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Tendon tissue engineering usingscaffold enhancing strategiesYang Liu1, H.S. Ramanath2 and Dong-An Wang1

1 Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore

637457, Republic of Singapore2 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 tissueengineering aims to address these lesions by integratingengineered, 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 cellularhybridization, interfacing improvement, and physicalstimulation.

Tendons are connective tissues that join muscles to bones.

By transmitting tensile forces and providing connective

flexibility, 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 shown in Figure 1 [1]. The spindle-shaped tendon

fibroblasts (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 annuallyin the 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 inflammatory responses [3–5]. Tendons areable to healnaturally, but theirpre-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 fibrils 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 [8–10]. 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 artificial 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-

ficial prostheses lack material durability and often lead tomechanical 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 modification,

growth factor cure, mechanical stimulation and contact

guidance. In this review, TTE scaffolding materials andrelevant 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 fulfill 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 were con-

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. ([email protected] ).

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

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damaged tissue with an artificial 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

proliferation and differentiation but 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 fibrils 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.

PGA was 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 unwovenPGA 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, PLA and PLGA all belong to

the group of poly-a-hydroxyesters, the cellular responses to

these materials, as well as their individual degradation

profiles, 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 tendon’s long axis. This hierarchical structure contributes to the mechanical competence of the tendon [1].

Review Trends in Biotechnology Vol.26 No.4

202



dation profile 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-modified 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 TTE applications. 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

sufficient 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 significantly 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 efficiency, a combination of

collagen gels with collagen fibres or sponges represents a

promising strategy. Juncosa-Melvin et al. [17] showed that

gel–collagen 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

significant 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, thereremain 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].


203



bone engineering. [19–21]. More recently, polysaccharides

have also been applied in the field 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-(1–4)-

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 specific 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 astudy 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

hybridization with negatively charged polysaccharides 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].Itisan

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 TTE scaffolds. Funakoshi et al. [25] demonstrated

that scaffolds composed of hybridized chitosan–hyaluronan

exhibited enhanced mechanical competence. Moreover, an

improved adhesion to patellar tendon fibroblasts was also

observed. In another study, Funakoshi et al. [26] reported

that the chitosan–hyaluronan 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 [27–29].

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 alginate–chitosan hybrid scaffold showed

significantly 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

sufficient, and specialized scaffold enhancing strategies

are employed, such as cellular hybridization, surface modi-fication, 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

fibroblasts [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 significantly

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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Therefore, there is a need for cells from more obtainable

sources. Dermal fibroblasts are attractive candidates

because they are easily accessible and their harvest nor-mally does not create significant adverse effects. A few

studies have investigated their potential for TTE. For

instance, Liu et al. [33] reported that an introduction of

dermal fibroblasts into PGA scaffolds could achieve similar

efficacy as a seeding of tenocytes. Yet the use of dermal

fibroblasts as seeding cells in TTE requires further inves-

tigation to evaluate and verify their effectiveness.

Surface modification

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 finally 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], whichbind to short peptides sequences, for example to Arg–Gly–

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 modification 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. fibro-

nectin, vitronectin and laminin) that contain RGD tripep-

tides can also facilitate the attachment of cells to the

scaffold surface. Among these adhesion proteins, fibronec-

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 findings Refs

PLGA Cellular hybridization: MSCs In vivo :

rabbit

model

Stif fness: 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 fibres

[13]

Collagen Cellular hybridization: MSCs In vivo :

rabbit

model

Maximum force: 17–25% (but

1.7 times greater than in

natural repairs)

No significant differences in cellular

organization or histological

appearance between engineered

tendons and naturally healed tendons

[16]

Stiffness:10–19% (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. Significantly improved cell

adhesion and collagen type I secretion

were also observed.

