-
Progress in Polymer Science 37 (2012) 1079 1104
Contents lists available at SciVerse ScienceDirect
Progress in Polymer Science
j ourna l ho me pag e: ww w.elsev ier .com/ locate /ppolysc
i
Additive manufacturing of tissues and organs
Ferry P.W. Melchelsa,b,1, Marco A.N. Domingosc,2, Travis J.
Kleina,3,Jos Maldaa,b,4, Paulo J. Bartoloc,5, Dietmar W.
Hutmachera,d,
a Institute of Hb Department oc Centre for RaGrande, Portugd
George W Wo
a r t i c l
Article history:Received 12 MReceived in re15 November Accepted
17 November 2011Available online 8 December 2011
Keywords:Additive manuBioprintingBiofabricationHydrogelsTissue
enginee
in combination with different biomaterials can be generated. The
level of control offered bythese computer-controlled technologies
to design and fabricate tissues will accelerate ourunderstanding of
the governing factors of tissue formation and function. Moreover,
it willprovide a valuable tool to study the effect of anatomy on
graft performance. In this review,
Contents
1. The ra2. Histor3. State-
3.1. 3.2. 3.3.
Correspon60 Musk Aven
E-mail addj.malda@umc
1 Tel.: +61 72 Tel.: +3513 Tel.: +61 74 Tel.: +31 85 Tel.:
+351
0079-6700/$ doi:10.1016/j.facturing
ring
we discuss the rationale for engineering tissues and organs by
combining computer-aideddesign with additive manufacturing
technologies that encompass the simultaneous depo-sition of cells
and materials. Current strategies are presented, particularly with
respect tolimitations due to the lack of suitable polymers, and
requirements to move the currentconcepts to practical
application.
2011 Elsevier Ltd. All rights reserved.
tionale . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . 1080ical overview .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . 1081of-the-art . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10832D patterning and direct cell manipulation . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . 1083Additive manufacturing techniques . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . 1084Biomaterials . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . 10863.3.1.
Scaffold materials . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . 10863.3.2. Hydrogels . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . 10873.3.3. Scaffold-free
tissue manufacture approaches . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 1088
ding author at: Queensland University of Technology, Institute
of Health and Biomedical Innovation, Chair Regenerative
Medicine,ue, Kelvin Grove, QLD 4059, Australia. Tel.: +61 7 3138
6077; fax: +61 7 3138 6030.resses: [email protected]
(F.P.W. Melchels), [email protected] (M.A.N. Domingos),
[email protected] (T.J. Klein),utrecht.nl (J. Malda),
[email protected] (P.J. Bartolo), [email protected]
(D.W. Hutmacher).
3138 0503; fax: +61 7 3138 6030. 244 569 441; fax: +351 244 569
444.
3138 6142; fax: +61 7 3138 6030.8 755 8078; fax: +31 30
2510638.
244 569 441; fax: +351 244 569 444.
see front matter 2011 Elsevier Ltd. All rights
reserved.progpolymsci.2011.11.007ealth and Biomedical Innovation,
Queensland University of Technology, 60 Musk Avenue, Kelvin Grove,
QLD 4059, Australiaf Orthopaedics, University Medical Center
Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlandspid and
Sustainable Product Development (CDRsp), Polytechnic Institute of
Leiria, Rua de Portugal Zona Industrial, 2430-028 Marinhaalodruff
School of Mechanical Engineering, Georgia Institute of Technology,
Atlanta, GA, USA
e i n f o
ay 2011vised form2011
a b s t r a c t
Additive manufacturing techniques offer the potential to
fabricate organized tissue con-structs to repair or replace damaged
or diseased human tissues and organs. Using thesetechniques,
spatial variations of cells along multiple axes with high geometric
complexity
-
1080 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
4. Challenges and current developments . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . 10894.1. Construct design . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10894.2. Hardware . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . 10904.3. Biomaterials
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . 1093
4.3.1. Degradation properties. . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . .
4.4. . . . . . . . 4.5. . . . . . . . 4.6. . . . . . . . .
5. Futur . . . . . . . .5.1. . . . . . . . .5.2. . . . . . . .
5.3. . . . . . . . . 5.4. or testi5.5. . . . . . . .
6. Concl . . . . . . . Ackno . . . . . . . Refer
Nomenc
2PP AM BLP CAD CT DA DMD ECM FDM HA HEMA LCST MA MMP NIPAAmPEG
PPO RP SFF SLA SLS SPECT STL TEC
1. The rati
The funing is to coand/or biolneering coregeneratioport
structuor matrix (olated) is exthe suppordifferentiat4.3.2.
Mechanical properties . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . .4.3.3. Hybrid structures . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Vascularization . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .Scale-up of the
AM process . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . .Regulatory and commercialization aspects . . .
. . . . . . . . . . . . . . . . . . .
e directions. . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Modular tissue assembly . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . Convergence of techniques . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . .Automation of pre- and post-manufacturing phases . . . . . . .
