional (3D) amalgamated fibrous
scaffold using electrospinning have been attempted to mimic the natural ECM structure
of a tendon or ligament, which is made up of a hierarchal structure of aligned collagen
fibers at both the micro- and nanoscales [
168
]. Among these scaffolds, electrospun aligned
biodegradable synthetic nanofibers and microfibers can have desirable mechanical proper-
ties to regenerate a neo-tissue resembling the native tendon and ligament with alignment,
migration, proliferation, and tenogenic differentiation of seeded cells [
169
]. The 3D electro-
spun fibrous scaffold could be prepared from aligned nanofiber yarns after electrospinning
using advanced textile techniques such as braiding and weaving. Different electrospun
PCL scaffolds were fabricated by rolling membranes prepared by melt electrospinning in
three bundles, which were subsequently braided and combined with a bone compartment
for the development of a bone-ligament-bone construct [
170
]. The orientation of human
mesenchymal stem cells (MSCs) was investigated
in vitro
and fiber alignment could ori-
entate the cells towards the axial direction of the fibers to highly express tendon-specific
genes, as compared with randomly oriented nanofibrous scaffolds where no cell orientation
could be found. The final construct with complex geometry could achieve mechanical
resilience under cyclic stretching. Considering the inherent low cellularity and vascularity
properties of tendon tissue, treatments with aligned nanofibers loaded with thymosin
beta-4 was used to improve the biocompatibility, which also significantly upregulated the
expression of tendon-specific gene markers, improved cell proliferation, and promoted
tenogenic differentiation of human ADSCs [
54
]. Combining ADSCs with human tenocytes
(HTs) and human umbilical vein endothelial cells (HUVECs), simultaneous tri-culture
of ADSCs/HTs/HUVECs provided aligned topographic and biomechanical cues to the
attached ADSCs.
The bioactive scaffolds utilized in tendon tissue engineering should be able to with-
stand excessive stresses in addition to encouraging the healing process of injured ten-
dons [
58
]. Aligned electrospun nanofibers can serve the purpose by providing a structural
feature simulating the nonlinear stiffening behavior of crimped collagen fibrils found in
tendon tissue and offer mechanical support similar to the highly anisotropic structure
found in tendon tissue [
168
,
171
]. As scaffolds containing well-aligned ultrafine fibers can
exhibit many of the mechanical characteristics of native tendon tissues, a 3D mat of aligned
fibers prepared by stable jet electrospinning was used to induce differentiation of human
pluripotent stem cells into the tendon lineage [
47
]. The structure of the scaffold can mimic
the microstructure and mechanical properties (Young’s modulus) of native tendons, and
seeded stem cells can differentiate into tenocyte-like cells for Achilles tendon regeneration
through the activated mechanic-signaling pathway. However, the maximal scaffold tensile
strength was only 14.2 MPa, which may restrict its application for tendon regeneration
due to the extreme load-bearing sustained by tendon tissues [
47
]. Following this line,
Shalumon et al. prepared a novel 3D fibrous scaffold using the electrospinning technique,
in which three suture-reinforced single yarns composed of aligned PCL fibers were braided
together to fabricate a multiple-yarn scaffold with good mechanical stability and excellent
tensile strength, as well as a fibrous surface topography for cell seeding (Figure 8) [
68
].
In vitro
culture using tendon-derived fibroblasts indicates that fiber alignment can promote
cellular proliferation and ECM synthesis, as well as expediting tendon maturation. The
cell/scaffold construct after
in vitro
culture can repair extensor digitorum tendon defects
to restore the tendon ECM structure. The use of a 3D electrospun fibrous scaffold as a graft
for tendon repair was reported recently [
172
]. An additively manufactured PCL tubular
stent was integrated with two layers of electrospun drug (vancomycin, ceftazidime, and
lidocaine)/PLGA and collagen/PCL nanofibrous membranes to treat Achilles tendon rup-
ture. The results from this study indicate the repaired tendon can show increased strength
in the stent/drug group compared with the stent group.

Nanomaterials 2023,13, 1847 20 of 35
Figure 8.
