Tendon tissue engineering using

scaffold enhancing strategies

Yang Liu
1
, H.S. Ramanath
2
and Dong-An Wang
1
1
Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore
637457, Republic of Singapore
2
Product and Process Development, Bioscaffold International Pte Ltd, Singapore 117525, Republic of Singapore
Tendon traumas or diseases are prevalent and debilitat-
ing lesions that affect the quality of life among popu-
lations worldwide. As a novel solution, tendon tissue
engineering aims to address these lesions by integrating
engineered, living substitutes with their native counter-
parts in vivo, thereby restoring the defective functions in
situ. For such a purpose, competent scaffolding materials
are essential. To date, three major categories of scaffold-
ing materials have been employed: polyesters, polysac-
charides, and collagen derivatives. Furthermore, with
these materials as a base, a variety of specialized meth-
odologies have been developed or adopted to enhance
neo-tendogenesis. These strategies include cellular
hybridization, interfacing improvement, and physical
stimulation.
Tendons are connective tissues that join muscles to bones.
By transmitting tensile forces and providing connective
exibility, they permit body locomotion and enhance joint
stability. The unique biomechanical properties of tendons
are mainly attributed to the high degree of organization of
the tendon extracellular matrix (ECM). Primarily consist-
ing of collagen type I, the ECM of tendons is arranged in a
hierarchy of bundles that have different dimensions and
which are aligned in a parallel manner in a proteoglycan
matrix, as showninFigure 1 [1]. The spindle-shaped tendon
broblasts (also known as tenocytes) are situated in longi-
tudinal rows and have numerous sheet-like cell extensions
reaching into the ECM. Injuries to tendons are quite com-
mon, resulting in more than 33 000 tendon repair proce-
dures annually inthe United States [2]. Tendoninjuries can
be acute or chronic. Acute injuries are primarily caused by
trauma, whereas chronic injuries are usually elicited by
repetitive mechanical loading below the failure threshold
and concurrent inammatory responses [35]. Tendons are
able to heal naturally, but their pre-injuryconditions are not
restored, owing to the development of scar tissues at the
wound site, the biomechanical properties of which are
inferior to uninjured tendon. The loss of mechanical compe-
tence is mainly due to a distorted ECM composition and a
misalignment of collagen brils in the scar tissue [1,6,7].
To improve the quality of repaired tendons, various
surgical repair techniques using sutures and soft tissue
anchors have been developed [810]. However, surgically
repaired tendons still possess inferior functionalities com-
pared with those of uninjured tendons.
Currently, some alternative therapies for tendon repair
exist, including biological grafts (e.g. autografts, allografts
and xenografts), permanent articial prostheses, and tis-
sue engineering. Biological grafts have several shortcom-
ings. They can induce donor site morbidity, they are only
available in limited amounts, and they can contribute to
disease transmission and tissue rejection. Permanent arti-
cial prostheses lack material durability and often lead to
mechanical failures later on. Tendon tissue engineering
(TTE) represents a more promising approach because,
through interdisciplinary engineering strategies, it aims
to promote full tendon regeneration per se, rather than
physically replacing tendons with partially functionalized
foreign substitutes. TTE typically involves a scaffold as a
temporary structure to support initial tissue growth. TTE
scaffolds can enhance tendogenesis by facilitating cell
proliferation, by promoting matrix production and by orga-
nizing the matrix into functional tendon tissues. Moreover,
tendogenesis can be further facilitated through approaches
such as cellular hybridization, surface modication,
growth factor cure, mechanical stimulation and contact
guidance. In this review, TTE scaffolding materials and
relevant enhancing strategies will be discussed.
Scaffolding materials for tendon tissue engineering
TTE aims to repair tendon lesions in situ by integrating
engineered, living substitutes with their native counter-
parts in vivo. For this purpose, competent scaffolding
materials are needed, and these ideally should fulll the
following requirements:
(i) Biodegradability with adjustable degradation rate.
(ii) Biocompatibility before, during and after degra-
dation.
(iii) Superior mechanical properties and maintenance of
mechanical strength during the tissue regeneration
process.
(iv) Biofunctionality: the ability to support cell prolifer-
ation and differentiation, ECM secretion, and tissue
formation.
