advances in tendon and ligament tissue engineering ...tendon and ligament tissue prosthesis material...

Review ArticleAdvances in Tendon and Ligament Tissue Engineering:Materials Perspective

Feras Alshomer ,1,2 Camilo Chaves,1,3 and DeepakM. Kalaskar1,4

1UCLCentre for Nanotechnology andRegenerativeMedicine, Division of Surgery & Interventional Science, University College London,London NW3 2PF, UK

2King Khalid University Hospital Surgery Department 37, Plastic and Reconstructive Surgery Section, Riyadh 11472, Saudi Arabia3Université Paris Sud, 63 Rue Gabriel Péri, 94270 Le Kremlin-Bicêtre, France4Institute of Orthopaedics and Musculoskeletal Science, Division of Surgery and Interventional Science, University College London,Royal National Orthopaedic Hospital, Brockley Hill, Stanmore, Middlesex HA7 4LP, UK

Correspondence should be addressed to Feras Alshomer; [email protected]

Received 6 May 2018; Revised 6 July 2018; Accepted 26 July 2018; Published 7 August 2018

Academic Editor: Amit Bandyopadhyay

Copyright © 2018 FerasAlshomer et al.This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction. Tendons are specialised, heterogeneous connective tissues, which represent a significant healthcare challenge afterinjury. Primary surgical repair is the gold standard modality of care; however, it is highly dependent on the extent of injuries. Tissueengineering represents an alternative solution for good tissue integration and regeneration. In this review, we look at the advancedbiomaterial composites employed to improve cellular growth while providing appropriate mechanical properties for tendon andligament repair. Methodology. Comprehensive literature searches focused on advanced composite biomaterials for tendon andligament tissue engineering. Studies were categorised depending on the application. Results. In the literature, a range of naturaland/or synthetic materials have been combined to produce composite scaffolds tendon and ligament tissue engineering. In vitroand in vivo assessment demonstrate promising cellular integration with sufficient mechanical strength. The biological propertieswere improved with the addition of growth factors within the composite materials. Most in vivo studies were completed in small-scale animal models. Conclusions. Advanced composite materials represent a promising solution to the challenges associated withtendon and ligament tissue engineering. Nevertheless, these approaches still demonstrate limitations, including the necessity oflarger-scale animal models to ease future clinical translation and comprehensive assessment of tissue response after implantation.

1. Introduction

Traumatic tendon and ligamentous injuries represent sig-nificant healthcare and economic challenges for the future.Notably, these injuries are estimated to affect 110 millionpeople in the United States [1], and incomplete repair isassociated with variable disabilities and chronic sequelae [2,3].

Tendon represents a specialised connective tissue inwhich collagen type I accounts for ∼80% of the net dryweight. In combination with proteoglycans and elastin, col-lagen permits high mechanical strength in tendons [4, 5].Furthermore, tendons display a unique structural hierarchywhere collagen molecules produce collagen fibrils, whichgroup together to form collagen fibres. The multicomposite

tendon units are composed of several collagen fibres, knownas tropocollagen (Figure 1) [3, 4, 6].

Ligaments are another form of viscoelastic connectivetissue, with a highly organised composition where collagens(types I, III, and V) constitute the bulk. Proteoglycans andchondroitin sulfate are also expressed, allowing the ligamenttissue to swell in aqueous environments [7]. In the body,the attachment of tendons and ligaments to bone involves atransition zone with unmineralised andmineralised fibrocar-tilage [3, 8]. Tendons can further attach to muscles throughfascia [4]. Defining the structural organisation of tendonsand ligaments has improved understanding of the way theseheterogeneous tissues function in synergy [9].

Surgical repair of tendons and ligaments via primarysuture or autologous transfer techniques are considered

HindawiJournal of MaterialsVolume 2018, Article ID 9868151, 17 pageshttps://doi.org/10.1155/2018/9868151

http://orcid.org/0000-0001-9282-3845https://doi.org/10.1155/2018/9868151

2 Journal of Materials

Collagen

FiberFibril

Fascicle

Tendon

Figure 1: Complex structural hierarchy of tendon. Unique struc-tural hierarchy in which collagen molecules represent the simplestforming structure of tendon with complex arrangement up totendon fascicles producing the final tendon tissue.

the gold standard modality of care. However, these solu-tions are often associated with challenges including reducedmechanical strength due to scar tissue formation, infections,donor site morbidity, and limited availability of autografts[10]. Furthermore, specialised physiotherapy protocols arerequired to improve the repair-site properties and limit scartissue formation [11].

To overcome this clinical issue, additional therapeuticoptions involving the use of synthetic prosthesis or tissueengineered biomaterial constructs have been investigated inthe literature [12]. Ranges of synthetic prosthetic deviceshave been described as tendon and ligament tissue substi-tutes. Nonabsorbable and biocompatible polyester polyethy-lene terephthalate (PET) was investigated as a potentialtendon and ligament tissue prosthesis material with suit-able mechanical and tissue integrative properties [13, 14].Other materials like polytetrafluoroethylene (PTFE) werealso investigated as ligament tissue substitutes or in ten-don augmentation grafts, for their biologically inert andstrong mechanical characteristics [15, 16]. However, limi-tations such as graft failure, poor durability, poor tissueintegration, and foreign body synovitis were observed withthese materials [17]. Tissue engineering represents an alter-native option with potential for proper tissue integration ofimplants.

Different synthetic or naturally derived materials havebeen investigated as scaffolds, which permit cell integrationand subsequent matrix deposition. Among the naturallyderived materials, collagen and chitosan have been exten-sively investigated for scaffold development due to theiroptimum biocompatibility and tissue integration potential[18]. Nevertheless, these materials exhibit limitations suchas low mechanical strength, batch variability, and difficultprocessing with latent immunogenicity [19].

Synthetic materials, including polylactic acid (PLA), pol-yglycolic acid (PGA), and polylactic-co-glycolic acid (PLGA)have been extensively investigated for tendon and ligamentrepair [20]. Synthetics exhibit several advantages linkedto large-scale manufacturing, limited disease transmission,controlled degradation, and better host tissue integration.There are also limitations associated with synthetic biomate-rials; cell integration is challenging without further materialtreatment, degradation-related products can be cytotoxic,and the materials are mechanically weaker than healthymusculoskeletal tissues [18, 21].

Novel tissue engineering approaches using compositematerials have also been described to mimic the complex,nonhomogenous environments in tendons and ligaments. Inthese composite materials, specialised cells have been com-bined with biomaterials to produce complex, heterogeneousscaffolds with controlled mechanical properties [1, 9]. Thisreview will present an overview of the different approachesdescribed to address the use of composite scaffolds for tendonand ligament tissue engineering. An in-depth critique ofthe mechanical properties, cell/ tissue integration, successcriteria, and limitations is discussed in detail.

2. Materials and Methodology

Comprehensive literature searches were conducted on thePubMed, Medline, Web of Science, and Google Scholardatabases. Various combinations of keywords were used inthe search process, including “tendon”, “ligament”, “tissue”,“engineering”, “hybrid”, “composite”, “scaffold”, “material”,“biomaterial”, “graft”, and “polymer”. Only publications inEnglish were included and there were no restrictions onyear of publication since no previous reviews were foundcovering the use of composite materials in tendon andligament tissue engineering simultaneously. All relevantmanuscripts were accessed and articles that combined tissueengineering approaches for both tendons and ligaments wereincluded. Articles that discussed different tissue engineeringapproaches of the tendon/ligament to bone interface werealso excluded due to the broadness of the topic and theimportant number of review articles on the subject [8, 22–24].