. . . . . Manufacturing of tissue-like constructs for drug
discovery and/In situ additive manufacturing . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . .
usion . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.wledgements . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . .ences . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.
lature
two-photon polymerizationadditive
manufacturingbiolaserprintingcomputer-aided designcomputed
tomographydiacrylatedigital mirror deviceextracellular matrixfused
deposition modelinghyaluronic acidhydroxyethyl methacrylatelower
critical solution temperaturemethacrylatematrix
metalloproteinases
N-isopropylacrylamidepoly(ethylene glycol)poly(propylene
oxide)rapid prototypingsolid freeform fabricationstereolithography
(apparatus)selective laser sinteringsingle-photon emission
CTstandard tessellation languagetissue-engineered construct
onale
damental concept underlying tissue engineer-mbine a scaffold or
matrix, with living cells,ogically active molecules to form a
tissue engi-nstruct (TEC) to promote the repair and/orn of tissues.
The scaffold (a cellular solid sup-re comprising an interconnected
pore network)ften a hydrogel in which cells can be encapsu-
pected to perform various functions, includingt of cell
colonization, migration, growth andion. Further, for their design
physicochemical
properties, be consideare of impoindividual pscaffold or
chemistry, of the consue formatautomated create scaffThese
havenologies, soaccording tufacturing scaffolds
wicomputer-atechniquesTogether wfor these tewith tunablphysical
proarea.
The lastactivity andsue engineresulted frolatable fromand under
efolds have osteochondstrategies ttissue enginthe bodys s
Neverthfrom both adeveloped sarily inferand efcienincreases inof
pre-fabdependent, . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .1093. . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . 1094
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . 1095. . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . 1095. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . 1096
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . 1098 . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . .1099
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . 1099. . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . 1099. . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . 1099ng . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . 1100. . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . 1100. . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . 1100. . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1100. .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . 1100
morphology and degradation kinetics need tored. External size
and shape of the constructrtance, particularly if it is customized
for anatient [1]. Besides the physical properties of a
matrix material (e.g. stiffness, strength, surfacedegradation
kinetics), the micro-architecturestructs is of great importance for
the tis-ion process [2]. In recent years, a number offabrication
methods have been employed toolds with well-dened architectures
[3,4,180].
been classied as rapid prototyping (RP) tech-lid freeform
fabrication (SFF) techniques, oro the latest ASTM standards,
additive man-(AM) techniques [5]. With AM techniques,th precise
geometries can be prepared [6], usingided design combined with
medical imaging
to make anatomically shaped implants [7].ith the development of
biomaterials suitablechniques, the automated fabrication of
scaffoldse, reproducible and mathematically predictableperties has
become a fast-developing research
few years have seen an upturn in economic successful application
of newly developed tis-ering products, which for the largest part
has
m identication of products that are trans-
bench to bedside with available technologyxisting regulatory
guidelines [8]. Cell-free scaf-shown clinical success, e.g. for
bone (Fig. 1),ral tissue repair, cartilage and skin [9]. Also,o
create new vasculature a critical aspect ofeering are being
developed by making use ofelf-healing capacity [10].eless,
cell-based therapeutics have largely failed
clinical and nancial perspective [12,13]. Thetissue engineering
products were not neces-ior to previous alternatives, but the
efcacycy were not sufcient to justify the associated
costs [14,15]. Manual cell seeding and culturingricated
scaffolds is time-consuming, user-
semi-efcient and, therefore, economically and
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1081
Fig. 1. ExampScaffold designCT images shoReproduced w
logistically an economicurrent tissof pre-fabri
mimic the upscale to address th
The usethese pointcontrolled cell/materia(Fig. 2). Thetissue
fabriby the spaextracellulature of a soearly stage
experimentapproach ations on thnot. Automable and rep[18]. Furthle
of cell-free clinical application of tissue engineering: calvarial
reconstructioed from medical CT imaging data and fabricated by
fused deposition modeling Bwing beginning bony consolidation of the
defect after 6 months.ith permission from (2011) Georg Thieme
Verlag KG [11].
not feasible to achieve clinical application atcal scale
[16,17]. Particular shortcomings of theue engineering paradigm
involving cell seedingcated scaffolds are the inabilities to:
cellular organization of natural tissues; (economically
feasible) clinical application;e issue of vascularization.
of additive tissue manufacturing addressess by the incorporation
of cells into a computer-fabrication process, thus creating livingl
constructs rather than cell-free scaffolds
fundamental premise of computer-controlledcation is that tissue
formation can be directedtial placement of cells themselves (and
theirr matrix), rather than by the spatial architec-lid support
structure alone. Although still at anof concept development and
proof-of-principles, it appears that endeavors following thisre the
most promising to deliver clinical solu-e longer term where
cell-free approaches can-ated tissue assembly opens up a route to
scal-roducible mass production of tissue precursorsermore,
implementing good manufacturing
practices (facilitated b
The aimcuss currenapplicationto the lack othe current
2. Historic
In the clproduced emass produthan tailor-ual labor, yor
requiremcal picture to create oaided
desigstandardizetechnologygroups, themostly outsprojects sudecade
fromn using polycaprolactone-calcium phosphate scaffolds. A..
Calvarial defect C. Defect after implantation of scaffold D.