(
a
) The fabrication and fibrous structure of three-dimensional (3D) electrospun fibrous
scaffolds. (
b
) The scanning electron microscopy images of suture-reinforced single yarn (SY) covered
with fibers (
A
,
C
), and multi yarn (MY) scaffolds from braiding of three SY (
B
,
D
). An optical image of
MY scaffold is shown in (E). Reproduced with permission from [68]. Copyright 2023. Elsevier.
3. Scaffold Functionalization for Tendon and Ligament Tissue Engineering
3.1. Biological Cues Using Growth Factors
Growth factors (GFs) represent the largest group of biomolecules that can induce
tenogenesis, and considerable studies have been undertaken to elucidate their roles during
tendon healing. GFs can belong to several families, including fibrous growth factor-2 (
FGF-2
or bFGF), transforming growth factors beta (TGF-
β
1,
β
2, and
β
3), vascular endothelial
growth factor (VEGF), connective tissue growth factors (CTGF), platelet-derived growth
factor (PDGF), and insulin-like growth factors-1 (IGF-1) [38,173,174]. GFs not only induce
tenogenic differentiation, but also enhance cell growth and expression of gene markers

Nanomaterials 2023,13, 1847 21 of 35
in tendons and ligaments [
175
]. In response to tendon tissue damage, GFs are released
and bind to external receptors on the cell membrane, resulting in intracellular pathways
for DNA synthesis and transcriptional expression. This can elicit a direct influence on
numerous cellular processes such as cell proliferation, chemotaxis, matrix synthesis, and cell
differentiation, all contributing toward the tendon-healing cascade [
176
,
177
]. Immediately
after tendon injury, activated platelets release GFs from injured tissues, followed by the GF-
driven inflammatory cascade to recruit inflammatory cells to the site of damage, followed
by more GF production, eventually exacerbating the inflammatory cascade [
173
]. The
tendon cells can align themselves next to the injury area, which helps to activate the cells
and produce GFs to promote tendon healing. The optimal milieu for induction tendon
healing thus demands the presence of numerous GFs with a precise ratio, and they should
be delivered in a well-orchestrated temporal pattern [
28
,
173
,
174
,
178
]. Although numerous
GFs have been identified for this purpose, the precise environment for signal transmission
to induce tenogenic differentiation is still largely unknown [56,149,179–181].
3.1.1. Basic Fibroblast Growth Factor (bFGF or FGF-2)
Basic fibroblast growth factor (bFGF), which belongs to the family of heparin-binding
growth factors, is well recognized for being a strong inducer of angiogenesis and cellular
migration. Although
in vivo
bFGF supplementation can affect early stage rat patellar
tendon healing, the prompt inactivation and limited plasma half-life have hindered this
effect at the healing site [
182
]. Considering this, GFs can be integrated into an electrospun
fibrous scaffold, which can act as a depot for the sustained release of GFs in addition
to replicating the natural ECM structure in ligaments and tendons [
180
,
183
,
184
]. For
instance, Sahoo et al. showed bFGF released from a PLGA fibrous scaffold can upregulate
the expression of ligament/tendon-specific genes and increase collagen production [
149
].
Similarly, Jayashree et al. reported that the combination of PCL/collagen/bFGF nanofiber
scaffolds with tenocytes can increase the expression of tendon-related markers such as
COL I
, COL III, and tenascin C [
56
]. Petrigliano et al. [
183
] investigated the effects of
bFGF in a bFGF-coated porous 3D polymer scaffold and reported results in terms of
cell morphology and gene expression [
183
]. The scaffolds were loaded with different
concentrations of bFGF, seeded with cells, and subjected to mechanical stimulation or
maintained in a static environment. They reported a dose-dependent stimulatory effect
from bFGF, which could be further enhanced in the presence of mechanical tensile strain.