3. Results and Discussion

3.1. Composite Scaffolds for Tendon and Ligament TissueEngineering. Tendon injuries present significant healthcarechallenges due to slow healing rates, loss of function, andscar tissue formation around trauma sites [12]. In severe cases,tissue engineered scaffolds have been fabricated to replace thelost tissues. These artificial grafts can be composed of naturalor synthetic biomaterials [49]. The ideal scaffold in tendontissue engineering should exhibit specific properties such ascontrolled degradation rates, appropriate mechanical proper-ties, nonimmunogenicity, and suturability. Additionally, easymass processing and fabrication, while mimicking the nativetissue environment, are desirable [12].

3.2. Synthetic CompositeMaterials. One example of materialsused for tendon tissue engineering involved the fabricationof composite, heterogeneous scaffolds from polyglycolic acid(PGA), and polylactic acid (PLA) [44]. The outer surfacewas composed of a knitted scaffold made from a 4:2 mixtureof PGA and PLA fibres. Internally, the construct containedlongitudinally arranged, unwoven PGA fibres. The wholeconstruct was folded and secured with sutures at each end toproduce a cord structure and was tested both in vitro and invivo. Results showed that the scaffold seeded with adipose-derived stem cells (ADSCs) promoted matrix depositionand the formation of mature collagen fibrils. These scaffolds

Journal of Materials 3

presented subphysiological mechanical properties. Althoughthe study showed promising results, assessment of tenogenicdifferentiation of ADSCs was not elaborated.

Another manufacturing technique was proposed byBaker B. M. et al., who investigated the use of coelectrospin-ning technique of different polymers to yield a compositescaffold with variable degradation techniques [28]. Theyevaluated the use of PCL fibres as slow absorbing elements,while PLGA (50:50 polylactic/ glycolic acid) or PCL/ PLGAfibres were applied for intermediate degradation rates in asingle scaffold. A water-soluble polyethylene oxide (PEO)was as a sacrificial fibre element with fast absorption ratesand aimed to increase the scaffold porosity with minimalinfluence on scaffold integrity. Mechanical assessment ofPCL/ PLGA/ PEO constructs showed a maximum stressyield of nearly 3.5MPa at 0.08% strain. The modulus wasaround 100MPa and the yield strain was 0.026. PCL/ PLGA-PCL/ PEO scaffolds showed a maximum stress yield of2MPa at 0.12% strain. Initial modulus of the material was25MPa and dropped to 12.5MPa after 63-day incubationin culture media. Strain increased from 0.065% to 0.12%after incubation. A 22% mass decrease of the compositescaffold after hydration was caused by the dissolution of thePEO component. However, themechanical properties changeafter hydration and dissolution of PEO are worth furtherexplanation.

It has been suggested that woven scaffolds are superiorfor tissue integration due to their interconnected porousstructures.Nevertheless, they require challenging cell seedingtechniques and complex cell delivery systems [4, 5]. Sahoo S.et al. investigated the use of woven scaffoldsmade fromPLGAor PLLA [29]. Both f scaffolds were coated with PCL, PLGAnanofiber, or collagen type I to yield composite scaffolds andwere seeded with porcine bonemarrow-derived MSCs. Somecollagen-coated scaffolds were seeded with human dermalfibroblasts to test cell seeding and integration efficiency.Results showed that PLLA-based woven scaffolds performedinferiorly in terms of cell attachment. This was linked to thehydrophobic nature of the PLLAmaterial. Furthermore, PCLcoating of both types of knitted scaffolds was associated withhighermechanical strength but reduced cell attachment. Thiswas also linked to greater hydrophobicity in PCL than in theother polymers.

As mechanical strength is particularly important, anapproach using PLAwith graphene nanoplatelets (GNP) andPLA with carboxyl functionalized carbon nanotubes (CNT-COOH) has been proposed by Pinto et al. [25, 26]. Invitro assessment of the composite was nontoxic to humanfibroblasts. In vivo assessment using mouse model did notshow any toxicity or local or systemic inflammatory response.The addition of nanofillers mentioned enhanced themechan-ical properties of PLA polymer films reaching Young’smodulus of 4.86±0.47 GPa for CNT-COOH scaffolds and4.92±0.15GPa for GNP scaffold groups. The tensile strengthhowever was mostly enhanced in the PLA/ CNT-COOHscaffolds reaching up to 72.22±1.52MPa. The authors did notinvestigate, however, the effect of in vivo implantation onthe associated mechanical properties mimicking real clinicalsettings.

3.3. Biological Composite Scaffolds. Collagen type I compositescaffold was also investigated to incorporate resilin-like pro-tein. Resilin is an arthropods protein with elastic and highlystretchable structure. Sanami, M., et al. [27] investigated thefabrication of such composite.The scaffoldwasmade throughextrusion process of composite solution containing Collagenand Resilin at different concentration into polyethyleneglycol buffer. Fibres were subsequently cross-linked using 4-arm poly(ethylene glycol) ether tetrasuccinimidyl glutaratesolution. Mechanical assessment showed that resilin in non-cross-linked collagen scaffold significantly reduced stressat break and Young’s modulus values, while significantlyincreasing break strain. Cross-linked collagen/resilin scaffoldshowed significant increase in stress and strain values and asignificantly decreased Young’s modulus values. This showsan interesting effect of resilin exhibiting its natural properties.In vitro, the scaffold produced supported 100% fibroblastproliferation and alignment compared to 80% in collagen-fibre control.

Scaffolds made from collagen and glycosaminoglycan(GAG) were recognised for their role in supporting cellu-lar proliferation and differentiation. However, this type ofscaffold lacks the mechanical characteristics required fortendon tissue engineering applications. Caliari et al., 2011,have proposed the concept of developing composite mate-rials in a core-shell fashion with the necessary mechanicalproperties [30]. The group fabricated scaffolds composedof highly porous, aligned isotropic GAG cores surroundedby strong, high density isotropic GAG membranes. Thecore-shell design was proposed to increase the mechanicalstrength of the scaffolds. The material was fabricated usingan evaporation technique to create membranes and freeze-drying to incorporate the core within the membrane shell.Dehydrothermal (DTH) cross-linking was used to increasematerial integration, as well as the mechanical properties. Invitro assessment using horse-derived tenocytes demonstratedgood cell attachment, proliferation, and cell viability up to14 days after seeding. Scaffolds exhibited high porosity andappropriate mechanical properties depending on the mem-brane thickness. Several factors however that need furtherinvestigation like cellular functional assessment (e.g., proteinexpression), nature and content of collagen after cell seeding,and the mechanical properties of the scaffold at differentstates (e.g., wet versus dry).

Chitosan is a naturally derived polysaccharide with excel-lent potential for tissue engineering applications. Due tothe biocompatible and cell adhesive properties of chitosan,the polysaccharide has been investigated for tendon tissueregeneration [9–11]. In one example, composite scaffoldsmade from chitosan and alginates were produced through aspinning/coagulation technique to yield an alginate-0.1% chi-tosan scaffold. Alginate is an anionic polysaccharide with cal-cium chain in which the integration of chitosan improves itsbiocompatibility and cell adhesive potential and decreases itsdegradation rate. In vitro assessment using rabbit patellar ten-don fibroblasts showed that alginate-0.1% chitosan scaffoldshad significantly higher cell adhesion and matrix depositioncompared to alginate-only and polyglactin 910 controls. Thealginate-0.1% chitosan material was evaluated mechanically