GMP), quality control and legislation arey the use of automated
processes.
of this comprehensive review article is to dis-t strategies of
AM-related tissue engineerings, particularly with respect to
limitations duef suitable polymers and requirements, to move
concepts to practical application.
al overview
assical picture of manufacturing, objects can beither
tailor-made on a one-by-one basis, or byction. Mass-produced goods
are much cheapermade products that usually involve skilled man-et
leave little room for customer specicationsents. With the advent of
AM, this classi-
has started to change. AM enables engineersbjects from
personalized specic computer-ns, while employing automated
processes andd materials as building blocks. Currently, AM
is still quite expensive for the personal userrefore, the
fabrication of self-designed objects isourced to companies, but
with fast-developingch as Fab@Home [19] it is realistic that in
a
now many households will have their personal
-
1082 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 2. Schem g of an omanufacturing ed in gemuscle cells in ct.
D. Im
AM equipmers, this wiobjects in a
With resindividual nbility to incThe combinis what msuch
persocells can beterning tectopographybe designedlatory
micrmicro-enviIn two dimeand physicacharacterizof knowledpatterned
sbeen showncell biologyassays [23]structure foby a numbretain
manculture in-blose many otured as a mlong knowncultures, wat
surface
is onlprove
of cono niching a
time liechniqatic elucidating the principle of additive tissue
manufacturing. A. Imagin of scaffolding structure (biodegradable
thermoplastic) and cells suspend
gel mimicking their native ECM. C. Manufactured 3D neo-tissue
constru
ent. As a 3D analogue to inkjet and laser print-ll allow users
to fabricate personally designedn inexpensive and automated
manner.pect to medical implants, patients might haveeeds, based on
specic anatomy or the possi-
lude autologous cells to enhance the treatment.ation of
automation and exibility in design
boneto imlevelin vivretain
A ing takes AM very suitable for the generation ofnalized
implants and devices. The behavior of
directed by tailoring their environment. Pat-hnologies can
control surface chemistry and
at scales smaller than a single cell. They can to mimic the
natural surroundings and regu-
o-environments of cells in vivo, or to modify theronment to
study the cellular response [2022].nsions, one has more control
over the chemicall properties on a small scale, and imaging
andation are simpler. Although a signicant bodyge on cell behavior
has been accumulated usingurfaces, two-dimensional (2D) techniques
have
to be insufcient for some new challenges of and biochemistry, as
well as in pharmaceutical. The importance of a three-dimensional
(3D)r in vitro experiments has been demonstrateder of studies [24].
For example, hepatocytesy of their liver-specic functions for weeks
inetween two layers of collagen gel, whereas theyf these functions
within a few days when cul-onolayer on the same gel [25]. Also, it
has been
that chondrocytes retain their phenotype in 3Dhereas they
dedifferentiate when cultured ons [26]. The vascularization of
tissue-engineered
of TECs is gevant breakbeen listed/ing with celfor nearly hwas
inventto manufacdates back of printingsimple homications tohas
since twell-underand biomatbeen develotion proces2004),
steredispensing modeling aor bioplottiAM directly[45].
Furtheillustrated tion in 2009(www.biofargan to obtain 3D digital
blueprint. B. Concurrent additivels: pre-adipocytes in
adipose-mimetic ECM gel and smoothplantation after mastectomy.
y possible in 3D [27]. The current challenge is 3D tissue
manufacture techniques to a highertrol at higher accuracies, aiming
to recreate thee with automated fabrication methods whileclinically
relevant production rate.ne starting from the invention of the rst
print-ues up to the current state-of-the art in AM
raphically illustrated in Fig. 3 (additional rel-throughs in
science and technology have alsoincluded). Although automated
processes deal-ls, peptides and biomaterials have been aroundalf a
century (e.g. the rst automated cell sortered already in 1965), the
rst reported attemptsture biological constructs including living
cellsless than a decade. Pioneering work in this kind
was done in the Boland laboratory, using aeofce desktop printer
with only minor mod-
deposit cells and proteins [28]. Inkjet printinghen been studied
and developed to a quitestood process capable of patterning viable
cellserials [29]. A number of AM techniques haveped or modied to
include cells in the fabrica-s, among which biolaserprinting
[31,39] (sinceolithography [3235] (since 2004) and robotic[3644]
(which is based on fused depositionnd also referred to as 3D ber
plotting (3DF)ng) (since 2005). Recently, the very rst use of
in vivo was reported (biolaserprinting, 2010)r, the exponential
growth of this new eld is
by the establishment of the journal Biofabrica- and the
International Society for Biofabricationbricationsociety.org) in
2010.
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1083
Fig. 3. His the intr
3. State-of
It shoultive tissue structures cthe designssions haveimposed
reself-supporsome of thestructs of cThese data time the
limopment.