3.1.2. Transforming Growth Factor Beta (TGF-β)
Transforming growth factor beta (TGF-
β
) has been found to be active in almost all
phases of tendon healing by guiding fibroblast migration, neovascularization, and ECM
protein production [
174
]. There are three known isoforms of mammalian TGF-
β
(
β
1,
β
2,
and
β
3), and in many circumstances they do not show distinguishable differences in their
effects on cell behavior. To enable binding of TGF-
β
to its receptors and activation of the
intracellular pathways, these isoforms are released as latent precursor molecules and are
activated by binding to three different membrane receptors on the surface of cells engaged
in the tendon/ligament healing process [
185
–
187
]. Tellado et al. seeded ADSCs onto
TGF-
β
2 functionalized biphasic silk fibroin scaffolds containing both anisotropic (ligament-
like) and isotropic (bone-like) pore structures [
175
]. They found that pore anisotropy and
TGF-
β
2 functionalization can synergistically increase the expression of tendon/ligament
gene markers, with up to a 4-fold increase in the anisotropic (ligament/tendon) region
of the scaffold. Such combination strategy of biological and structural cues on stem cell
fate may be a promising way for ligament-to-bone regeneration. Chang et al. developed
PLLA/PEG electrospun fibrous scaffolds and studied the effects of TGF-
β
supplementation
on long-term matrix deposition. The TGF-
β
could transform the fibroblast phenotype from
proliferative to synthetic and stimulate matrix deposition and enhance collagen production,
but showed minimal effects on cell growth [
188
]. A TGF-
β
3-immobilized chitosan/PCL
nanofiber scaffold was investigated by Leung et al. for tendon tissue engineering, in

Nanomaterials 2023,13, 1847 22 of 35
which rapid and effective differentiation of human BMSCs into tenogenic progenitors was
demonstrated [
145
]. Similarly, Roth et al. studied the bioactivity of scaffold-associated
TGF-
β
3
in vitro
by physically adsorbing TGF-
β
3 to decellularized digital flexor tendon
scaffold [
189
]. Using scaffold-associated TGF-
β
3 or free TGF-
β
3 in cell culture medium,
similar cell conformation change and tendon-specific gene expression was found, and up-
regulation of tenascin C and downregulation of other matrix molecules such collagen IA1
and IIIA1 was found, indicating that the bioactivity of TGF-
β
3 is preserved after binding to
the scaffold [
189
]. The TGF-
β
1 has been shown to promote cellular migration and prolifera-
tion as well as enhancing the production of COL I and COL III in flexural tendon-derived
cells [
190
]. Although all three isoforms of TGF-
β
affect the formation of ECM proteins in
mesenchymal cells and tenocytes, the TGF-
β
1 stimulates the production of collagen IA1
and IIIA1 the most [
191
]. However, other studies have found that this effect mediated by
TGF-
β
1 is restricted to tendons only, and other tissues have a different temporal and spatial
gene response when stimulated with TGF-
β
1 [
187
]. However,
Maeda et al.
showed that
an acute tendon transection injury can result in destabilized organization of tendon ECM
structure, with the excessive release of TGF-
β
and ultimately the death of tenocytes [
192
].
This mortality of tenocytes can be prevented by inhibiting the TGF-
β
1 activity. Although
a number of
in vitro
and
in vivo
studies have shown that inhibiting TGF-
β
1 production
may prevent adhesion formation and expand the range of motion in flexor tendons, some
studies have also demonstrated a decrease in the mechanical strength of healed tendon
tissues [193,194].
3.1.3. Platelet-Derived Growth Factor BB (PDGF-BB)
Platelet-derived growth factors (PDGFs) are a class of dimeric polypeptide isoforms
that are made up of three structurally identical subunits. Because of their well-known
chemotactic, mitogenic, and angiogenic properties, PDGFs have been studied as a GF to help
tendon healing after damage or rupture to tendons [
195
]. The PDGF-BB is the most effective
chemotactic factor for mesenchymal stem cells when compared to other dimer isoforms (AB,
AA, CC, and DD) of PDGF. This has elicited interest centered on the effective delivery of
PDGF-BB for wound healing and tissue regeneration with diverse biomaterial formulations.
Furthermore, PDGF can promote the production of other GFs, such as insulin-like growth
factors, to aid in tendon repair [
174
,
195
]. A PDGF-BB immobilized heparin/fibrin hydrogel
can coat PLGA fibrous scaffolds with sustained GF release behavior, which is beneficial for
the cellular activity of ADSCs
in vitro
and the repair of intrasynovial flexor tendon defects in
a canine model [
151
]. The cellularity and vascularity of the injured tendons were increased
following the transplantation of a cell/PDGF-BB-incorporated scaffold construct [
151
].