and exhibited lower tensile strength and strain at failure thanthe polyglactin group [33]. Chitosan was also fabricated withhyaluronic acid followed by wet spinning and hybridisationto increase the mechanical properties of the construct.Different hyaluronic acid concentrations were investigatedand showed that the combination of chitosan with 0.1%hyaluronic acid showed the best cell attachment. The finalcomposite constructs were sterilised using ethylene oxide gasprior to in vitro evaluation with rabbit patellar fibroblasts.Mechanical properties of the materials were reduced withinthe first 2 hours after seeding but the consequent moduluswas maintained for 28 days of culture. Cell proliferationquantificationusingDNAcontent analysis showed significantimprovement in chitosan-0.1% hyaluronic acid compositescompared to other chitosan-based scaffolds. Todetermine theclinical potential of the composite scaffolds, authors assessedthe chitosan-0.1% hyaluronic acid composites in vivo bytreating rabbit rotator cuff injuries with cell-seeded scaffolds[31]. The scaffolds were cultured with rabbit patellar tendonfibroblasts for 4 weeks prior to implantation. Additionally,authors tested the potential for ligament tissue engineeringusing a rabbit medial collateral ligament injury model. Theyused scaffolds seeded with fibroblasts adapted from rabbitsAchilles tendon for 2 weeks prior to implantation. Forthe tendon model, results indicated collagen deposition incell-seeded scaffolds with significant improvement in themechanical properties from 4 to 12 weeks after implantation.For the ligament-engineering model, authors showed a lackof tissue integration with the bony tunnel attachment with60% recovery in failure load compared to healthy ligament.Additional research on the chitosan-hyaluronic acid scaffoldshas aimed to understand the effects ofmechanical stimulationon fibroblasts response [35]. It was found that the applicationof 90-degree rotations and 5% stretch at 0.5Hzwas associatedwith increased expression of fibromodulin, and collagens Iand III. No further assessment of the overall mechanicalinfluence of the cultured scaffolds under dynamic conditionswas made.

Additionally, composite scaffolds made from extruded,cross-linked bovine type I collagen and chondroitin-6-sulfatewere fabricated [36]. The collagen-based constructs under-went a series of cross-linking steps using carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), andN-hydroxysuccinimide (NHS). Cross-linking was combinedwith freeze-drying to incorporate a porous chondroitin-6-sulfate. The final constructs had open and interconnectedporosity with an average pore size of 100𝜇m and axi-ally aligned collagen fibres. Mechanical assessment showedthat the cross-linked composite scaffolds could withstand61.94±15.54 N, compared to 11.75±2.62 N without cross-linking. The maximum strain was shown to be 30.17±7.17%with cross-linked fibres, while it was 15.40±3.22% with-out. Composites with cross-linked fibres presented a tensilestrength of 1.55±0.30MPa compared to 0.24±0.08MPa fornon-cross-linked fibres. It is important to mention thatscaffold porosity is inversely related to mechanical strengthand further studies are required to evaluate the cell responseof such construct for application in tendon and ligamenttissue engineering.

Gelatin was also utilised to fabricate a composite scaffoldfor tendon tissue engineering applications. Coelectrospin-ning of aligned poly-𝜀-caprolactone (PCL) and methacry-lated gelatin (mGLT) followed by photo-cross-linking wasused in the fabrication process [34]. Scaffold films were firstproduced and then were seeded with ADSC, after whichphoto-cross-linking of 5 layers using UV radiation was madeto produce a multilayered scaffold mimicking the naturaltendon structure. In vitro assessment showed biocompatibil-ity of produced composite in which ADSCs were orientedalong the longitudinal access of aligned fibre construct andexpressed tendon-related markers (scleraxis and tenascin-C)after being stimulated with TGF𝛽-3. Mechanical assessmentof cell-seeded cross-linked scaffolds however was far inferiorfor potential clinical application (details in Table 1).

Other groups have combined collagen type I with silkto improve scaffold properties for tendon applications [37].Sericin protein was extracted from silk fibres producedby Bombyx mori, and mixed with collagen solution to fillthe gaps in the silk fibre net. Furthermore, adherence andmechanical strength were increased though DTH cross-linking.Mesenchymal stem cells derived fromhuman embry-onic stem cells (hEC-MSCs) were used in in vitro and invivomodels and showed that themechanically loaded knittedsilk/ collagen microsponge scaffolds can induce tenogenicdifferentiation in mesenchymal stem cells and support ten-don regeneration up to 360 days after implantation (Table 1).

In an additional study, the same composite scaffoldswere tested with the supplementation of recombinant humanstromal cell-derived factor-1 alpha (rhSDF-1 alpha), to thecollagen type I sponges [38]. SDF-1 is a chemokine thatpromotes cell recruitment and enhances tissue regeneration.In a murine Achilles tendon model, the authors showed thatthis approach permits higher expression of collagen one weekafter implantation, indicating an accelerated onset of tendonhealing. Further, the mechanical properties were marginallyhigher than in scaffolds without rhSDF-1 alpha treatment.

In order to improve the mechanical properties andtendon regeneration, Chen X. et al. utilised the same com-posite scaffolds with hEC-MSCs [39]. Cells were geneticallyengineered to overexpress the Scleraxis (SCX) gene. SCX is atranscription factor identified for its role as a tenocytemarkerand upstream regulator of tendon-related genes. In vitro andin vivo results showed enhanced tendon regeneration andbetter quality neotendon formation, compared to the previ-ous studies with the same scaffolds. Importantly, there wasless osteogenic, chondrogenic, and adipogenic differentiationof the SCX-hEC-MSCs compared to nongenetically modifiedMSCs. Mechanical properties in a murine Achilles tendoneight weeks after implantation were inferior to native tendontissue. This research highlights that scaffold properties areimportant for tendon tissue engineering, but factors like cell-types used can influence mechanical and histological results.

3.4. Synthetic and Biological Composite Scaffolds. Othershave also focused on incorporating PLGA with silk derivedmaterials for tendon tissue engineering applications [40]. Acomposite scaffold comprised degummed silk microfiberscoated electrospun PLGA.The authors degummed silk fibres


Table1:Summaryo

fthe

literaturerelated

tothea

pplications

ofhybrid

biom

aterialintend

ontissuee

ngineerin

g.∗

indi

cate

stha

tapp

roximaten

umberswe

reextractedfro

msupp

liedfig

ures

since

noexactreference

values

arem

entio

nedin

thetext.PD

S:po

ly-dioxano

ne;SCX

,scleraxis;

TGF𝛽

,transform

inggrow

thfactor

beta;hEC

S-MSC

s,mesenchym

alste

mcells

deriv

edfro

mhu

man

embryonics

tem

cells;rhSDF1,recom

binant

human

strom

alcell-deriv

edfactor

1;PG

A,poly-(la

ctide-co-glycolid

e);P

CL,poly-caprolactone;bFG

F,basic

fibroblastgrowth

factor.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

PGA

/PLA

com

posi

te.[

24]

Invitro(A

DSC

s)In

vivo

(rab

bit)

Afte

rimplantatio

nin

Achillestendo

n,Te

nsile

stre

ngth

:4.88±

8.07

MPa

No

repo

rtof

othe

rmec

hani

calp

rope

rtie

s.

(i)Grossly,

theimplantedcell-seeded

scaffoldwas

integrated

with

the

nativ

etissue

interfe

rence,with

asmoo

thsurfa

cecord-like

shapew

ithlessno

ticeabler

emaining

materialafte

r45we

eks.

(ii)T

issue

adhesio

ns,described

grossly

tobe

lesscomparedto

control.

(iii)Paralleland

morem

aturec

ollagenfib

ersa

ndlong

itudinally

alignedcells

arep

resentsthancontrol.

P(L

LA-C

L)/C

olla

gen

I.[2

5]In

vitro(Tenocytes)

Youn

g’sm

odul

us∗

Non

seeded

abou

t2.2MPa.

Cellseededabou

t3MPa.

Tens

ilest

reng

th∗

Non

seeded

abou

t3MPa.

Cellseededabou

t4.5MPa.