(v) Processability: the ability to be processed to form
desired constructs of complicated structures and
shapes, such as woven or knitted scaffolds etc.
Historically, tendon-like mechanical properties werecon-
sidered the primary requirement for a TTE scaffolding
material. However, because TTE is aimed at the regener-
ation of a functional neo-tissue rather than at replacing
Review
Corresponding author: Wang, D.-A. (DAWang@ntu.edu.sg).
0167-7799/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2008.01.003 Available online 4 March 2008 201
damaged tissue with an articial prosthesis, a mechanical
property of the scaffold that is similar to that of tendons is
not strictly required. By contrast, a superior mechanical
strength remains essential to ensure that the structural
integrity of the scaffold during tendogenesis is maintained.
Moreover, the interfacial interactions between the scaffold-
ing material and the cells are crucial, and scaffolding
materials should therefore offer a bio-functionality that
stimulates regenerative responses of cells at the molecular
level. Ideally, the scaffold should not only promote cell
proliferationand differentiationbut also restore the natural
ECM composition and histological structure of tendon.
To date, three major categories of scaffolding materials
have been employed. These are polyesters, polysacchar-
ides, and collagen derivatives.
Polyesters
A vast majority of biodegradable polymers for TTE appli-
cations are polyesters, such as polyglycolic acid (PGA),
polylactic acid (PLA) and their copolymer polylactic-co-
glycolic acid (PLGA), as illustrated in Figure 2a-c. These
polymers are attractive, because their degradation pro-
ducts, glycolic acid and lactic acid, are natural metabolites
that are normally present in the human body. Moreover,
their good mechanical properties as well as their outstand-
ing processability further increase their appeal.
PLGA scaffolds have been reported to improve tendon
regeneration considerably. Ouyang et al. [11] found that
knitted PLGA scaffolds augmented the tendon healing,
both histologically and mechanically. These scaffolds facili-
tated production of collagen type I and type III brils and
therefore contributed to the improved mechanical proper-
ties. In another study, carried out by Cooper et al. [12],
PLGA was also selected as the scaffolding material.
PGAwas also reported as a scaffolding material feasible
for TTE applications. Cao et al. [13] developed a PGA
scaffold that could successfully restore the mechanical
capacity of tendons in a hen model. Moreover, Wei et al.
[14] found that woven PGA scaffolds were particularly
suitable for TTE because they surpassed the unwoven
PGA in mechanical performance and at the same time
degraded more slowly.
Furthermore, efforts have also been directed towards an
understanding of the similarities and disparities of differ-
ent polyesters. Although PGA, PLAand PLGAall belong to
the group of poly-a-hydroxyesters, the cellular responses to
these materials, as well as their individual degradation
proles, appear to be very different. Lu et al. [15] compared
scaffolds based on three different materials, PGA, poly-L-
lactic acid (PLLA) and PLGA. Although the PGA-based
scaffolds showed the highest initial strength, they sud-
denly lost mechanical strength owing to the bulk degra-
Figure 1. Schematic illustration of the hierarchical structure of tendon. The tendon has a multi-unit hierarchical structure composed of collagen molecules, fibrils, fibre
bundles, fascicles and tendon units that run parallel to the tendons long axis. This hierarchical structure contributes to the mechanical competence of the tendon [1].
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Trends in Biotechnology Vol.26 No.4
202
dation prole of PGA, and this resulted in a matrix dis-
ruption and a loss of integrity. Regarding cellular
responses, it was reported that, when using PLLA and
PLGA, the morphology of attached cells resembled that of
tendons and ligaments, whereas the best cell proliferation
was reported for surface-modied PLLA scaffolds. Despite
these advantages, there still remain several limitations of
polyesters (Box 1).
Collagen derivatives
In addition to polyesters, collagen derivatives have been
intensively investigated for use in TTEapplications. Given
that tendon ECMs are mainly composed of type I collagen,
scaffolds based on collagen derivatives are highly biocom-
patible. Moreover, collagen derivatives also exhibit
superior bio-functionality in that they better support cell
adhesion and cell proliferation than do other materials
such as polyesters.
