Advances in Tendon and Ligament Tissue Engineering ...tendon and ligament tissue prosthesis material...
Transcript of Advances in Tendon and Ligament Tissue Engineering ...tendon and ligament tissue prosthesis material...
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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
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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
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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
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4 Journal of Materials
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
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Journal of Materials 5
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.
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6 Journal of Materials
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.
-
Journal of Materials 7
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.
-
8 Journal of Materials
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.
-
Journal of Materials 9
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.
-
10 Journal of Materials
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.
-
Journal of Materials 11
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.
-
12 Journal of Materials
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.
-
Journal of Materials 13
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
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14 Journal of Materials
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
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Journal of Materials 15
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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