(i)Sign

ificantlyhigh

ercellproliferatio

nin

then

anoyarnscaffold

comparedto

otherscaffo

ldsa

ndcontrol.

(ii)S

EMshow

edspindle-shaped

cellin

both

nano

yarn

andaligned

nano

fiber

scaffold,whilepo

lygonaland

rand

ompatte

rncells

are

foun

din

ther

ando

moriented

fibers.

(iii)Ex

pressio

nof

tend

onspecificE

CM(ty

peIc

ollagen,

type

IIIcollagen,

decorin

,tenascin-C,

andbiglycan)w

assig

nificantly

high

erat14

days

inthen

anoyarngrou

p.

P(L

LA-C

L)/C

olla

gen

Ina

noya

rn.[

26]

Invitro(TDSC

s)In

vivo

(mou

se)

Mec

hani

cals

tres

s:Dynam

icgroupwas

59.58±7.8

1MPa

Staticgroupwas

43.18±6.58

MPa

Controlgroup

was

32.43±

5.27%MPa

Youn

g’sm

odul

us:

Dynam

icgroupwas

51.99±

7.16MPa

Staticgroupwas

34.76±4.75

MPa.

Controlgroup

was

23.30±3.83

MPa

TDSC

swereu

sedin

scaffoldseedingwith

both

dynamicandsta

ticcultu

recond

ition

s.In

vitro:

(i)TD

SCssho

wedelon

gatedfib

roblast-likem

orph

olog

ywith

asig

nificantincreaseincellcoun

tindynamicgrou

pat14

days.

(ii)M

orec

ellinfi

ltrationanddensem

atrix

indynamicgrou

p.(iii)PC

Rconfi

rmed

Tend

onrelatedmRN

Aexpressio

nmorein

dynamicgrou

p.(iv

)Western

blottin

gshow

edsig

nificantincreaseinthep

rotein

expressio

nlevelsof

Collagens

Iand

III,and

tenascin-C

inthe

dynamicgrou

p.In-vivo:

(i)Sign

ificantlylowe

rnum

bero

fcellsat12

weeksw

ithgreaterm

atrix

depo

sitionandlong

itudinalspind

le-shapedcells

indynamicgrou

pcomparedto

others.

(ii)C

ollagencontentw

ashigh

estindynamicgrou

preaching

77.76±

6.82%of

norm

alrabb

itpatellartendo

n(174.31±13.89𝜇g/mg).

(iii)CollagenIexpressionwas

Sign

ificantlyhigh

erin

dynamicgrou

p.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

PLA

/Col

lage

n-Ie

lect

rosp

unbu

ndle

s[27

]In

Vitro(Tenocytes)

Com

posites

caffo

ldsw

erem

adefrom

blends

containing

PLA/C

oll-7

5/25

(w/w

).Yo

ung’s

mod

ulus

was

98.6±12.4MPa

Max

imum

stre

sswas

14.2±0.7MPa

(i)Seeded

Teno

cytessho

wnto

exhibitgoo

dcelladhesio

nprofi

leand

moree

long

ated

morph

olog

ythatwas

bette

roverP

LA/C

oll-5

0/50

blends

than

other.

PLA

/gra

phen

enan

opla

tele

ts(G

NP)

and

PLA

/car

boxy

l-fu

nctio

naliz

edca

rbon

nano

tube

s(C

NT-

CO

OH

)[2

8,29

]

Invitro(fi

broblast)

Invivo

(mou

se)

Youn

g’sm

odul

usPL

Acontrol:3.99±0.42

GPa

PLA/C

NT-COOH:4.86±0.47

GPa

PLA/G

NP:

4.92±0.15

GPa

Tens

ilest

reng

thPL

Acontrol:59.90±4.93

MPa

PLA/C

NT-COOH:72.22±1.5

2MPa

PLA/G

NP:

58.56±3.99

MPa

(i)Bo

thprod

uced

scaffolds

supp

ortedfib

roblastsmetabolicactiv

ityandproliferatio

ntillfi

nalassessm

entp

oint

(72hrs).

(ii)Invivo

assessmentsho

wedlack

ofanylocalorsystemic

inflammatoryr

espo

nseu

singN-acetylglucosaminidase(NAG

)and

nitricoxide(

NO)serum

levels.

(iii)Noassociated

hepatotoxicityin

histo

logica

ssessm

ent.

(iv)H

istologicassessmento

fexplanted

scaffoldshow

edform

ationof

thin

capsulea

roun

dtheimplantw

ithho

mogenou

sgranu

latio

ntissue.

PCL/

colla

gen-

PLLA

/col

lage

n[3

0]In

vitro(m

yoblasts,

fibroblast)

Tens

ilest

reng

thwas

0.5058±0.2130

MPa

Max

imum

stra

inwas

18.49%±8.210

Youn

g’sm

odul

uswas

7.339±2.131M

Pa

(i)Statisticallyhigh

erviability

ofbo

thmyoblastand

fibroblastsin

all

region

softhe

scaffold.

(ii)S

caffo

ldcouldsupp

ortthe

form

ationof

myotubesthatise

ssentia

lforn

ormalmuscle-tend

onjunctio

nform

ation.

Alig

ned

PLLA

nano

fiber

/Lay

ered

chito

san-

colla

gen

hydr

ogel

/Alg

inat

eout

erco

atin

g.[3

1]

Invitro(Tenocytes)

Max

imum

Forc

eto

brea

kFo

runcoated2and3layersscaffold:

7.89±

1.5Nand7.4

5±0.3N

,Fo

rgelcoated

2and3layersscaffolds:4.76±

0.23Nand6.49±0.09N.

No

repo

rtof

othe

rmec

hani

calp

rope

rtie

s.

(i)Alginatec

oatin

gwas

associated

with

significantly

lessattached

proteins

than

control.

(ii)B

othcoated

andun

coated

scaffoldmaintain50%of

their

substancea

fterincub

ationwith

PBScontaining

104un

its/m

llysozym

esolutio

n.(iii)Alamar

blue

andDNAconcentrationassessmentsho

wedhigh

cellu

larv

iability,metabolicactiv

ity,and

proliferatio

nup

to7days

after

seeding.

(iv)S

eededscaffolds

were

show

nto

supp

ortcellulara

lignm

ent.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

Elec

tros

pun

colla

gen

Ina

nofib

er/c

olla

gen

mic

rofib

er[3

2]

Invitro(fi

broblast)

Invivo

(rab

bit)

Before

implantatio

n:M

axim

umlo

adwas

28.33±2.19

N.

Max

imum

stre

sswas

2.69±0.47

MPa

Max

imum

stra

inwas

61.34±4.71

%Yo

ung’s

mod

ulus

was

43.81±

4.19

KPa.

60days

after

implantatio

nto

Achillestendo

n:M

axim

umlo

adwas

abou

t63.72

N.

Max

imum

stre

sswas

abou

t9.84N/m

m.

Max

imum

stra

inwas

abou

t16.35%

Youn

g’sm

odul

uswas

abou

t0.62N/m

m.

Invitro:

(i)Sign

ificantlyhigh

ercellviability

inthea

lignedhybrid

scaffold

comparedto

others.

(ii)S

EMandim

mun

ofluo

rescence

proven

superio

ralignm

ento

ffib

roblaststogether

with

cellproliferatio

nwith

closec

ell-to-cell

contactinthea

lignedhybrid

scaffoldcomparedto

others.

Invivo:

(i)Sign

ificant

increase

inthen

umber,density,and

alignm

ento

fthe

collagendepo

sitionwith

maturee

lasticfi

bers.

(ii)S

ignificantincreaseinthen

umbera

ndmaturity

ofteno

blastsand

teno

cytes.