3.1. 2D pat
In vitro 3are now emture assaysoversimpli(which do of the humity
of envirmanufacturlaserprintinaimed at thdiscuss pathave been p
Workinghas more cties on a smimaging) iscan spatialat the
micterning tecsurface by functionalizple cell-adbound sign
niqueingle cof phthe eq
usesg an e
silicortuallyferredganic ces harrounin vivto studps hav
cells.rthermalterneing dbut ste cell-lithog
whereoverhtory of additive manufacturing and its application in
tissue engineering;
-the-art
d be understood that, technologically, addi-manufacture is still
in its infancy. Hydrogelontaining viable cells have been produced,
but
have been simple and isotropic, the dimen- been limited to a few
millimeters and thequirement for mechanical properties has beenting
or handleable. Fig. 4 shows the results of
most advanced attempts to fabricate living con-ells and
hydrogels with automated processes.sets show the potential of AM,
yet at the sameitations and the embryonic stage of its devel-
terning and direct cell manipulation
D models based on engineered human tissueserging as a viable
alternative to 2D cell cul-
(which often give false predictions due to aned cell
environment) and in vivo experiments
not necessarily capture the important aspectsan condition, and
are limited in the possibil-
the uon stage with raphycurinon aof vitransor orsurfaral sucells
ed stamliving
Fusive are bcies, on thphotophy, onto onmental control).
Nevertheless, some tissue
e techniques such as inkjet printing and bio-g have emerged from
technologies that initiallye manufacturing of 2D systems. Here, we
brieyterning and cell manipulation techniques thaterformed in
2D.
in 2D has several specic advantages. Oneontrol over the chemical
and physical proper-aller scale, and characterization
(particularly
easier. Using patterning technologies, onely control surface
chemistry and topographyrometer level or even below. Most 2D
pat-hniques involve the fabrication of a patternedphotolithography,
followed by the selectiveation of the patterned surface with for
exam-hesive peptides, cell-repellent polymers oraling molecules.
Photolithography provides
ofce printmanipulatiovidual cellsin refractivtweezers)
cell-by-celltion of preccontacts.
Surface techniques direct cellthat those purpose. Hultimately
sion to be organs.oduction of technologies and major scientic
ndings.
ability to study cell-substrate interactionsells in conned
areas. A specic disadvan-otolithography is the high cost
associateduipment and cleanroom facilities. Soft lithog-
elastomeric (soft) stamps by casting andlastomer (typically
poly(dimethoxy siloxane))n master [48]. With these stamps
patterns
any compound (including proteins) can be onto most surfaces,
without the use of UVsolvents. Using this collection of
techniques,ve been designed that mimicked the natu-dings and
regulatory micro-environments ofo, and micro-environment have been
modi-y the cellular response [2022]. Elastomerice even been
employed to directly pattern
ore, technologically simpler and less expen-atives for
cleanroom-based photolithographyeveloped. These mostly have lower
accura-ill high enough to engineer an environmentsize level.
Examples are LCD-based projectionraphy [50] and transparency-based
lithogra-
masks are obtained by simply printing patternsead projector
sheets with a high-resolution
er [51]. Another technique that allows directn of cells is
laser-guided direct writing. Indi-
in suspension are guided (based on differencese indices) by
directed laser-light (opticalto be deposited onto solid surfaces
[49]. The
deposition theoretically allows the genera-ise patterns of
cells, inducing specic cellcell
patterning and direct cell manipulationhave proven to be useful
tools to study
material interactions and we concludewill remain to be applied
for this specicowever, the designed micro-environmentsneed to be
expanded into the third dimen-useful for the manufacturing of
tissues and
-
1084 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 4. Exampand hepatocytgelatin/chitosathe web
versioReproduced w
3.2. Additiv
With AMcan be consfashion, as otraditional mof tissue aensures
mimaterial, nThe use of 3tissues, whtreatment clevel of
coconstructs, up and staobject throles of bioprinted structures. A,
B, C: layer-by-layer fabrication of gelatin/alginatees in
gelatin/alginate/chitosan (white). D: fusion of printed cell
aggregates for sn hydrogel structures 1 month post-dispensing. (For
interpretation of the referen of the article.)ith permission from
(2009) Elsevier [46] and [40,47] copyright 2005, 2009. Re
e manufacturing techniques
techniques, objects from 3D model data setstructed by joining
material in a layer-by-layerpposed to a subtractive manner in which
mostanufacturing methodologies operate. In terms
nd organ manufacturing, the additive naturenimal waste of scarce
and expensive buildingamely cells, growth factors and
biomaterials.D model data enables fabrication of customizedich is a
conditio sine qua non for patient-speciconcepts. Further, AM
techniques offer a highntrol over the architecture of the
fabricatedguarantee reproducibility and enable scale-ndardization.