To improve the biomechanical properties of repaired tendons by delivery of GFs at the
injured site, Evrova et al. incorporated PDGF-BB into double-layered coaxially electrospun
tubes made from biodegradable polyester/urethane block copolymer [
196
]. The PDGF-
BB-incorporated scaffold was used as a graft to regenerate tendon tissue and to improve
tendon healing in an Achilles tendon full laceration model in rabbits. Three weeks after
implantation, PDGF-BB delivered from the scaffolds did not lead to the hyperproliferation
of pro-fibrotic cells and upregulation of
α
-smooth muscle actin expression at the wound
site. Nonetheless, the tensile strength of the healed tendons increased 2-fold using scaffold-
based delivery of PDGF-BB, and increased production of COL I and COL III but decreased
production of fibronectin was found.
A summary of the scaffold-based delivery of growth factors for tendon and ligament
tissue engineering is provided in Table 3.

Nanomaterials 2023,13, 1847 23 of 35
Table 3.
Summary of scaffold-based delivery of growth factors for tendon and ligament tissue engineering.
Scaffold Tissue Type Growth
Factor Effects References
Knitted silk scaffold Ligament/tendon bFGF Upregulate gene expression of
ligament/tendon [149]
PCL/collagen fibrous scaffold Achilles tendon bFGF Enhanced cellular proliferation
and tenogenic gene expression [56]
Gelatin/PCL/heparin scaffold Achilles tendon bFGF
Upregulated COL I and tenascin C,
improved mechanical properties [38]
Electrospun PLLA/PEO
scaffold Ligament TGF-βImproved mechanical properties [188]
Biphasic silk fibroin scaffold Ligament-to-bone TGF-β2
Upregulated tendon/ligament
markers, enhanced cell
proliferation, and ECM synthesis
[175]
None Archiles Tendon TGF-β1Upregulated gene expression of
procollagen type I and type III [190]
Decellularized superficial
digital flexor
tendon scaffold
Tendon TGF-β3
Upregulation of tenascin C and
downregulation of decorin gene,
enhanced tendon regeneration
[189]
Hydrogel-coated
heparin-PLGA fibrous scaffold
Tendon PDGF-BB Enhanced cell proliferation,
increased tensile strength [151]
Electrospun coaxially PU
tubular scaffold Tendon PDGF-BB Upregulated COL I and COL III,
downregulated fibronectin [196]
3.2. Physical Cues Using Cyclic Tensile Mechanical Stimulation
In tendon and ligament tissue engineering, a growing emphasis is placed on dynamic
mechanical stimulation during cell culture, by culturing the cell/scaffold construct in a
bioreactor under cyclic tensile loading. It is reported that mechanical stimulation is one
of the most important physical cues affecting cell proliferation, gene expression, ECM
formation, collagen fiber alignment, and tissue remodeling in tendons and ligaments [
197
].
Moreover, by conveying mechanical tensile strain to cells, the mechanical signals may
activate the adhesion receptors on cell surface and trigger downstream intracellular path-
ways [
198
]. However, the effects of mechanical stimulation on cells are usually influenced
by the magnitude of strain, duration time, and frequency of tensile loading, and all these fac-
tors influence the phenotypic gene expression of mechanically loaded cells during
in vitro
culture [
199
]. Furthermore, different kinds of mechanical stimulation might play a different
role during cell differentiation. For instance, mechanical compression is advantageous for
both osteogenic differentiation and chondrogenic differentiation, but mechanical stretching
has been frequently used for tendons to induce tenogenic differentiation [
199
]. As the main
mechanical stimulus for the formation and development of tendons is tension-induced me-
chanical stretching; the biophysical signal provided from mechanical stretching is expected
to provide physical cues to improve the functions of engineered tendons. To this end, the
in vitro
mechanical stimulation of cells seeded in a scaffold before implantation may have
significant implications for treating tendon or ligament injuries, and damaged tendons or
ligaments will be regenerated better using this biomechanical signal. However, it should
be noted that mechanical stretching may also result in unexpected consequences, such
as increasing the diameter of the scaffold, causing elongation of the scaffold, or reducing
the mechanical properties of the scaffold. Furthermore, cells, especially stem cells, may
undergo early differentiation and apoptosis if they are subjected to excessive mechanical
tensile loading [
200
,
201
]. It should also be noted that mechanical stretching has been shown
to be highly related to the strain value, and only a particular range of strain can promote
tenogenic differentiation [202].