(iii)Sign

ificantlylowe

rperitend

inou

sadh

esions,m

usclefi

brosis,

and

atroph

yandinflammatoryc

ellsfoun

din

thetreated

tend

ons

comparedto

controls.

Elec

tros

pun

colla

gen

Ina

nofib

er/c

olla

gen

mic

rofib

er/

PDS

shee

ts.[33]

Invivo

(rab

bit)

Afte

r60days

ofim

plantatio

nto

Achilles

tend

on:

Max

imum

load∗was

abou

t74.02

NM

axim

umst

ress∗was

abou

t11.3

7MPa

Youn

g’sm

odul

us∗was

abou

t0.754

MPa

(i)Sign

ificantlyhigh

erfib

rillogenesis

after

PDStre

atment.

(ii)S

ignificantly

high

ercollagenfib

rilsw

erefou

ndin

theP

DStre

ated

scaffolds

comparedto

ther

egular

one.

(iii)Sign

ificant

increase

intheloadto

failu

reandload

toyieldpo

int

with

PDStre

atment.

(iv)S

ignificantly

improved

peritendino

usadhesio

ns,m

usclefi

brosis,

andatroph

yscores

thatarec

omparableintheP

DStre

ated

and

nontreated

collagenscaffolds.

Cor

e-sh

ellC

olla

gen

type

I/G

lyco

sam

inog

lyca

n.[34]

Invitro(Tenocytes)

Tens

ilem

odul

usfo

rdry

mem

bran

eswas

abou

t636±47

MPa

to693±20

MPa

Tens

ilem

odul

usfo

rhyd

rate

dm

embr

anes

was

30MPa.

Tens

ilem

odul

usfo

rdry

alig

ned

core

scaff

oldranges

from

833±236to

829±165

KPa

(i)Sign

ificant

increase

inthen

umbero

fcellsatday1afte

rseeding

inthec

ore-shellcom

positec

omparedto

core

alon

escaffo

lds.

(ii)H

igherm

etabolicactiv

ityob

served

atday7in

thec

orea

lone

scaffoldthatissta

tistic

allysig

nificant.

(iii)Nosig

nificantd

ifference

inthem

etabolicactiv

ityandcell

numberb

etwe

enthetwo

grou

psat14-day

perio

d.

Col

lage

n-I/

nano

carb

onfib

ers

[35]

-Mechanicalassessm

entinwe

tcon

ditio

nYo

ung’s

mod

ulus

was

840±140MPa

Tens

ilest

reng

thwas

70±8MPa

(i)Nocellwo

rkwas

presented.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

Col

lage

nty

peI/

Resi

lin[3

6]In

vitro(Fibroblasts)

Max

imum

stre

sswas

34.63±9.7

5MPa

Max

imum

stra

inwas

0.21±0.04

MPa

Youn

g’sm

odul

uswas

49.48±10.71M

Pa

(i)Hum

anadultfi

broblasts

were

used

inthea

ssessm

entp

rocess.

(ii)Th

eautho

rsshow

edthatadditio

nof

resilin

andtheu

seof

poly(ethyleneg

lycol)either

tetrasuccinimidylglutarated

idcomprom

isethec

ellulara

ctivity.

(iii)Th

escaffo

ldprod

uced

managed

tosupp

ortcellularp

roliferation

and100%

cellu

lara

lignm

entafte

r7days

ofseedingcomparedto

80%

incollagencontrol.

poly

-𝜀-c

apro

lact

one(

PCL)

and

met

hacr

ylat

edge

latin

(mG

LT)

[37]

Invitro(A

DSC

s)M

axim

umlo

adwas

abou

t0.25N

.N

ore

port

ofot

herm

echa

nica

lpro

pert

ies.

(i)Teno

genicd

ifferentia

tionwas

indu

cedthroug

hdifferentiatio

nmedium

having

DMEM

,2%FB

S,P/S,and10

ng/m

lTGF-𝛽3for7

days

after

seeding.

(ii)S

eededconstructswe

reshow

nto

exhibitelong

ated

morph

olog

yas

wellas

expressio

nof

teno

genicm

arkers(scleraxisandTenascin-C

).(iii)Stackedscaffoldwas

show

nto

have

adequatepo

rosityforc

ell

diffu

sionanddifferentiatio

n.

Seri

cin

extr

acte

dkn

itted

silk

/cro

ss-li

nked

colla

gen

type

Im

icro

spon

ges[38]

Invitro

(hES

C-MSC

s)In

vivo

(mou

se)

4we

eksa

fterimplantatio

nto

Achillestendo

nof

cell-seeded

scaffolds:

Max

imum

load

was

65.24±9.5

8N

Max

imum

stre

sswas

6.72±0.90

MPa

Stiff

nesswas

28.26±2.95

N/m

mYo

ung’s

mod

ulus

was

34.91±

5.08

MPa

Invitro:

(i)Goo

dcellattachment,proliferatio

n,andspreadingat14

days'

perio

d.(ii)S

cx.,collagenI,andcollagenIII

expressio

nwe

resig

nificantly

high

erin

dynamicgrou

pthan

thec

ontro

l.In

vivo

assessmenta

fter4

weeks

ofim

plan

tatio

n:(i)

Grossly,

well-integrated

constructw

ithnativ

etendo

ns.

(ii)V

iablec

ellsarep

resentssho

wingspindle-shaped

morph

olog

y.(iii)Sign

ificantlyhigh

ercollagenI,III,V𝛼1,V𝛼2,andTG

F𝛽1in

cell-seeded

scaffoldcomparedto

cellfre

escaffo

ld.

(iv)N

eocollagenwas

replacingexogenou

sone

with

morec

ontent,

andmaturem

orph

olog

yin

cell-seeded

scaffoldcomparedto

cellfre

escaffold.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

Seri

cin

extr

acte

dkn

itted

silk

/cro

ss-li

nked

colla

gen

type

Im

icro

spon

ges/

rhSD

F-1a

lpha

[39]

Invitro(fi

broblast)

Invivo

(rat)

4we

eksa

fterimplantatio

nin

Achillestendo

n:M

axim

umlo

adwas

68.5±18

NM

axim

umst

resswas

7.02±1.7

MPa

Stiff

nesswas

39.0±6.6N/m

mYo

ung’s

mod

ulus

was

45.3±10.4MPa

Invivo

assessmenta

fter4

weeks

ofim

plan

tatio

n:(i)

Morefi

brob

lasts

-like

cells

andlessinflammatorycells

arefou

ndearly

after

implantatio

n(4

days,1

week).

(ii)M

oreo

rganized

continuo

uscollagenfib

ersa

refoun

dwith

ahigh

erconcentrationof

collagentype

I.(iii)Goo

dvascular

compo

nentsw

ithlessinflammatorycells

are

present.

(iv)S

ignificantly

high

erexpressio

nof

Collagens

Iand

III,decorin

intheS

DF-1scaffo

ldsa

tallassessmentp

eriods.

(v)L

arge

fibrilso

fAchilles

tend

onsformed

intheS

DF-1scaffo

lds

(41.6±5.5nm

)com

paredto

control(37.1±2.9nm

).

Seri

cin

extr

acte

dkn

itted

silk

/cro

ss-li

nked

colla

gen

type

Im

icro

spon

ges/

SCX

engi

neer

edce

lls[40]

Invitro

(hES

C-MSC

s)In

vivo

(mou

se)

4we

eksa

fterimplantatio

n:Yo

ung’s

mod

ulus

was

60.63±17.6MPa

Max

imum

stre

sswas

8.73±2.15

MPa

8we

eksa

fterimplantatio

n:Yo

ung’s

mod

ulus

was

71.3±12.4MPa

Max

imum

stre

sswas

10.1±2.2MPa

Invitro:

(i)SC

Xtre

atmentincreases

collagenIexpressionwith

enhanced

cell-sheetformation.