[25]

Chitosan/alginate

hybrid

Cellular hybridization:

Tenocytes

In vitro – Significantly enhances cell adhesion

capacity of hybrid scaffolds;

predominant ECM component

deposited: collagen type I

[32]

PGA Cellular hybridization: dermal

fibroblast and tenocytes

In vivo :

porcine

model

Tensile strength of fibroblast

engineered tendons: 74%;

tensile strength of tenocyte

engineered tendons: 76%

Parallel collagen fibre alignment in

fibroblast – and tenocyte – engineered

tendons

[33]

PLGA Cellular hybridization: MSCs;

surface modification: electro-

spun nanofibres

In vitro – Nanofibre coating enhanced cellular

adhesion, 6.5-fold at day 2

[47]

PLGA Cellular hybridization: dermal

fibroblasts; mechanical

stimulation: uniaxial strain at1 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

significantly greater in the

cyclic strained samples than in

untreated controls


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molecular-weight glycoprotein that binds to integrins and

also to ECM components, such as collagen, fibrin and

heparan sulfate [40]. The presence of fibronectin might

therefore have a positive effect on cellular responses and

tissue regeneration, and indeed it was reported that fibro-

nectin is upregulated during tendon formation and woundhealing [41,42]. Tsuchiya et al. investigated the efficacy of

surface coating of fibronectin in promoting cellular

adhesion [43]. The authors showed that coating of 96-well

plate surfaces with fibronectin tremendously enhanced

cellular attachment, and nearly all MSCs that were seeded

also adhered to the fibronectin-coated surface within

30 minutes, whereas surfaces modified 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 modification

with fibronectin greatly bolsters cellular attachment to

PLGA and PLLA scaffolds. Furthermore, Qin et al. [44]

used a coating of fibronectin to effectively increase

adhesion strength of human embryonic tenocytes.

More recently, with the further development of nano-

technology, coating scaffolds with electro-spun nanofibres

has become a novel alternative for enhancing cell adhesion

[45]. A surface modification with nanofibres 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 fibroin

nanofibres to promote cell adhesion and production of type

I collagen of human fibroblasts. Moreover, Sahoo et al. [47]

showed that a coating of electro-spun nanofibres signifi-

cantly enhanced cellular attachment, proliferation and

matrix production on a knitted PLGA scaffold (Figure 4).These studies indicate that a coating with nanofibres can

be an attractive choice for surface modification 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

from being 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 fibroblast 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].


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 [50–52], the carti-

lage-derived morphogenetic proteins (CDMP)-1, -2 and -3

[53–55] and the vascular endothelial growth factor (VEGF)

[56–58].

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 appliedcyclic 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 fibrils,which is a challenging task.

It is well known that fibroblasts can align and deposit

ECM axially in response to topographical cues provided by

scaffold surfaces, such as microgrooves or microchannels

[65–67]. 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 fibres that contained eight open grooves. Fibro-

blasts were found to proliferate within these grooves andoriented 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

fibrils 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 filled 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].


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, TTE offers in

situ restoration of defective functions by integrating in

vitro engineered, living substitutes with their nativecounterparts 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 modification, 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

modification, growth factor cure and mechanical stimu-

lation have proved to have distinct merits when applyed

individually, and the synergistic effects resulting fromcombinations of them could be explored in future works.

Despite the encouraging results, several challenges still

remain for TTE: first, 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

tissue quality;third, there is a significant gap between the in

vitro stage and theinvivo stage ofTTE;lastbut notleast,our

current knowledge about the effectsof regulatory factors(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 resultsof in vitro studies to aid the active and intensive investi-

gation of TTE at the in vivo stage; and finally, 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.

AcknowledgementGrant ARC 10/16, Ministry of Education, Singapore and Startup Grant,

College of Engineering, Nanyang Technological University, Singapore.

References

1 Wang, J.H.C. (2006) Mechanobiology of tendon. J. Biomech. 39, 1563–

1582

2 Langer, R. andVacanti, J. (1993)Tissue engineering. Science 260,920–

926

3 Stratz, T. et al. (2002) Local treatment of tendinopathies: a comparison

between tropisetron and depot corticosteroids combined with local

anesthetics. Scand. J. Rheumatol. 31, 366–369

4 Tang, J.B. (2006) Tendon injuries across the world: Treatment. Injury

37, 1036–1042

5 Hess, G.P. et al. (1989) Prevention and treatment of overuse tendon

injuries. Sports Med. 8, 371–384

6 Sharma, P. and Maffulli, N. (2005) Tendon injury and tendinopathy:

healing and repair. J. Bone Joint Surg. Am. 87, 187–202

7 Reddy, G.K. et al. (1999) Matrix remodeling in healing rabbit Achilles

tendon. Wound Repair Regen. 7, 518–527

8 Dona, E. et al. (2003) Biomechanical properties of four circumferential

flexor tendon suture techniques. J. Hand Surg. [Am.] 28, 824–831

9 Zobitz, M.E. et al. (2001) Tensile propertiesof suturemethods for repair

of partially lacerated human flexortendon in vitro. J. Hand Surg. [Am.]