Nanomaterials 2023,13, 1847 24 of 35
Wang et al. studied the effect of uniaxial and biaxial mechanical loading on tendon-
derived stem cells (TDSCs) by applying a 6% biaxial or uniaxial cyclic strain at 0.25 Hz,
8 h/day
, for 6 days to enhance tenogenic differentiation [
203
]. They reported better differ-
entiation of TDSCs toward the tenogenic lineage by applying uniaxial loading, judging
from tendon-specific marker gene expression and protein production, while biaxial loading
causes TDSCs to differentiate into the osteogenic, adipogenic, and chondrogenic lineages.
Engebretson et al. also demonstrated that mesenchymal stem cells seeded on human
umbilical vein (HUV) under mechanical stimulation at 2% strain, 30 min/day, 0.5 Hz can
improve the cellularity in a cell/HUV construct by 37% and the ultimate tensile strength
by 33% in 14 days [
204
]. Although 1% to 15% strain has been shown to be beneficial for
tendon differentiation, the strain is usually controlled within 4–8% based on the physiologi-
cal strain experienced by a tendon
in vivo
. In earlier studies, dynamic stretching at 10%
strain could increase the expression of tendon-related genes such as COL I, scleraxis, and
tenascin C. However, further increasing the strain to 15% resulted in decreased production
of COL I and tenascin C [
198
,
205
]. Consistent with the trend, Chen et al. found the genes
and proteins related to tendons, such as COL I and tenascin C, were expressed more in
aligned fibrous scaffolds seeded with tenocytes and stimulated at 6% strain [
152
]. Similarly,
Jayashree et al. reported that a multiscale PCL–collagen braided scaffold could increase
cellular proliferation and tenogenic marker expression of seeded tenocytes with 5% strain,
0.5 Hz, and 3 h/day mechanical stimulation [
56
]. Recently, Shalumon et al. showed how
different strain rates at 3% and 5% can affect cell growth, gene expression, and protein syn-
thesis in PCL multi-yarn scaffolds [
68
]. Compared to static cultures, dynamic culture with
5% strain mechanical stimulation can improve the rates of cell proliferation and tenogenic
differentiation. Xu et al. revealed that mechanical stimulation under different amplitudes
and frequencies leads to distinct effects on TDSCs seeded in electrospun nanoyarn scaffolds,
including cell proliferation and tenogenic differentiation [
206
]. These effects were most
pronounced at 0.5 Hz and at 4% cyclic tensile strain. In addition, the signaling pathway
analysis showed that although cyclic tensile strain impaired the ECM–receptor interaction,
this physical cue strongly upregulated the genes that encoded the regulators of transcrip-
tional activities and transcriptional factors and aided in cell proliferation and differentiation.
This transcriptome analysis may provide new insights into the signaling networks and the
molecular mechanism when TDSCs are under tensile loading in a fibrous scaffold.
A PCL nanofibrous woven scaffold was used for the tri-culture of human ADSCs/human
tenocytes (HTs)/human umbilical vein endothelial cells (HUVECs) under cyclic uniaxial
elongation along the direction of nanofibers using 4% strain at 0.5 Hz, 2 h/day, for a total
of 12 days. The mechanically stimulated cell/scaffold constructs under dynamic stretch
can significantly enhance tenogenic differentiation with increased production of tendon-
related proteins and upregulated expression of tendon-specific genes [
169
]. Using uniaxial
stretching, Nam et al. cultured BMSCs in a silicon chamber and uniaxially stretched the cells
under different strains
in vitro
. They found that most tendon-related genes and proteins
were expressed under 4, 8, or 12% strain rate and at 0.5 or 1 Hz frequency [
207
]. The highest
cell proliferation rate was found at 4% strain/1 Hz, and the highest collagen production and
tenogenic gene expression were found at 8% strain/1 Hz, but no significant increase in both
cell proliferation and tenogenic differentiation were found by further increasing the strain
or frequency. In general, high strain can induce early cell differentiation and apoptosis, but
it can also change the mechanical properties of the scaffold, where excessive elongation can
lead to enlarged pore size. In contrast, a strain that is too low might not be able to induce
the intended stimulatory effects. The ideal strain rate might also be different in different
loading systems under different operating conditions, which demands careful study to
tailor to each circumstance [
205
,
208
]. However, in scaffold-based physical signaling, most
of the stretching frequencies used are lower than 1 Hz in tendon tissue engineering.