(ii)D

ecreased

capacityof

adipogenic,cho

ndrogenic,andosteogenic

differentiatio

nof

thec

ellswith

moretenogenicdifferentiatio

n.In

vivo:

(i)Goo

dproliferatio

nandearly

matrix

depo

sitionin

theS

CXcells

comparedto

control.

(ii)Increased

CollagenIa1,Ia2,andtend

onrelatedtranscrip

tion

factor

Eya2

inSC

Xcells

comparedto

control.

(iii)Morefi

brob

last-

likespind

le-shapedcells

andfewe

rimmun

ogenic

cellinfiltrates

intheS

CXcells

grou

p.(iv

)Highere

xpressionof

biglycan

intheS

CXcells

grou

p,indicatin

gmorem

aturee

ndogenou

scollagen.

Kni

tted

silk

coat

edw

ithel

ectr

ospu

nco

llage

n-I/

poly

uret

hane

(PU

)nan

ofibe

rs[4

1]

Invitro(Fibroblast)

Max

imum

stre

sswas

13.5±1.5

MPa

Youn

g’sm

odul

uswas

21.7±4MPa

(i)Hum

anfib

roblastswe

reseeded

oncompo

sites

caffo

ldto

testfor

viability.

(ii)A

ssessm

entw

asmadethrou

ghAlamar

Blue

assayandshow

edthatsamples

with

high

ercollagencontenth

adhigh

ercellu

larv

iability

profi

le(C

OL75/PU

25)thanotherg

roup

s.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

NSi

lkco

ated

with

Poly

capr

olac

tone

(PC

L)or

Poly

(3-h

ydro

xybu

tyra

te)

(P3H

B)na

nofib

ers[

42]

Invitro(Fibroblast)

Max

imum

load

97.6±11.4Nforsilk

fibroin/P3H

B110

.5±6.6Nforsilk

fibroin/PCL

No

repo

rtof

othe

rmec

hani

calp

rope

rtie

s.

(i)Hum

anfib

roblastswe

reseeded

oncompo

sites

caffo

ldandshow

edgood

cellu

larv

iabilityandno

toxicity(assessm

entfor

3days).

Deg

umm

edSi

lk-fi

broi

nm

eshe

s/al

igne

dSi

lk-fi

broi

n[43]

Invitro(M

SCs)

Max

imum

load

ford

ynam

iccu

lture

cond

ition

Alignedscaffold

144.44±5.03

N(7

days)

172.08±6.28

N(14

days)

Rand

omScaffold

122.35±3.67

N(7

days)

138.67±9.2

2N(14

days)

Stiff

ness

Alignedscaffold

24.33±1.4

0N/m

m(7

days)

26.93±2.40

N/m

m(14

days)

Rand

omScaffold

17.48±0.93

N/m

m(7

days)

23.07±

2.54

N/m

m(14

days)

No

repo

rtof

othe

rmec

hani

calp

rope

rtie

s.

(i)Highlysig

nificantcellviabilityin

thea

lignedscaffoldcomparedto

ther

ando

mon

eatb

othdynamicandsta

ticcond

ition

s.(ii)C

onsistent

cellu

larp

roliferationin

both

aligned(dyn

amicand

static

)and

dynamicrand

omscaffold.

(iii)Higherc

ollagendepo

sitionin

thed

ynam

icalignedscaffold

comparedto

thes

tatic

onea

ndther

ando

mscaffoldgrou

ps.

(iv)D

ynam

iccultu

reim

proved

theh

istologicalassessmento

fboth

alignedandrand

omscaffoldwith

morec

ellelong

ationandmatrix

depo

sition.

(v)H

ighere

xpressionlevelofcollagenI,tenascin-C

,and

teno

mod

ulin

inthea

ligneddynamicscaffolds

comparedto

others.

Kni

tted

PLG

A-P

LLA

/coa

ting

with

(1)P

CL.

(2)P

LGA

nano

fiber

.Ty

peIc

olla

gen[44]

Invitro(BMSC

s)

Max

imum

load

Uncoated68.4±5.37

N.

PCLcoated

63.1±4.52

N.

PLGAcoated

56.3±6.61

N.

Collagencoated

59.5±8.3N.

Stiffness

Uncoated9.1±2.38

N/m

m.

PCLcoated

4.3±0.91

N/m

m.

PLGAcoated

5.8±0.70

N/m

m.

Collagencoated

5.7±0.48

N/m

m.

No

repo

rtof

othe

rmec

hani

calp

rope

rtie

s.

(i)Effi

cientcellseeding

onPL

GAnano

fiber

coated

and

collagen-coated

scaffolds.(80-89%

and61-69%

,respectively

).(ii)S

ignificantly

high

ercellproliferatio

nbetween2n

dand7t

hcultu

redays

ofPL

GAnano

fiber

coated

knitted

PLGAscaffolds.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

PLG

Ana

nofib

er/S

ilkm

icro

fiber

[45]

Invitro(BMSC

s)

Max

imum

load

ofun

seeded

scaff

olds

75.3±4.79

Non

day0,

and61.5±3.43

Non

day21

Max

imum

load

ofrolledseeded

scaff

olds

68.2±6.72

Non

day21

Stiffnessofun

seeded

scaff

olds

4.8±0.52

N/m

mon

day0,

and5.9±0.54

N/m

mon

day21

Stiffnessofrolledseeded

scaff

olds

5.5±0.30

N/m

mon

day21

No

repo

rtof

othe

rmec

hani

calp

rope

rtie

s.

(i)Dualsurface

seeded

scaffolds

show

edsig

nificantly

high

ercell

proliferatio

nratesc

omparedto

singles

urface

seeding.

(ii)Increased

cellproliferatio

nbetween14

days

and21

days

after

cultu

re.

(iii)Ro

lled-up

scaffolds

hadno

nsignificantly

lowe

rcellproliferation

rates.

PLG

Ana

nofib

er/b

FGF

/Silk

mic

rofib

er[46]

Invitro

(mesenchym

alprogenito

rcell)

3we

eksa

fterc

ulture

ofrolledscaffolds:

Max

imum

load

Unseeded

scaff

olds∗we

reabou

t61.5

NSeeded

bFGF-fre

escaffolds∗

were

abou

t68.2

N Seeded

bFGFscaff

olds∗we

reabou

t82.7N

Stiffness

Unseeded

scaff

olds∗we

reabou

t5.92N/m

mSeeded

bFGF-fre

escaffolds∗

were

abou

t5.53

N/m

mSeeded

bFGFscaff

olds∗we

reabou

t6.97

N/m

m

(i)Sign

ificantlyhigh

ercellviability

intheb

FGFscaffolds

compared

tobF

GF-fre

escaffo

lds.

(ii)S

ignificantly

high

ercollagens

Iand

III,fi

bron

ectin

,and

biglycan

14days

after

cultu

rein

theb

FGFscaffolds

comparedto

bFGFfre

escaffolds.

(iii)Sign

ificantlyhigh

ercollagencontentintheb

FGFscaffolds

comparedto

bFGFfre

escaffo

ldsb

y3r

dwe

ekaft

ercultu

re.


Table1:Con

tinued.

CO

MPO

SITE

/HYB

RID

MO

DEL

SCA

FFO

LDPR

OPE

RTIE

SC

ELL/

TISS

UE

INTE

GRA

TIO

N

Alg

inat

e/0.

1%ch

itosa

n[47]

Invitro(fi

broblast)

Tensile

strengthwas

235.2±

8.5MPa

Max

imum

strainwas

12.3±0.3%

(i)Sign

ificantlylowe

rnum

bero

funatta

ched

cells

inalginate–chitosangrou

pcomparedto

polyglactin

andalginatealon

e.(ii)F

ibroblastswe

respread

onthep

olym

erfib

ers.