The rst step to produce a 3Dugh AM is the generation of the
corresponding
computer mor importenumber of ior animal bphy,
magnesingle-photimaging [53STL le, whmodels. Bematically
slreproducedSeveral wetechniquesscaffolds fo
AM techmanipulati/brinogen containing adipose-derived stem cells
(in pink)caffold-free vascular tissue engineering. E: hepatocytes
innces to color in this gure legend, the reader is referred to
printed by Permission of SAGE.
odel either by the aid of 3D CAD softwared from 3D scanners
[52]. There are a largemaging methods for data acquisition of
humanody parts, such as X-ray computed tomogra-tic resonance
imaging, ultrasound echoscopy,on gamma rays (SPECT) and
bioluminescence56]. The CAD model is then tessellated as anich is
currently the standard le for facettedfore manufacturing, the STL
model is mathe-iced into thin layers (sliced model), which are
into a physical 3D object by the AM device.ll-developed and
commercially available AM
have been employed to design and fabricater tissue engineering
applications (Table 1).nologies produce 3D parts by spatially
directedon of materials in several possible ways:
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1085
Table 1Description of four common, commercially available AM
techniques that are often employed in the preparation of tissue
engineering scaffolds.
depositare melt
the 3Dtely. Theaintaineerial to
A): Wit throughses usu
thermal, chprocesses, which it unIn chemicais xed
byMechanicalcells or mamers are mmodes are c
In generhighest restion methoand biolasemerization and
exogentechniques Melt extrusion/fusedlaments or granules by a
computer, to formand hardens immedianext layer, must be mthe
thermoplastic mat
Stereolithography (SLmulti-layer procedurepolymer. These
proces
method is a mask-based methby irradiating through a patteusing a
focused UV beam prod
Inkjet printing: The process dover the surface of a powder
bformed. A piston lowers the pover the surface of the
previourepeated until the 3D object is
Selective laser sintering (SLSto selectively heat powder mashape
of each cross-section ofAfter each layer is solidied, tnew layer of
powder is suppli
emical, mechanical and/or optical. In thermalthe material is
formed into an object afterdergoes a thermal transition to x the
shape.l-based processes, the manufactured shape
a chemical reaction (often polymerization). processes rely on
the physical deposition ofterials, and in optical processes cells
or poly-anipulated using light. Often several processingombined in
an AM technique (Table 2).al, techniques that use optics can
achieve theolutions. Examples of accurate optical fabrica-ds are
stereolithography, laser direct writingrprinting. Additionally,
photo-initiated poly-can be used for safe encapsulation of cellsous
growth factors into hydrogels. Thermalsuch as selective laser
sintering or fused
deposition requiring sube adaptedMechanicalfabrication cells
such atridge oric
Stereolitmost accurapplied forfabrication Although
itcomparablebecause to enables haand/or cellsion modeling (FDM): By
this process, thin thermoplasticed by heating and guided by a
robotic device controlled
object. The material leaves the extruder in a liquid form
previously formed layer, which is the substrate for thed at a
temperature just below the solidication point ofassure good
interlayer adhesion
h this process 3D solid objects are produced in a the selective
photo-initiated cure reaction of a
ally employ two distinct methods of irradiation. The 1st
od in which an image is transferred to a liquid polymerrned
mask. The 2nd method is a direct writing processuces polymer
structures.
eposits a stream of microparticles of a binder materialed,
joining particles together where the object is to beowder bed so
that a new layer of powder can be spreads layer and then
selectively joined to it. The process is
completely formed.
): This technique uses a laser emitting infrared
radiation,terial just beyond its melting point. The laser traces
the
the model to be built, sintering powder in a thin layer.he
piston over the model retracts to a new position and aed using a
mechanical roller.
modeling are not compatible with cells ifpra-physiological
temperatures, but they can
for processing thermosensitive hydrogels. processes often allow
for including cells in theprocess, as long as shear stresses
induced ons by deposition through a needle or inkjet car-e are
sufciently low.hography is the oldest, most developed andate of all
AM technologies, and it has been
several biomedical applications including theof TECs with
encapsulated living cells [68].
is one of the few techniques with accuracies to the size of a
cell, its use has not been favoreddate, a system has not yet been
developed thatndling of different compositions of materials.
Pioneering work on tissue manufacture has
-
1086 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104Ta
ble
2Char
acte
rist
ics
of
AM
tech
niq
ues
that
are
use
d
for
the
pre
par
atio
n
of
cell-lad
en
const
ruct
s
and
cell-fre
e
scaf
fold
s
for
tiss
ue
engi
nee
ring.
Proc
essing
mod
es
are
indic
ated
by
t for
ther
mal
pro
cess
ing,
c for
chem
ical
,m
for
mec
han
ical
and
o f
or
optica
l,
wher
e
mod
es
in
brac
kets
are
option
al.