Indeed, a collective finding from previous studies underlines that a mechanical stretch-
ing frequency of 1 Hz may be the optimal value to generate a favorable cellular response,
such as an exceptionally high degree of cell proliferation and tenogenic differentiation,

Nanomaterials 2023,13, 1847 25 of 35
and a diminished beneficial effect was found beyond this value. Nevertheless, a lower
frequency may be better when considering the quality of regenerated tendons or ligaments.
Additionally, cells can be progressively adapted to the stimulus during the rest intervals,
resulting in a reduction in the impact of the applied mechanical stimulation. By including a
rest interval, the mechanical sensitivity of stretched cells may be restored and result in a
more favorable impact. Several studies found that shorter duration and lower frequency
were associated with higher cell proliferation rates. The highest cell proliferation rate was
found with mechanical stretching at 0.5 or 1 Hz and 2 or 3 h/day, which resulted in upreg-
ulated tenogenic marker expression and high-quality neo-tendon tissue formation under
mechanically stimulated
in vitro
[
68
,
146
,
169
,
200
,
206
]. In general, it might be challenging to
determine which dynamic stretching parameter is associated with the strongest impact on
tenogenic differentiation. Therefore, using a bioreactor that offers the possibility to control
several tensile stretching parameters simultaneously is preferred for examining the effect
of each stretching parameter individually. Nonetheless, a neo-tendon construct demands
appropriate biomechanical stimulation with well-tuned parameters in a biomimetic milieu
simulating a native tendon/ligament in the scaffold. To this end, different imaging tech-
niques such as high-resolution X-ray tomography, fluorescent microscopy, and scanning
electron microscopy may be used to confirm changes of cellular shape and orientation dur-
ing dynamic culture. Using these techniques, a study by Sensini et al. showed preferential
axial distribution of human fibroblasts in electrospun aligned PLLA/collagen fiber bundles
by stimulating at 5% strain and 1 Hz frequency for 1 h [
209
]. Table 4provides a summary
of the scaffold-based delivery of physical cues from cyclic tensile loading in a bioreactor for
tendon and ligament tissue engineering.
Table 4.
Summary of scaffold-based delivery of physical cues from cyclic tensile loading in a bioreactor
for tendon and ligament tissue engineering.
Scaffold Cell Type Parameters Effects References
PCL–Collagen braided
fibrous scaffold Tenocytes 5% strain, 0.5 Hz,
3 h/day
Up-regulated tenogenic
gene expression [56]
Human umbilical
vein (HUV) MSCs 2% strain, 0.5 Hz,
30 min/day Increased tensile strength [204]
Porous PLCL scaffold Tenocytes 10% strain, 0.25 Hz,
6000 cycle/day
Increased cellularity,
mechanical properties, and
tenogenic gene expression.
[198]
Collagen sponge scaffold BMSCs
15% strain, 1 Hz, 3 days
cyclic stretching
Increased cellularity, but
COL I and tenomodulin
expression did not
significantly change.
[205]
PCL/HA/PRP core–shell
nanofibrous scaffold Tenocytes 6% strain, 1 Hz,
3 h/day
Increased gene expression of
COL I and tenascin c. [152]
PCL multi-yarn
fibrous scaffold
Tendon-derived
fibroblasts
3% and 5% strain,
0.5 Hz, 3 h/day
Enhance cell proliferation
and ECM synthesis rates,
fasten tendon maturation.
[68]
P(LLA-CL)/Collagen
scaffold TDSCs 2, 4, and 8% strain; 0.3,
0.5 and 1 Hz
Up-regulated gene encoding
regulator of
transcriptional activities
[206]
PCL nanofibrous
woven scaffold ADSCs/HTs/HUVECs 4% strain, 0.5 Hz,
2 h/day
Increased tenomodulin, COL
I, tenascin C, scleraxis, and
vascular endothelial growth
factor (VEGF)
gene expression.
[169]

Nanomaterials 2023,13, 1847 26 of 35
Table 4. Cont.