(iii)Prom

inentcollagentype

Iprodu

ction14

days

after

cultu

rewas

moreo

nthes

caffo

ldsurfa

ceby

immun

estainingwith

noclear

visualizationof

both

typesIIa

ndIII

collagen.

Chi

tosa

n/0

.1%hy

alur

onic

acid

[48]

Invitro(fi

broblast)

Invivo

(rat)

(i)In

vitr

o:Tensile

strength:

Before

seedingwas

213.3±10

MPa

2hrsa

fterseeding

was

60±6.7MPa

14days

after

seedingwas

66.7±6.8MPa

28days

after

seedingwas

65.1±6.6MPa

Max

imum

strain:

Before

seedingwas

3.2±0.6%

(ii)I

nvi

vote

ndon

mod

el:

Tangentm

odulus

forc

ell-seededscaff

old:

4we

eksa

fterimplantatio

n:abou

t58±MPa

12we

eksa

fterimplantatio

n:abou

t85±MPa

Invi

volig

amen

tmod

el:

Max

imum

load

forc

ell-seededscaff

old:

12we

eksa

fterimplantatio

n:abou

t110±10

N

Invitroup

to28

days

after

cellseeding:

(i)Grossob

servationof

ECM

prod

uctio

nby

light

microscop

y.(ii)P

rominentcollagentype

Iprodu

ction14

days

after

cultu

remore

onthes

caffo

ldsurfa

ce.

Invivo

tend

onmod

el:

(i)Ty

peIc

ollagenisseen

onlyin

cell-seeded

scaffold.

Invivo

ligam

entm

odel:

Non

e.


to achieve a more efficient sericin removal. This resulted insmoother fibre surfaces and preserved the generalmechanicalproperties of the silk. In vitro studies using rabbit bonemarrow-derivedMSCs revealed good cell viability dependingon the seeding technique applied (single or dual surfaceseeding) and whether scaffolds had a flat or rolled/ cylindricalmorphology. Rolled structures had lower cell proliferationcompared to the other constructs but, overall, the electrospunPLGA polymer provided a large surface area for cell prolifer-ation. To increase tenocyte differentiation of bone marrow-derivedMSCs, authors reported amodified protocol inwhichthe scaffold has a 1-week release of basic fibroblast growthfactor (bFGF) [45]. bFGFwas blendedwith PLGAand bovineserum albumin prior to electrospinning with knitted silkfibres. Results showed that incorporating bFGF was associ-ated with upregulation of tenogenic markers during MSCdifferentiation. Collagen expression was also increased, andthis contributed to improved mechanical properties of thescaffold. The combined effect of growth factor incorporationtogether with dynamic culturing conditions would be ofinterest for its effect over MSC differentiation and overallconstruct incorporation.

Sharifi-AghdamM. et al. [46] have investigated a compos-ite scaffold consisting of knitted silk coated with electrospuncollagen-I/polyurethane nanofibres. The main aim of thisapproach was to incorporate a polyurethane polymer layerto increase the attachment of collagen to silk fibres. Invitro testing using seeded human fibroblast showed thatcomposite scaffold maintained adequate cellular metabolicactivity. Assessment of mechanical properties showed anultimate stress profile reaching 13.5±1.5MPa and Young’smodulus of 21.7±4MPa. However, further assessment ofseeded constructs for their biocompatibility and cell functionwas not investigated.

Investigative work showed the incorporation of silk withnanofibres derived either from Polycaprolactone (PCL) orPoly(3-hydroxybutyrate) (P3HB) [41].The composite scaffoldwas fabricated through electrospinning process of differentpolymer material over twisted silk fibroin fibres. The mainaim of this approach was to incorporate nanofibrous struc-tures onto the composite scaffold to increase the surfaceto volume ratio and therefore increase cellular attachment.Authors showed a composite scaffold with no toxic effect ofseeded fibroblasts with good cellular viability up to day 3after seeding. Mechanical assessment of fabricated constructshowed amaximum load of 97.6±11.4 N for silk fibroin/P3HBand 110.5±6.6 N for silk fibroin/PCL with no statisticaldifference in between. Authors, however, did not furtherinvestigate the effect of cellular functions or matrix produc-tion or the effect of cell seeding on associated mechanicalproperties.

Others have focused on the modification of silk toproduce scaffolds for tendon regeneration [42]. Degummedsilk fibroin meshes were integrated with electrospun alignedsilk fibroin cores [42]. In vitro assessment with rabbit MSCsinvestigated the effects of aligned fabrication techniques,compared to random fabrication, under static and dynamicculture conditions. Results showed that the effects ofmechan-ical stimulation on MSCs was intensified during culture

on aligned silk fibroin scaffolds. The dynamic effect wasapplied in both translational and rotational movement tomimic in vivo environment. This enhanced cellular pro-liferation and remodelling, with an overall improvementin mechanical properties. Apart from the material used,presence of the aligned scaffold core was proven to beessential for these positive effects when compared to thesame scaffolds without alignment. Several authors haveshowed a similar effect when topography was introducedto the surface of a polymeric scaffold with improvement incellular alignment and collagen content as well as the expres-sion of different tendon-related extracellular matrix proteins[43, 50].

Elsewhere, collagen has been combined with variouspolymers through a range of manufacturing techniques tosimulate the heterogeneous nature of tendon tissues. Col-lagen type I was electrospun with synthetic poly(L-lactide-co-caprolactone) at a 10:90 ratio, respectively [47]. Fibreswere twisted into nanoyarn to produce 150 𝜇mthick scaffolds.Scaffolds aligned randomly or in nanofibers were tested forporosity, surface morphology, and adhesion of tenocytes.Results showed improved cell proliferation in nanoyarnscaffolds, but with poor mechanical properties not useful forfuture clinical applicability.

In a follow-up study, tendon-derived stem cells (TDSCs)were harvested from rabbit patellar tendons and seeded ontothe fibrous scaffolds [48]. In vitro and in vivo assessmentunder static and dynamic conditions revealed that the com-posite scaffolds seeded with TDSCs were a promising meansfor neotendon formation. Furthermore, mechanical dynamicstimulation of the cell-seeded constructs could significantlypromote tendon regeneration compared to static cultureconditions.

Sensini A. et al. have investigated an electrospun bun-dled scaffold containing PLA and collagen type I [51]. Thisscaffold had sufficient mechanical properties with blendscontaining PLA/collagen 75:25 reaching aYoung’smodulus of98.6±12.4MPa as spun bundles and 205.1±73.0MPa after 14days of immersion inPBS.Maximumstress was 14.2±0.7 MPaas spun bundles and 6.8±0.6MPa after 14 days in PBSimmersion. Tenocytes were metabolically active with goodcellular alignment more on blends containing PLA/collagen50:50 ratio.

Collagen type I has been integrated also with other com-posite synthetic polymers blends. Polycaprolactone (PCL)/collagen and poly L-lactide (PLLA)/collagen were evalu-ated as potential composite materials for tendon-musclejunction tissue engineering [52]. Triphasic scaffolds werefabricated with regions primarily composed of PCL/collagenin one part, PLLA/collagen on the other part, and a mix-ture of both composites in the middle. All constructs hadgood biodegradability and biocompatibility. PCL supportedmyoblast growth due to its low stiffness profile and thehigher stiffness of PLLA encouraged fibroblast prolifera-tion. Scaffolds were manufactured using electrospinningtechnique combined with glutaraldehyde cross-linking andcollagen to increase mechanical strength and cell attach-ment, respectively. In vitro, scaffolds presented good cellintegration, viability, and formation of myotubes as those


found in tendon-muscle junctions in vivo. Aligned col-lagen scaffolds were produced with surface cover havingpolydioxanone (PDS) nanoplates [32]. Assessment in rabbitAchilles tendon demonstrated the same biocompatibility asthe previously tested electrospun collagen scaffolds. Furtherdetailed assessment of the composite with and without PDSshowed significant improvement in water uptake and release.Histological evaluation showed good tenocyte alignment,neotendon formation, and an initial increase in the inflam-matory cell response. The addition of PDS sheets resultedin decreased peritendinous adhesions with higher numbersof mature tenocytes and increased collagen fibril alignment.Improved mechanical properties of the construct were alsoobserved compared to scaffolds made of collagen fibres only[32, 50].