26, 821–827

10 Gordon, L. et al. (1998) Flexor tendon repair using a stainless steel

internal anchor — Biomechanical study on human cadaver tendons.

J. Hand Surg. [Br.] 23, 37–40

11 Ouyang, H.W. et al. (2003) Knitted poly-lactide-co-glycolide scaffold

loaded with bone marrow stromal cells in repair and regeneration of

rabbit Achilles tendon. Tissue Eng. 9, 431–439

12 Cooper, J.A. et al. (2005) Fiber-based tiss ue-engineered scaffold for

ligament replacement: design considerations and in vitro evaluation.

Biomaterials 26, 1523–1532

13 Cao, Y. et al. (2002) Bridging tendon defects using autologous tenocyte

engineered tendon in a hen model. Plast. Reconstr. Surg. 110, 1280–

1289

14 Wei, X. et al. (2005) Use of polyglycolic acid unwoven and woven fibers

for tendon engineering in vitro. Key Eng. Mater. 288–289 (Advanced

Biomaterials VI), 7–10

15 Lu, H.H. et al. (2005) Anterior cruciate ligament regeneration using

braided biodegradable scaffolds: in vitro optimization studies.


16 Awad, H.A. et al. (2003) Repair of patellar tendon injuries using a cell-

collagen composite. J. Orthop. Res. 21, 420–431

17 Juncosa-Melvin, N. et al. (2006) The effect of autologous mesenchymal

stem cells on the biomechanics and histology of gel-collagen spongeconstructs used for rabbit patellar tendon repair. Tissue Eng. 12, 369–

379

18 Gentleman, E. et al. (2006) Development of ligament-like structural

organization and properties in cell-seeded collagen scaffolds in vitro.

Ann. Biomed. Eng. 34, 726–736

19 Suh, J.K.F. and Matthew, H.W.T. (2000) Application of chitosan-based

polysaccharide biomaterials in cartilage tissue engineering: a review.


20 Wang, L. et al. (2003) Evaluation of sodium alginate for bone marrow

cell tissue engineering. Biomaterials 24, 3475–3481

21 Li, Z. et al. (2005) Chitosan-alginate hybrid scaffolds for bone tissue

engineering. Biomaterials 26, 3919–3928

22 Kumar, M.N.V.R. et al. (2004) Chitosan chemistry and pharmaceutical

perspectives. Chem. Rev. 104, 6017–6084

23 Bagnaninchi, P.O. et al. (2007) Chitosan microchannel scaffolds for

tendon tissue engineering characterized using optical coherencetomography. Tissue Eng. 13, 323–331

24 Toole, B.P. (2001) Hyaluronan in morphogenesis. Semin. Cell Dev. Biol.

12, 79–87

25 Funakoshi, T. et al. (2005) Novel chitosan-based hyaluronan hybrid

polymer fibers as a scaffold in ligament tissue engineering. J. Biomed.

Mater. Res. A 74A, 338–346

26 Funakoshi, T. et al. (2005) Application of tissueengineering techniques

for rotator cuff regeneration using a chitosan-based hyaluronan hybrid

fiber scaffold. Am. J. Sports Med. 33, 1193–1201

27 Kuo, C.K. and Ma, P.X. (2001) Ionically crosslinked alginate hydrogels

as scaffolds for tissue engineering: Part 1. Structure, gelation rate and

mechanical properties. Biomaterials 22, 511–521

28 Rowley, J.A. et al. (1999) Alginate hydrogels as synthetic extracellular

matrix materials. Biomaterials 20, 45–53


208



29 Genes, N.G. et al. (2004) Effect of substrate mechanics on chondrocyte

adhesion to modified alginate surfaces. Arch. Biochem. Biophys. 422,

161–167

30 Madihally, S.V. and Matthew, H.W.T. (1999) Porous chitosan scaffolds

for tissue engineering. Biomaterials 20, 1133–1142

31 Hsu, S.H. et al. (2004) Chitosan as scaffold materials: Effects of

molecular weight and degree of deacetylation. J. Polym. Res. 11,

141–147

32 Majima, T. et al. (2005) Alginate and chitosan polyion complex hybrid

fibers for scaffolds in ligament and tendon tissue engineering. J.