Collagen gel has been reported to augment the quality of
tendon repair. Given that collagen gel does not possess
sufcient mechanical strength, it is often accompanied by a
high-strength component. For instance, Awad et al. [16]
studied collagen gels in combination with a polyglyconate
suture for patellar tendon repair. The biomechanical prop-
erties of the resulting tendon tissues were signicantly
better than those of naturally healed tendons, yet still
much inferior to those of uninjured tendons.
Compared with collagen gel, collagen sponges exhibit
greater mechanical competence. Given that collagen gels
exhibit superior cell-seeding efciency, a combination of
collagen gels with collagen bres or sponges represents a
promising strategy. Juncosa-Melvin et al. [17] showed that
gelcollagen sponge constructs could greatly enhance func-
tional tendogenesis. Another study, conducted by Gentle-
man et al., [18] provided further evidence that a
combination of collagen gels and sponges could bolster
development of tendon-like tissue. Despite this superior
bio-functionality and biocompatibility, collagen also suf-
fers from several limitations (Box 2).
Polysaccharides
Polysaccharides have been underutilized in biomedical
engineering and have only in recent decades attracted
signicant attention as possible biomaterials. Tradition-
ally, polysaccharides have been considered as scaffolding
materials for hard-tissue regeneration. For instance, poly-
saccharides such as chitin, chitosan, alginate and agarose
have been employed to fabricate scaffolds for cartilage and
Figure 2. Chemical structures of bio-macromolecules used in tissue engineering. (a) PLA, (b) PGA, (c) PLGA and (d) Chitosan.
Box 1. Limitations of polyesters as TTE scaffolding
materials
Despite their advantages, polyesters also suffer from several
limitations. First, owing to their hydrophobic nature, poly-a-hydro-
xyesters do not support a high level of cell adhesion [69,70], which
is the initial and crucial step to engineer functional tendons.
Fortunately, this limitation can be overcome by means of surface
modification with adhesive agents such as fibronectin [44]. Second,
although degradation products of PGA, PLA and PLGA are natural
metabolites, they are also acidic. The presence of these metabolites
in large concentrations can therefore give rise to significant
systematic or local reactions [71,72]. When the sizes of scaffolds
are smaller, the occurrence of such adverse biological reactions is
greatly reduced. Therefore, in general, polyesters are more apt for
repair of smaller defects, which need smaller scaffolds.
Box 2. Limitations of collagen as a TTE scaffolding material
Despite its superior bio-functionality and biocompatibility, there
remain several limitations to collagen. First, its processability is
limited. As a result, the degree to which collagen scaffolds can be
characterized is restricted. Second, the batch-to-batch variety in
collagen constructs makes a reliable reproduction of these scaffolds
difficult. Third, the mechanical strength of collagen scaffolds is
much lower than other materials such as the polyesters. Thus, there
remain concerns that collagen scaffolds cannot withstand mechan-
ical stress over time when implanted in the challenging mechanical
environment of the tendon [36]. Cross-linking collagen with agents
such as 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC)
enhances its mechanical strength, yet only in a moderate fashion
[18]. Finally, because collagen is a natural biopolymer, it is more
prone to induce antigenic and immunogenic reactions, although the
probability is considered to be low [73].
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203
bone engineering. [1921]. More recently, polysaccharides
have also been applied in the eld of soft tissue engineer-
ing, and chitosan in particular has been used to regenerate
tendons.
Chitosan, a deacetylation product of chitin, is a linear
polysaccharide composed of randomly distributed b-(14)-
linked D-glucosamine (the deacetylated unit) and N-acetyl-
D-glucosamine (the acetylated unit), as shown in Figure 2d.
In contrast to the hydrophobic polyesters PLA and PGA,
chitosan is hydrophilic and therefore exhibits better cell
adhesion and proliferation characteristics [19]. Moreover,
the N-acetylglucosamine moiety present in chitosan is a
structural feature that is also found in glycosaminoglycan,
which is involved in many specic interactions with growth
factors, receptors and adhesion proteins. Chitosan as a
glycosaminoglycan analogue might therefore also exhibit
similar bio-functionality. Furthermore, chitosan can create
highly porous structures that make it especially suitable for
a scaffolding material used in TTE [22].