Tech
niq
ue
Proc
essing
mod
es
Acc
ura
cy
(m
)
Mat
eria
ls
Cel
ls
Adva
nta
ges
Disad
vanta
ges
Ref
s
Inkj
et
printing
(ther
mal
orpie
zo-e
lect
ric)
t/m
, (c)
201
00
Liqu
ids,
hyd
roge
ls
Yes
Use
of
existing
chea
pte
chnol
ogy,
multip
leco
mpos
itio
ns
Low
visc
osity
pre
vents
build-u
p
in
3D, l
owst
rengt
h[2
8,57
64]
3D
printing
m, (
c)
50
Poly
mer
s,
cera
mic
s
No
Multip
le
com
pos
itio
ns
Req
uires
pow
der
, cel
l-unfrie
ndly
envi
ronm
ent
[65
67]
Ster
eolith
ogra
phy
(incl. t
wo-
phot
onpol
ymer
izat
ion)
o,
c
0.5
50
Hyd
roge
ls, p
olym
ers,
cera
mic
-com
pos
ites
Yes
Hig
h
accu
racy
Singl
e
com
pos
itio
n, r
equires
phot
o-cu
rabl
em
ater
ial
[32
35,6
870
]
Lase
r
direc
t
writing
o
20
Cel
ls
in
med
ia
Yes
Singl
e
cell
man
ipula
tion
No
stru
ctura
l suppor
t,
scal
ability
[49]
Direc
t
writing
m, c
1
Poly
elec
trol
ytes
Not
yet
Hig
h
accu
racy
Req
uires
solv
ents
, cel
l-unfrie
ndly
envi
ronm
ent,
scal
ability
[71,
72]
Mel
t
extr
usion
(includin
g
FDM
)
t,
m
100
Ther
mop
last
ics,
com
pos
ites
No
Tech
nol
ogic
ally
sim
ple
Req
uires
stro
ng
la
men
t an
d
hig
h
tem
p.
[73,
74]
Rob
otic
dispen
sing
m, (
t), (
c)
100
Hyd
roge
ls, p
olym
ers,
cera
mic
-com
pos
ites
Yes
Multip
le
com
pos
itio
ns
Rel
ativ
ely
low
accu
racy
[36
44]
Sele
ctiv
e
lase
r
sinte
ring
o,
t
50
Poly
mer
s,
cera
mic
s
No
Req
uires
pow
der
, cel
l-unfrie
ndly
envi
ronm
ent
[75,
76]
Bio
lase
rprinting
o,
t
10
Liqu
ids
Yes
Hig
h
accu
racy
at
hig
hsp
eed
Low
visc
osity
pre
vents
build-u
p
in
3D
[30,
31]
Rob
otic
asse
mbl
y
m
5
Rig
id
solids
Not
yet
No
hea
t,
ligh
t
orre
action
requ
ired
Expen
sive
mac
hin
ery
[77]
been done using inkjet and laser printing. However, overthe last
few years the focus has been mostly on the roboticdispensing of
hydrogels with encapsulated cells. With thisclass of techniques,
highly viscous cell suspensions or liq-uid gel precthrough a method
is used, in covarieties ofpumps, extnological cthe
relativebioprinterscally couldautomated robotic gripbuilding bloing
blocks cell types.
3.3. Biomat
Over therials have fabrication (natural anites [82]. Tbeen
procea few excecally for useand reprodwith the anthese matehigh
tempeconducive gaining incr[83].
3.3.1. ScaffoScaffold
from polymites). To obtechniquesing, gas foatechniquesscaffold
arcprocessed bthe oldest aa photo-curscaffolds fr(meth)acrytone)
[85], poly(ethylea diluent thposites havparticles inceramic
strstructures ing out of tceramic [89ursors are dispensed from
cartridges or syringesnozzle and deposited as strands (Fig. 2).
Theversatile in terms of materials that can bentrolling the
environmental conditions and in
dispensing mechanisms (pneumatic, syringeruder screws). The
versatility and limited tech-omplexity are perhaps the main reasons
forly wide commercial availability of dispensing. A less-developed
method that technologi-
be applied to make living constructs in anmanner is robotic
assembly. High-precisionpers can assemble pre-fabricated
microscalecks into larger structures [78], and these build-
could potentially be pre-seeded with different
erials
last two decades, several biodegradable mate-been used and
developed for the design andof scaffolds and matrices, including
polymersd synthetic) [79,80], ceramics [81] and compos-he polymeric
and ceramic materials that havessed by AM to prepare scaffolds have
all, withptions, been modied or synthesized speci-
with a single AM technique, enabling accurateucible fabrication
of well-dened architecturesticipated physicochemical properties.
However,rials typically require process parameters (e.g.,rature,
solvents, lack of water) that are not
to direct inclusion of cells. Hydrogels are thuseasing interest
for the manufacturing of tissues
ld materialss for tissue engineering are mostly prepareders,
ceramics, or their combination (compos-tain an interconnected pore
network many
have been employed including porogen leach-ming and
phase-separation/freeze-drying. AM
however offer a higher degree of control overhitecture [3], and
a range of materials can bey AM techniques (Table 2).
Stereolithography,nd most developed of AM techniques, requiresable
material. It has been employed to prepareom poly(propylene
fumarate) [84] and fromlated poly(trimethylene carbonate co
caprolac-poly(lactide) [86], polycaprolactone [87] andne glycol)
[3235], mostly in the presence ofat can be either reactive or
unreactive. Com-e been prepared by mixing in small ceramic
the stereolithography resin [88], and pureuctures were realized
by preparing compositewith high ceramic loading, followed by
burn-he polymer while simultaneously sintering the].