Orthop. Sci. 10, 302–30733 Liu, W. et al. (2006) Repair of tendon defect with dermal fibroblast

engineered tendon in a porcine model. Tissue Eng. 12, 775–788

34 Pittenger, M.F. et al. (1999) Multilineage potential of adult human

mesenchymal stem cells. Science 284, 143–147

35 Satija, N.K. et al. (2007) Mesenchymal stem cells: molecular targets for

tissue engineering. Stem Cells Dev. 16, 7–23

36 Juncosa-Melvin, N. et al. (2006) Effects of cell-to-collagen ratio in stem

cell-seeded constructs for Achilles tendon repair. Tissue Eng. 12, 681–

689

37 Hynes,R.O. (1992)Integrins– versatility, modulation, andsignalingin

cell-adhesion. Cell 69, 11–25

38 Bokel,C. andBrown, N.H. (2002)Integrinsin development: moving on,

responding to, and sticking to the extracellular matrix. Dev. Cell 3,

311–321

39 Chen, J.S. et al. (2003) Human bone marrow stromal cell and ligament

fibroblast responses on RGD-modified silk fibers. J. Biomed. Mater. Res. A 67, 559–570

40 Lyon, M. et al. (2000) Elucidation of the structural features of heparan

sulfate important for interaction with the Hep-2 domain of fibronectin.

J. Biol. Chem. 275, 4599–4606

41 Repesh, L.A. et al. (1982) Fibronectin involvement in granulation-

tissue and wound-healing in rabbits. J. Histochem. Cytochem. 30,

351–358

42 Lehto, M. et al. (1990) Fibronectin in the ruptured human achilles-

tendon and its paratenon - an immunoperoxidase study. Ann. Chir.

Gynaecol. 79, 72–77

43 Tsuchiya, K. et al. (2001) Effects of cell adhesion molecules on adhesion

of chondrocytes, ligament cells and mesenchymal stem cells. Mater.

Sci. Eng. C 17, 79–82

44 Qin, T.W. et al. (2005) Adhesion strength of human tenocytes to

extracellular matrix component-modified poly(DL-lactide-co-

glycolide) substrates. Biomaterials 26, 6635–664245 Li, W.J. et al. (2002) Electrospun nanofibrous structure: A novel

scaffold for tissue engineering. J. Biomed. Mater. Res. 60, 613–621

46 Min, B.M. et al. (2004) Electrospinning of silk fibroin nanofibers and its

effect on the adhesion and spreading of normal human keratinocytes

and fibroblasts in vitro. Biomaterials 25, 1289–1297

47 Sahoo, S. et al. (2006) Characterization of a novel polymeric scaffold for

potential application in tendon/ligament tissue engineering. Tissue

Eng. 12, 91–99

48 Sahoo, S. et al. (2006) FGF-2 releasing nanofibrous scaffolds for tendon

tissue engineering (poster presentation). Tissue Eng. 12, 1053

49 Costa, M.A. et al. (2006) Tissue engineering of flexor tendons:

Optimization of tenocyte proliferation using growth factor

supplementation. Tissue Eng. 12, 1937–1943

50 Klein, M.B. et al. (2002) Flexor tendon healing in vitro: Effects of

TGF-beta on tendon cell collagen production. J. Hand Surg. [Am.]

27, 615–62051 Kashiwagi, K. et al. (2004) Effects of transforming growth factor-beta 1

on the early stages of healing of the achilles tendon in a rat model.

Scand. J. Plast. Reconstr. Surg. Hand Surg. 38, 193–197

52 Anaguchi, Y. et al. (2005) The effect of transforming growth factor-beta

on mechanical properties of the fibrous tissue regenerated in the

patellar tendon after resecting the central portion. Clin. Biomech.