Scaffold Cell Type Parameters Effects References
Knitted silk-collagen
sponge scaffold Human ESCs 10% strain, 1 Hz,
2 h/day
Upregulated expression of
tendon-related genes
scleraxis, COL I and COL III
[210]
P(LLA-CL)/Col scaffold Rabbit TDSCs 4% strain, 0.5 Hz,
2 h/day
Enhanced tendon-specific
gene and protein expression [146]
3D PCL scaffold Human BMSCs 10% strain, 0.33 Hz Increased COL I, COL III,
and scleraxis expression. [211]
Polyurethane disk scaffold Human fibroblasts 10% strain, 0.25 Hz,
8 h/day
Increased cell proliferation,
COL I, transforming growth
factor β1 (TGF-β1), and
connective tissue growth
factor (CTGF) expression.
[212]
Plain knitted silk scaffold Rabbit TDSCs 2% strain, 1 Hz
Promote tenogenic
differentiation from COL III
and decorin production
[136]
Collagen sponge scaffold Rabbit MSCs 2.4% strain, 1 Hz,
8 h/day
Increased COL I gene
expression and linear
stiffness of construct
[213]
4. Conclusions and Outlook
Regeneration of tendon and ligament tissues using the tissue engineering principle
remains a challenging issue in orthopedic research. Current treatment strategies are un-
able to fully restore the functions of injured tendons and ligaments to their native states,
and repair of injured tendons and ligaments continues to be a clinical problem. Using
seeded cells in scaffolds supplemented with scaffold-based biological and physical cues,
the tendon/ligament tissue engineering approach may provide a solution to this prob-
lem, by providing a milieu for cell proliferation as well as for phenotype maintenance of
tendon-derived cells or for tenogenic differentiation of stem cells. However, the tissue
engineered tendon or ligament should be endowed with improved mechanical properties
and functions over naturally healed tendons or ligaments. By combining biomimicry
and manufacturing flexibility, electrospinning is undoubtedly one of the most promising
methods to fabricate scaffolds for tendon or ligament tissue engineering. Although nano-
to macro-scale scaffolds are discussed in this paper without clear distinction, a novel elec-
trospun 3D scaffold mimicking the hierarchical structure of a tendon or ligament must be
developed by integrating the beneficial physico–chemical properties offered by natural
and synthetic polymers. The growth factors play a crucial role during regeneration of
tendons/tendons; therefore, growth factors related to the development of these tissues
must be introduced into the scaffold with controlled release characteristics to provide
biochemical cues during the regenerative process. Furthermore, a novel scaffold design
for scaffold-based gene therapeutics or delivery of exosome may be a promising approach
toward this end and is expected to produce positive outcomes in the future. Mechanical
stimulation can provide physical cues to cells loaded in a scaffold, which appears to be an
important route to generating functional tendon or ligament tissue in a bioreactor under
dynamic culture. However, the operation parameters during cyclic tensile loading, such as
strain, frequency, and duration time, must be investigated and optimized. With additional
refinement and the combination of these components, tissue engineering will undoubtedly
be a very convincing option for tendon and ligament repair. However, the type of cells
for seeding in a scaffold should be carefully chosen based on the types of tissue to be
regenerated, which is expected not only to enhance tissue healing but also to provide
acceptable mechanical performance at the tissue level. The development of a feasible cell
delivery system that can temporally and spatially reproduce the normal physiology of

Nanomaterials 2023,13, 1847 27 of 35
native tendons or ligaments may require further investigation; however, the inception of
new techniques followed by substantial research for optimization will undoubtedly direct
them toward clinical applications in due course.
Author Contributions:
Conceptualization, D.T.G., C.-H.C., K.T.S. and J.-P.C.; writing—original draft
preparation, D.T.G.; writing—review and editing, J.-P.C.; visualization, D.T.G. and K.T.S.; resources,
C.-H.C., H.-H.K. and J.-P.C.; supervision, C.-H.C. and J.-P.C.; funding acquisition, C.-H.C., H.-H.K.
and J.-P.C. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Ministry of Science and Technology, Taiwan, ROC
(MOST109-2314-B-182-013-MY3); and Chang Gung Memorial Hospital, Taiwan, ROC (CMRPD2K0131).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
We acknowledge the technical supports provided by the Microscopy Center and
Instrumentation Center, Chang Gung University, and the Microscope Core Laboratory, Chang Gung
Memorial Hospital, Linkou.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
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