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1087
Selective laser sintering has been used to prepareporous
polycaprolactone (PCL) scaffolds, with or withoutadditional calcium
phosphate particles [76]. FDM-basedtissue engineering research has
revolved around thispolymer as illofacial arengineeringmodel in
shthetic and respectivelyDirect writiat much higstatic
interafabricate wall mentionscaffolds, tconditions cells or
cellHowever, reof scaffold mechanicalmated andstructures aproven
its is expectedadapted, aninclusion of
3.3.2. HydrHydroge
while remaistic three-large numbpolymer chtors that demaking
thepharmacy adesign and
As a resuenvironmengels are ussynthetic. Nitself) are gintrinsic
prited tunabilhydrogels bbiofunctionhybrid gelsare gainingmore
recennaturally deand dextranmethacrylalinking in cThe methacalso
for synto more na[92]. The indensities noalso allowsbehavior, d
modication of naturally derived hydrogels allows forcombination
of their intrinsic biofunctionality with thetunability of many
properties through these synthetic com-ponents. On the other hand,
synthetic gels are increasingly
func suchth fact-links
additas a b
that hde ble cellg the olution
denion. Th
sufcilet forrial, i.efrom ly quichape o
crossical, bthermhis croise ceanical
shaperemen
castinity can
the e
sides gel ha
tissue eeringare 3Drders octing minanletely
[93]. Hhydrogn peradation [98]-degr
ation asulatiare oof ach
size ted pr00]. Fremodydrog. Desisculariwell, leading to
clinical application in the max-ena [11] and the establishment of
bone tissue
concept based on a large long bone defecteep [27]. 3DP has been
applied to both syn-
biopolymers (polylactide [65] and starch [66],), as well as
ceramics (hydroxyapatite [67]).ng, a process similar to robotic
deposition buther resolutions achievable through to electro-ctions
and coagulation, has been employed toell-dened silk broin scaffolds
[72]. Althoughed materials are suitable for fabrication of
he toxicity of their precursors or processingoften still does
not allow the co-deposition of-laden hydrogels in the manufacturing
process.cent developments have shown a convergence
fabrication and cell deposition, combining the support of a
scaffold structure with the auto-
controlled placement of cells. These hybridre discussed in
detail in Section 4.3.3. AM hasvalue for the preparation of
scaffolds, and it
that current materials and processes will bed new ones will be
put into place to allow the
cell-laden hydrogels in the fabrication process.
ogelsls are polymeric networks that absorb waterining insoluble
and preserving their character-dimensional structure. This is
because of theer of physical or chemical links between theains.
Hydrophilicity is one of the main fac-termine the biocompatibility
of hydrogels, thusm attractive for application in medicine ands
drug and cell carriers, and specically for thefabrication of TECs
[90].lt, they can provide embedded cells with a 3Dt similar to that
in many natural tissues. Hydro-
ually classied as either naturally derived oraturally derived
gels (often derived from ECMenerally good cell support materials,
but haveoblems, such as batch-to-batch variation, lim-ity and
possibility of disease transfer. Syntheticear none of these
disadvantages, but often lackality. Besides these two classes of
hydrogels,
having both natural and synthetic components increased interest
in tissue engineering, andtly, in additive tissue manufacture. For
example,rived hydrogels such as gelatin, hyaluronic acid
have been functionalized with methacrylate ormide groups to
enable (photo-initiated) cross-ombination with robotic dispensing
[44,91].rylate chemistry that was used here and beforethetic
polymers, is versatile and can be appliedturally derived hydrogels,
including alginatetroduction of chemical cross-links at controlledt
only enables xation of printed shapes, but
tailoring of mechanical properties, swellingegradation kinetics
and so forth. The chemical
beingnentsgrowcross
Inboth CellsinclumuscDurinsor sinto agelatto bedropmatecells
ativethe sally achemby a and
tprommechcatedrequiwhenporoswhenhigh.
Behydroand enginthey are orestripredocompplaceinto
datioDegrdrivephotomigrencapcells aim meshsecregel [1tion, the
hoccurin vationalized with biologically active compo- as
cell-adhesive peptides, covalently boundors, heparan sulphate, and
protease-cleavable[93].ive tissue manufacturing, hydrogels are
useduilding material and as a cell delivery vehicle.ave been viably
encapsulated within hydrogelsroblasts, chondrocytes, hepatocytes,
smooths, adipocytes, neuronal cells and stem cells [94].AM of 3D
tissue constructs, a hydrogel precur-
with suspended cells needs to be processeded, designed shape
that is subsequently xed byerefore, the viscosity of the suspension
needsently high to overcome surface tension-drivenmation, to enable
drawing of thin strands of. create well-dened shapes, and to
prevent
settling during the fabrication process. A rel-k gelation is
subsequently required to retainf the fabricated structure. This
gelation is usu--linking reaction initiated either by light, by ay
hydrophobic or complexation interactions, oral transition. Both the
shaping of the constructss-linking reaction obviously should not
com-ll viability. Another requirement is adequate
properties to retain the designed and fabri-. Most manufacturing
processes impose stricterts on the mechanical properties of the
gels thang and molding. Large structures with included
only be accurately and reproducibly preparedlastic modulus and
gel strength are sufciently
these constraints related to manufacturing, thes to meet the
demands for cell encapsulationdevelopment. Most hydrogels used in
tissue
are chemically cross-linked, which means networks of polymer
chains with meshes thatf magnitude smaller than cells. This has a
largeeffect on the mobility of encapsulated cells;tly cell
migration, as well as proliferation is
arrested until degradation of the gel takesowever, degradation
sites can be incorporatedels, allowing for cell-mediated matrix
degra-
mitting migration and proliferation [9597].n of the matrix can
also be hydrolytically, or even light-driven through
incorporatedadable linkers [99]. Cell proliferation andre not
always essential in the initial stage afteron; in cartilage tissue
engineering, for example,ften encapsulated at high densities with
theieving high matrix production. Here, still theis important as it
inuences the diffusion ofoteins and glycosaminoglycans throughout
theor the engineering of tissues where prolifera-eling and
vascularization are required (Fig. 2),el should allow space for
these processes togned macroporosity in the construct can
aidzation, as demonstrated by branched vascular
-
1088 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 5. A. Proc alent caand calcium c indow fsodium algina of
velodelity of the Reproduced w 8/1758
networks btissue [46].