The bio-functionality of chitosan, such as supporting of
cellular attachment and proliferation, and the ability to
induce cells to produce ECM has been demonstrated. In a
study conducted by Bagnaninchi et al. [23], porous chitosan
scaffolds with microchannels were designed to engineer
tendon tissues.
In addition, the hybridization of chitosan with other
polysaccharides as TTE scaffolding materials has also been
explored. An instant merit of such hybridization is the
combination of the desirable properties of both components.
Moreover, the cationic nature of chitosan facilitates its
hybridizationwithnegatively chargedpolysaccharides such
as alginate and hyaluronan [22].
Hyaluronan (HA) is a uniformly repetitive linear GAG
composed of disaccharides of glucuronic acid and N-acetyl-
glucosamine: [-b(1,4)- GlcUA- b(1,3)-GlcNAc-]
n
[24]. It is an
essential component of ECM. Anionic hyaluronan interacts
with other macromolecules, such as link proteins and pro-
teoglycans, to facilitate tissue morphogenesis, cell
migration, differentiation and adhesion [24], whereas
cationic chitosan can elicit electrostatic interactions with
anionic glycosaminoglycans and other negatively charged
species [22]. Hybridization of hyaluronan and chitosan is
expected to augment the mechanical properties and bioac-
tivities of TTEscaffolds. Funakoshi et al. [25] demonstrated
that scaffolds composed of hybridized chitosanhyaluronan
exhibited enhanced mechanical competence. Moreover, an
improved adhesion to patellar tendon broblasts was also
observed. In another study, Funakoshi et al. [26] reported
that the chitosanhyaluronan scaffold improved the biome-
chanical properties of the regenerated tendon tissue in the
rotator cuff and bolstered production of collagen type I.
Alginate, another type of polysaccharide that can be
used for hybridization with chitosan, is an anionic poly-
saccharide composed of homopolymeric regions of guluro-
nic acid and mannuronic acid interspersed with mixed
sequences (M-G blocks). Because it contains D-glucuronic
acid as the main sugar residue in the repeat unit, alginate
is often considered to have similar biological activity to
glycosaminoglycans. However, owing to its anionic nature,
cell adhesion to alginate is often unsatisfactory [2729].
Adding cationic chitosan to alginate would therefore aug-
ment the bio-functionality of the scaffold because the ionic
interactions between alginate and chitosan are expected to
facilitate retaining and recruiting of cells and growth
factors, as well as cytokines [30,31]. Majima et al. [32]
reported that an alginatechitosan hybrid scaffold showed
signicantly enhanced cell adhesion to tenocytes. More-
over, in a similar fashion to composition of tendon ECM,
the predominant ECM component deposited on these scaf-
folds was collagen type I.
It is well known that saccharides play crucial roles in
cell signalling and immune recognition, but their detailed
mechanisms are far from being well understood. Thus, it is
anticipated that, as the biochemical signalling is further
elucidated, polysaccharides as scaffolding materials could
achieve great triumphs in the future.
Scaffold enhancing strategies
Tissue engineering scaffolds can promote and support
tendon regeneration and enhance repair tissue quality.
Yet, in most cases, mere application of scaffolds is not
sufcient, and specialized scaffold enhancing strategies
are employed, such as cellular hybridization, surface modi-
cation, growth factor cure, mechanical stimulation and
contact guidance. Table 1 presents the experimental
details of selected promising TTE studies, including their
scaffolding materials and relevant enhancing strategies.
Cellular hybridization
The concept of cellular hybridization comprises the intro-
duction of therapeutic cells into the scaffolds to encourage
repair of damaged tissues. Pre-seeding of cells on scaffolds
has been shown to improve the biochemical composition,
histological structure, and biomechanical properties of
repaired tissues [2,11,13]. To date, several types of cells
have been employed for pre-seeding in TTE, such as
mesenchymal stem cell (MSCs), tenocytes, and dermal
broblasts [16,25,33].