(Bristol, Avon) 20, 959–965

53 Forslund, C. et al. (2003) A comparative dose-response study of

cartilage-derived morphogenetic protein (CDMP)-1,-2 and-3 for

tendon healing in rats. J. Orthop. Res. 21, 617–621

54 Forslund, C. and Aspenberg, P. (2003) Improved healing of transected

rabbit Achilles tendon after a single injection of cartilage-derived

morphogenetic protein-2. Am. J. Sports Med. 31, 555–559

55 Virchenko, O. et al. (2005) CDMP-2 injection improves early tendon

healing in a rabbit model for surgical repair. Scand. J. Med. Sci. Sports

15, 260–264

56 Bidder, M. et al. (2000) Expression of mRNA for vascular endothelial

growth factor at the repair site of healing canine flexor tendon. J. Orthop. Res. 18, 247–252

57 Boyer, M.I. et al. (2001) Quantitative variation in vascular endothelial

growth factor mRNA expression during early flexor tendon healing: an

investigation in a canine model. J. Orthop. Res. 19, 869–872

58 Wang, X.T. et al. (2005) Tendon healing in vitro: modification of

tenocytes with exogenous vascular endothelial growth factor gene

increases expression of transforming growth factor beta but

minimally affects expression of collagen genes. J. Hand Surg. [Am.]

30, 222–229

59 Screen, H.R.C. et al. (2005) Cyclic tensile strain upregulates collagen

synthesis in isolated tendon fascicles. Biochem. Biophys. Res.

Commun. 336, 424–429

60 Yamamoto, E. et al. (2005) Effects of the frequency and duration of

cyclic stress on the mechanical properties of cultured collagen fascicles

from the rabbit patellar tendon. J. Biomech. Eng. 127, 1168–1175

61 Moe, K.T. et al. (2005) Cyclic uniaxial strains on fibroblasts-seededPLGA scaffolds for tissue engineering of ligaments. In Third

International Conference on Experimental Mechanics and Third

Conference of the Asian Committee on Experimental Mechanics:

Proceedings of the SPIE Vol. 5852 (Quan, C. et al, eds), pp. 665–

670

62 Qin, T.W. et al. (2005) A new construction model of engineered tendons

under mechanical strain in vitro. Key Eng. Mater. 288–289 (Advanced

Biomaterials VI), 19–22

63 Juncosa-Melvin, N. et al. (2007) Mechanical stimulation increases

collagen type I and collagen type III gene expression of stem cell-

collagen sponge constructs for patellar tendon repair. Tissue Eng. 13,

1219–1226

64 Juncosa-Melvin, N. et al. (2006) Effects of mechanical stimulation

on the biomechanics and histology of stem cell-collagen sponge

constructs for rabbit patellar tendon repair. Tissue Eng. 12, 2291–

230065 den Braber, E.T. et al. (1998) Orientation of ECM protein deposition,

fibroblast cytoskeleton, and attachment complex components on

silicone microgrooved surfaces. J. Biomed. Mater. Res. 40, 291–300

66 Walboomers, X.F. et al. (1999) Contact guidance of rat fibroblasts on

various implant materials. J. Biomed. Mater. Res. 47, 204–212

67 Vernon,R.B. et al. (2005) Microgrooved fibrillar collagen membranes as

scaffolds for cell support and alignment. Biomaterials 26, 3131–3140

68 Lu, Q. et al. (2005) Novel capillary channel fiber scaffolds for guided

tissue engineering. Acta Biomater. 1, 607–614

69 van Wachem, P.B. et al. (1985) Interaction of cultured human

endothelial cells with polymeric surfaces of different wettabilities.


70 Wan, Y-Q. et al. (2003) Biodegradable poly(L-lactide)-poly(ethylene

glycol) multiblock copolymer: synthesis and evaluation of cell

affinity. Biomaterials 24, 2195–2203

71 Bostman, O.M. and Pihlajamaki, H.K. (2000) Adverse tissue reactionsto bioabsorbable fixation devices. Clin. Orthop. Relat. Res. 371, 216–

227

72 Bostman, O. and Pihlajamaki, H. (2000) Clinical biocompatibility of

biodegradable orthopaedic implants for internal fixation: a review.


73 Lynn, A.K. et al. (2004) Antigenicity and immunogenicity of collagen.

J. Biomed. Mater. Res. B Appl. Biomater. 71, 343–354


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