PLLA was also utilised to fabricate a composite scaffoldin which electrospinning process was used to form analigned nanofibres that was later on cross-linked to chitosan-collagen hydrogel mimicking the extracellular matrix ofnative tendons [53]. The scaffold was rolled and coatedon the outer surface with alginate gel aiming to producean antiadhesion layer around the construct. The scaffoldsupported cellular alignment and proliferation of tenocyteswith no toxic effect. Additionally, adsorption tests showedsignificantly less attached proteins on the coated surfacecompared to the noncoated one. Mechanical assessmentof produced scaffold showed no effect of coating processon tensile strength of produced scaffold with an effect onthe layer number having a value around 2MPa for 2-layercoated and uncoated scaffolds whereas it was around 6MPafor uncoated and 4MPa for coated 3-layer scaffolds. Thedegradation profile of the scaffold was also investigated andshowed that scaffolds maintained 50% of their substance at21 days after incubation with PBS containing 104 units/mllysozyme solution.

E.C. Green et al. [54] investigated the fabrication ofcollagen-I/nanocarbon fibres composite scaffold for potentialtendon tissue engineering application. Fibres were madethrough gel-spinning process with the use of a filling load of0.5 and 5wt%.Thiswas followed by fibre elongation at a strainrate of 0.02mm/s and subsequent glutaraldehyde (GA) cross-linking. Material characterization showed a yielded fibreconstruct similar to native tendon collagen with enhancedmechanical properties.

3.5. Similarities and Dissimilarities between Tendons andLigaments. Tendons and ligaments have similar structureswith different fundamental properties and functions. Ten-dons are fibrous inelastic structures that connect muscles tobones within joints while ligaments are fibrous but flexiblestructures important for supporting bone and cartilage. On astructural level, both connective tissues are dense with vari-able cellular and proteomic elements. Mechanical analysis ofhuman tendons and ligaments showed that the maximumtensile strength ranges from 4.4 to 660MPa depending ondifferent locations [55, 56]. The maximum strain of theseconnective tissues was shown to range between 18 and 30%.Young’smoduluswas shown to range between 0.2 and 1.5 GPa[57, 58]. Structurally, tendons are predominantly composed

of a collagen type I matrix containing tenocytes andtenoblasts. In contrast, ligaments contain glycosaminoglycanand lower levels of collagen compared to tendon tissue,with fibroblasts being the main cellular element (Figure 2)[59, 60]. Kharaz Y. et al. compared the extracellular matrixcomposition of both, natural and tissue engineered, tendonsand ligaments [61]. Results showed that, although tissueengineered constructs share the same composition with thenative tissue in variable proportions, fundamental differencesexist. Specific proteins, such as asporin and tenomodulin,were limited to tendons, while versican, proteoglycan 4,and SOD3 were ligament-specific (Figure 1). Identifyingdifferences in structural protein expression indicates thattissue engineering approaches should aim to replicate thesedistinctions at the proteomic level. To date, this has not alwaysbeen the case, and the terms tendon and ligament are oftenused interchangeably in the literature. This is predominantlybecause the two connective tissues exhibit similar functionaland mechanical properties [12]. An important concept toremember is that cell source is fundamental in dictatingthe type of the matrix produced [61]. This highlights theintrinsic cell memory that is different between tendon andligament. In order to tissue engineer these specific tissues,it is important to consider their functional differences sothat biomimicry can be achieved. This will eventually helpin enhancing integration and function to be restored whenused to replace injured tendon or ligaments. From this, it isclear that although the two tissues share several features, adifference exist.

4. Conclusion and Future Directions

This review summarises the current strategies based oncomposite biomaterials for tendon and ligaments tissueengineering. This approach represents a way to address theheterogeneous nature shared between tendon and ligaments.Although both structures share similar mechanical prop-erties, their constituting cell-types and extracellular matrixcompositions are different. Currently, for tendon tissue engi-neering, several challenges need to be addressed. First, thenecessity for a single standard evaluation identifying themechanical requirements for successful tendon regenerationis lacking. Crucially, this should incorporate various forms ofmechanical assessment (including strain and rotation) withdifferent tissue engineering approaches to mimic the naturalenvironment. This is difficult to achieve and implementbecause of the heterogeneous nature of tendons. The secondchallenge is to address and control the response of thesurrounding tissue to the implanted scaffold. To date, this hasbeen limited to observation of the intrinsic healing responseduring in vivo applications. Additionally, most of the studiesinvestigating the use of composite materials for tendon andligament tissue engineering utilise small animals like rabbitsrather than larger models like sheep or horses [62]. Suchlimitations are based on significant weight and mechanical-related difference between in vivo models and native humantissues. Furthermore, the ability to fix the produced constructin situ (with suture, anchors) and shelf life availability shouldbe considered whenever a model is being tested. In fact


TendonLigament

Ligaments act to connect bones together around joints while tendons act to connect muscles to bones

Cell type Tenocytes (Fibroblast like cells).Fibroblasts.

Native ECM

EngineeredconstructsECM

MechanicalProperties

-Less collagen content. -Glycosaminoglycan. -More water content.

-Asporin.-Tenomodulin.

-Abundant collagen content. -Proteoglycan.-Elastin.

- Versican.- Proteoglycan 4. - SOD3.

-Maximum tensile strength ranges from 4.4 up to 660 MPa for both tissue types. -Maximum strain of both structures ranges between 18 and 30%.-Estimated Young’s modulus ranges between 0.2 and 1.5 GPa for both.

Figure 2: Comparison between natural and tissue engineered tendon and ligament constructs. Unique cellular and extracellular matrixcomposition that is different between the two types of tissues is important for future clinical translation of tendon and ligament research.SOD3, superoxide dismutase;MPa, mega-Pascal; GPa, giga-Pascal.

the interface scaffold-tendon or scaffold-bone is the placeat risk of rupture after implantation rather the scaffoldsitself.

The described challenges constitute a potential issue forfuture clinical application of tissue engineered tendon andligament solutions. Further research is required on the rolesof composite materials in order to mimic the appropriatestructural, mechanical, and functional characteristics of ten-don and ligaments. While synthetic materials are being usedcurrently in clinical practice for tendon and ligament repairs,it will be essential to mechanically match their propertiesto native tissue. To mimic the mechanical properties oftendon is particularly important as it would allow fasterphysiotherapy and therefor reduce scar tissue formationwhich can evolve to articular stiffness. Moreover, this willenhance integration and healing at injury site. The use ofcertain growth factors such as basic fibroblast growth factor(bFGF) [45] can also be employed during surgery to enhancetissue integration. However this requires a strict control andregulated environment. Since advanced composite materialscan be tailored to match different mechanical properties ofnative tissue, they can be incorporated with growth factorsand employed with and without cells, providing large numberof possibilities for their clinical use based on site and extentof injuries.

Conflicts of Interest

None of the authors have any commercial associations orfinancial relationships that would create a conflicts of interestwith the work presented in this article.

Acknowledgments

This study was funded by Royal Free Charity (Award no.167275).

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advances in tendon and ligament tissue engineering ...tendon and ligament tissue prosthesis material...

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