MSCs are multipotent stem cells that can replicate as
undifferentiated cells and can also differentiate to form
lineages of mesenchymal tissues, such as bone, cartilage,
fat, tendon, muscle, and marrow stroma [34]. These cells
have the unique feature of self-renewal, which is the
ability to proliferate while avoiding apoptosis and differ-
entiation [35]. Moreover, MSCs can be easily obtained from
sources such as bone marrow. Thus, it might be possible to
harvest human autologous MSCs for future clinical appli-
cations [34]. The feasibility of MSCs for TTE has been
demonstrated. For instance, Juncosa-Melvin et al. [17,36]
reported that seeding MSCs into collagen gels tremen-
dously augmented the mechanical properties and histology
of regenerated tissue (Figure 3). Ouyang et al. [11] also
demonstrated that introduction of MSCs enormously
enhanced the biomechanical competence of tissue repair.
Tenocytes are another choice as cellular components for
TTE constructs, becuase they are the primary cell-type
residing in tendons. Cao et al. [13] demonstrated that the
introduction of tenocytes into PGA scaffolds signicantly
augmented the mechanical competence of engineered ten-
dons. However, one major obstacle for the use of tenocytes
remains: the harvest of autologous tenocytes can cause
major donor-site morbidity.
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Trends in Biotechnology Vol.26 No.4
204
Therefore, there is a need for cells from more obtainable
sources. Dermal broblasts are attractive candidates
because they are easily accessible and their harvest nor-
mally does not create signicant adverse effects. A few
studies have investigated their potential for TTE. For
instance, Liu et al. [33] reported that an introduction of
dermal broblasts into PGAscaffolds could achieve similar
efcacy as a seeding of tenocytes. Yet the use of dermal
broblasts as seeding cells in TTE requires further inves-
tigation to evaluate and verify their effectiveness.
Surface modication
The adhesion of cells on the scaffolds is the initial and
crucial step of TTE. A successful tendogenesis requires a
large number of cells to adhere to the scaffold, to proliferate
and to nally organize the matrix into a functional tendon.
Cell adhesion to the scaffolds varies and is mediated by cell
surface receptors present, such as integrins [37,38], which
bind to short peptides sequences, for example to ArgGly
Asp (RGD). A direct grafting of RGD sequences onto scaf-
folds can therefore enhance cell adhesion. For example,
Chen et al. [39] showed that RGD modication of silk
scaffold increased the cell density by 250%, and the pro-
duction of collagen type I, as an indicator of tendon ECM
formation, was increased by 410%.
Alternatively, a coating of adhesion proteins (e.g. bro-
nectin, vitronectin and laminin) that contain RGD tripep-
tides can also facilitate the attachment of cells to the
scaffold surface. Among these adhesion proteins, bronec-
tin has been most widely used. Fibronectin is a high-
Table 1. Experimental details of selected TTE studies
Scaffolding material Scaffold enhancing strategy Model
system
Mechanical parameters of
resulting tendons (100%
equals normal tendons)
Other major ndings Refs
PLGA Cellular hybridization: MSCs In vivo:
rabbit
model
Stiffness: 87% Engineered tendons composed of
collagen types I and type III
[11]
Modulus: 62.6%
PGA Cellular hybridization:
tenocytes
In vivo:
hen
model
Tensile strength: 83% Longitudinal alignment of tenocytes
and collagen bres
[13]
Collagen Cellular hybridization: MSCs In vivo:
rabbit
model
Maximum force: 1725% (but
1.7 times greater than in
natural repairs)
No signicant differences in cellular
organization or histological
appearance between engineered
tendons and naturally healed tendons
[16]
Stiffness:1019% (but 1.8
times greater than in natural
repairs)
Collagen Cellular hybridization: MSCs In vivo:
rabbit
model
Maximum force: 50% Engineered tendons of high initial cell-
seeding density damaged by excessive
cell traction forces
[36]
Stiffness: 64%,
Maximum stress: 85%
Modulus: 93%
Chitosan Cellular hybridization:
tenocytes;
Contact guidance:
microchannels (diameter:
250 mm)
In vitro Growth and alignment of tenocytes,
along the channels, as well as ECM
production
[23]
Chitosan/hyaluronan
hybrid
Cellular hybridization:
Tenocytes
In vitro Enhanced tensile strength of hybrid
scaffolds. Signicantly improved cell
adhesion and collagen type I secretion
were also observed.
[25]
Chitosan/alginate
hybrid
Cellular hybridization:
Tenocytes
In vitro Signicantly enhances cell adhesion
capacity of hybrid scaffolds;
predominant ECM component
deposited: collagen type I
[32]
PGA Cellular hybridization: dermal
broblast and tenocytes
In vivo:
porcine
model
Tensile strength of broblast
engineered tendons: 74%;
tensile strength of tenocyte
engineered tendons: 76%
Parallel collagen bre alignment in
broblast and tenocyte engineered
tendons
[33]
PLGA Cellular hybridization: MSCs;
surface modication: electro-
spun nanobres
In vitro Nanobre coating enhanced cellular
adhesion, 6.5-fold at day 2
[47]
PLGA Cellular hybridization: dermal
broblasts; mechanical
stimulation: uniaxial strain at
1 Hz and 0.1 Hz
In vitro Cyclic strain led to increased mean
nuclei length and orientation of the
cells parallel to the straining axis.
Alignment was greater at the lower
frequency
[61]
Collagen Cellular hybridization: MSCs;
mechanical stimulation: cyclic
strain at 0.0033 Hz for 8 h/day
In vivo:
rabbit
model
Maximum force: 70% Cellular alignment of stimulated and
non-stimulated tendon repairs was
similar to that of normal tendons
[64]
Stiffness: 85%
Maximum stress: 70%
Modulus: 50%
All parameter were
signicantly greater in the
cyclic strained samples than in
untreated controls
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205
molecular-weight glycoprotein that binds to integrins and
also to ECM components, such as collagen, brin and
heparan sulfate [40]. The presence of bronectin might
therefore have a positive effect on cellular responses and
tissue regeneration, and indeed it was reported that bro-
nectin is upregulated during tendon formation and wound
healing [41,42]. Tsuchiya et al. investigated the efcacy of
surface coating of bronectin in promoting cellular
adhesion [43]. The authors showed that coating of 96-well
plate surfaces with bronectin tremendously enhanced
cellular attachment, and nearly all MSCs that were seeded
also adhered to the bronectin-coated surface within
30 minutes, whereas surfaces modied with other protein
such as type I collagen, type II collagen, vitronectin and
poly-L-lysine could only immobilise 40%of the cells [43]. In
addition, Lu et al. [15] showed that surface modication
with bronectin greatly bolsters cellular attachment to
PLGA and PLLA scaffolds. Furthermore, Qin et al. [44]
used a coating of bronectin to effectively increase
adhesion strength of human embryonic tenocytes.
More recently, with the further development of nano-
technology, coating scaffolds with electro-spun nanobres
has become a novel alternative for enhancing cell adhesion
[45]. A surface modication with nanobres can resemble
ECM structures and can result in a high surface area to
volume ratio. In addition, a high degree of porosity and a
wide range of pore size distribution can be achieved. For
instance, Min et al. [46] used electro-spun silk broin
nanobres to promote cell adhesion and production of type
I collagen of human broblasts. Moreover, Sahoo et al. [47]
showed that a coating of electro-spun nanobres signi-
cantly enhanced cellular attachment, proliferation and
matrix production on a knitted PLGA scaffold (Figure 4).
These studies indicate that a coating with nanobres can
be an attractive choice for surface modication in TTE
applications.
Growth factor cure
Growth factors are a group of naturally occurring proteins
that are important for regulating a variety of cellular
responses. Involved in almost every stage of the healing
process, they stimulate cellular proliferation, differen-
tiation, and matrix deposition as well as tissue ingrowths.
Although their exact mechanisms and pathways are far
frombeing completely understood, it is evident that growth
factors play crucial roles in successful tendogenesis. The
incorporation of growth factors into TTE scaffolds is there-
fore a promising strategy to promote tendon regeneration.
For instance, Sahoo et al. [48] fabricated a scaffold that
releases basic broblast growth factor (bFGF) to facilitate
TTE. Also, Costa et al. [49] reported that the delivery of
platelet-derived growth factor-BB(PDGF-BB), insulin-like
growth factor-1 (IGF-1), or bFGF, could enhance tenocyte
proliferation in TTE in vitro. Furthermore, this study also
Figure 3. Histological illustration of tendon tissues obtained with different repair methods. (a) Neo-tissues from MSC-seeded collagen constructs result in a parallel cellular
alignment along the tendon axis. The number of cells in the neo-tissues is moderately increased in comparison with control tendon (as shown in (c). (b) Neo-tissues
obtained from acellular collagen constructs show a more random cellular alignment. (c) Natural tendon midsubstance shows highly parallel cellular alignment [17].
Figure 4. The effect of nanofibre coating on cell population. (a) Nanofibre coating of PLGA scaffold results in a dense cell population, as observed by confocal microscopy of
live cells. (b) Uncoated scaffolds yield a significantly lower cell density [47].
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Trends in Biotechnology Vol.26 No.4
206
revealed a synergistic effect when multiple growth factors
were supplied, and the highest cell proliferation was
observed with a combination of IGF-1, PDGF-BB and
bFGF.
In addition, there are other growth factors that have
been reported to enhance tendon healing in orthopaedic
research. Although these growth factors have not been
employed in TTE yet, potentially they could be applied
in the future. Possible candidates include the transforming
growth factors b (TGF-b) -1, -2 and -3 [5052], the carti-
lage-derived morphogenetic proteins (CDMP)-1, -2 and -3
[5355] and the vascular endothelial growth factor (VEGF)
[5658].
Mechanical stimulation
Prior to implantation, TTE constructs often undergo a
certain period of in vitro cultivation, which is traditionally
carried out under mechanically static culture conditions.
However, in their natural cellular environment, tenocytes
are continuously subjected to various mechanical loads
(mainly tension) exerted by muscular contraction, body
movement or other external forces. Externally applied
cyclic strain under in vitro conditions has enormous effects
on various functions of tenocytes, such as their metab-
olism, proliferation, orientation and matrix deposition
[59,60]. Moreover, simulation of the biomechanical
environment in the body also helps to establish in vitro
TTE models, which serve as the foundation of in vivo
studies. In recent years, mechanical stimulation, especi-
ally cyclic strain, has been applied in the engineering of
tendon tissues.
It is known that cyclic strain can affect cell morphology
and induce uniaxial cellular alignment. Moe et al. [61]
observed that cyclic strain stimulation enhanced the cel-
lular alignment and changed the cellular shape (Figure 5).
Also, Qin et al. [62] found that cyclic strain promotes cell
proliferation, matrix deposition and increased collagen
production. In another study, Juncosa-Melvin et al. [63]
showed that the application of cyclic strain elevated the
gene expression levels of collagen type I. Finally, cyclic
strain can enhance mechanical competence of the regen-
erated tendons [64]. The authors of this study found that
values for maximum force, linear stiffness, maximum
stress, and linear modulus for repaired tendons were close
to those of natural patellar tendon. In terms of restoration
of key biomechanical parameters, these constructs appear
to be the best engineered tendons obtained so far.
Contact guidance
A major hurdle for successful TTE is to restore the highly
organized structure of ECM, which contributes to the
unique biomechanical properties of the tendons. Successful
mimicking of the ECM structure requires both axial align-
ment of cells and parallel arrangement of collagen brils,
which is a challenging task.
It is well known that broblasts can align and deposit
ECM axially in response to topographical cues provided by
scaffold surfaces, such as microgrooves or microchannels
[6567]. This phenomenon is commonly referred to as
contact guidance, and it provides a means to facilitate
tissue growth within a highly organized ECM structure
such as that present in tendons.
Recently, several pioneering studies applied contact
guidance in TTE applications with limited success. For
example, Lu et al. [68] created scaffolds with capillary
channel bres that contained eight open grooves. Fibro-
blasts were found to proliferate within these grooves and
oriented themselves in the direction of the grooves. ECM
proteins such as collagen and laminin were deposited
within the grooves parallel to the groove direction [68].
Furthermore, Bagnaninchi et al. [23] fabricated a porous
chitosan scaffold with microchannels for TTE.
Incorporating contact guidance into the design of TTE
scaffolds might enable the creation of an engineered ten-
don tissue that would exhibit a high level of ECM organ-
ization. However, only preliminary success has been made,
and the reconstruction of functional tendons by contact
guidance still has a long way to go. To date, there remain
several limitations to this strategy. First, a morphological
resemblance of the engineered tendons to that of natural
tendons does not necessarily imply functional competence.
Thus mechanical assessments of these engineered tendon
tissues are required to verify their biomechanical capabili-
ties. Second, although axial alignment of cells and ECM
brils occurs within the grooves or channels, the quality of
cell proliferation and resulting ECM production need to be
addressed systematically. The observation that the micro-
grooves or microchannels are only partially lled insinu-
ates that the quality of regenerated tissues might be rather
poor in terms of their biomechanical properties. Third,
Figure 5. Effect of cyclic strain on engineered tendon tissues, as observed after hematoxylin and eosin (H&E) staining: (a) unstrained samples. (b) Cells underwent cyclic
strains at a frequency of 0.1 Hz. (c) Cells underwent cyclic strains at a frequency of 1 Hz. With the application of cyclic strain, the shape of the cells changed from polygon
shapes to spindle-like shapes. Furthermore, cells showed a clear tendency to align in the direction of the straining axis. More cells were aligned in 0.1 Hz frequency straining
(b) than in 1 Hz frequency staining (c). The resulting tissues shown in (b) were therefore more similar to natural tendons [61].
Review Trends in Biotechnology Vol.26 No.4
207
despite the claimed capability of oxygen and nutrient
transport within these microgrooved scaffolds, the oxygen
and nutrient transport within such scaffolds still remains a
challenge, especially in larger scaffold sizes [68].
Conclusion
As a novel solution to address tendon lesions, TTEoffers in
situ restoration of defective functions by integrating in
vitro engineered, living substitutes with their native
counterparts in vivo. To achieve this, three major
categories of scaffolding materials have been employed:
polyesters, polysaccharides, and collagen derivatives. To
further enhance neo-tendogenesis, various scaffold enhan-
cing strategies have been employed, such as cellular
hybridization, surface modication, growth factor cure,
mechanical stimulation and contact guidance. Among
these strategies, cellular hybridization plays an essential
role in engineering functional tendon tissues, whereas the
success of contact guidance in TTE is still limited. Surface
modication, growth factor cure and mechanical stimu-
lation have proved to have distinct merits when applyed
individually, and the synergistic effects resulting from
combinations of them could be explored in future works.
Despite the encouraging results, several challenges still
remain for TTE: rst, there is currently no scaffolding
material that simultaneously offers superior biocompatibil-
ity, bio-functionality, mechanical property and processabil-
ity; second, the hybridization of therapeutic cells and TTE
scaffolds is not yet satisfactory, often resulting in a low rate
of cellular adhesion, uneven ECM deposition and inferior
tissuequality; third, there is asignicant gapbetweenthe in
vitrostageandtheinvivostageof TTE; last but not least, our
current knowledgeabout theeffectsof regulatoryfactors (i.e.
growth factors, mechanical signals) in the tendon regener-
ation process is still limited; it is based mainly on empirical
observations rather than on a thorough understanding of
the underlying mechanisms and pathways. Pinpoint exploi-
tation of these factors for TTE is therefore hindered.
Looking towards the future, breakthroughs in the fol-
lowing areas are expected, and these could possibly over-
come the above mentioned challenges: development of
advanced scaffolding materials that display ideal charac-
teristics for TTE; progress in the exploitation of stem cells
for TTE, which would enable functional tendon regener-
ation from autologous cell sources; development of nano-
technology that further improves the architecture, surface
properties and cellular hybridization of TTE scaffolds;
synergism of multiple strategies that further enhances
the quality of engineered tendon tissues; using the results
of in vitro studies to aid the active and intensive investi-
gation of TTE at the in vivo stage; and nally, advances of
biology and physiology that reveal the underlying prin-
ciples of tendon tissue regeneration. With progress in these
areas, TTE can become a viable clinical option, and it is
anticipated that, by then, TTE will contribute to the expe-
ditious and full reconstruction of functional tissue that is
as competent as a natural tendon.
Acknowledgement
Grant ARC 10/16, Ministry of Education, Singapore and Startup Grant,
College of Engineering, Nanyang Technological University, Singapore.
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