A particing using calong with both accurateria imposof
form-stacross-link dand proliferto be low. Fprintable bnate, has
oboth printiing windowdened former conceninuence othe
bioprinexample inof the printmicrosyringTwo procesmicroposititinct
hydroand the dsemi-quanthigh delitycally investthe depositseveral
yeademonstratlar AM systparametersquantitativ
Most atfar have utthan AM (Tmers speciexplored tolimitations
ow. O for AMhermoispens
whichugh m
the gedie wlves inerminainker fvalen
introds of cullows ical cr
grouposed te) A-bs of a cks no(LCST)y derivoto-p
lity.essing window for bioprinting of alginate hydrogels
cross-linked by divoncentrations. Cell culture imposes maximum
values, leaving a small wte/calcium at two distinct alginate
concentrations, showing the inuencenal shape.ith permission from
(2009) ASME [102] and (2009) IOP [103] (doi:10.108
ecoming an integral part of a manufactured
ular challenge in additive tissue manufactur-ell-laden hydrogels
is to develop a polymerprocessing conditions that are appropriate
forte printing and cell culture. Often, these cri-e opposing
requirements. For accurate printingble structures, high polymer
concentrations andensities are desired, whereas for cell
migrationation and subsequent ECM formation both needor example, a
currently used naturally derivediopolymer, namely
calcium-cross-linked algi-nly a small processing window of in
whichng and cell culture are possible: the bioprint-
(Fig. 5A). This bioprinting window can be other hydrogel systems
by varying the poly-tration and cross-link density and assessing
then printability and support for cell culture. Oftenting window
will be small, if at all present. The
Fig. 5B shows a semi-quantitative assessmentability of alginate
gels with a pressure-assistede, in the form of a delity phase
diagram.sing parameters are varied, the velocity of theoners and
the extrusion pressure, at two dis-gel precursor viscosities (or
concentrations),
windmadeThe tfor dture,althocess,cells dissothe ttide lfor
cobeenweekthat achemsamecomplactablockA-bloture partlfor
phstabielity of the resulting structure is assessed on aitative
scale ranging from a blob structure to a
structure. The same group has also systemati-igated the effect
of shear stress endured duringion on cell viability and function
[101]. Afterrs of predominantly proof-of-principle studiesing the
(bio)printability of a gel with a particu-em, researchers are
increasingly optimizing gel
and processing conditions in systematic ande ways.tempts of
additive tissue manufacturing soilized hydrogels designed for
purposes otherable 3). However, the development of poly-cally for
AM of cell-laden constructs has been
a limited extent, and may help overcome theof current gels and
expand the bioprinting
Photo-crgelation halated dextrhyaluronic stability duity
enablesxed subseand spacingfor LCST-gehydrogels tincrease th
3.3.3. ScaffoA relativ
endeavor tblocks to lcium ions. Printing imposes minimum values
for alginateor bioprinting. B. 3D phase diagram of microfabrication
ofcity of the micropositioners and extrusion pressure on the
-5082/1/4/045002).
ne of the few examples of a hybrid gel tailor- is based on a
PEGPPOPEG block copolymer.
sensitive block copolymer conveniently allowsing a cell
suspension at ambient tempera-
solidies upon collecting at 37 C. However,ost cells remain
viable during the plotting pro-l does not support cell viability in
culture; allithin a few days, while the thermogel slowlyto the
culture media [42]. By functionalizingl hydroxyl units of PEGPPOPEG
with a pep-ollowed by a methacrylate group, a mechanismt
cross-linking, as well as biodegradability haveuced, resulting in
increased viability over 3lture [104]. A similar approach of a
synthetic gelfor both thermal gelation as well as
UV-initiatedoss-linking was recently demonstrated by the
[36]. The polymer is an ABA block copolymerof
poly(N-(2-hydroxypropyl)methacrylamidelocks and hydrophilic
poly(ethylene glycol) B-molecular weight of 10 kDa. The
hydrophobict only induce lower critical solution tempera--behavior
employed for printing, but are alsoatized with methacrylate groups
that allowsolymerization for increased strength and
shapeoss-linkable gels that do not exhibit thermalve also been
printed. In one example, methacry-an was mixed with high-molecular
weightacid to obtain high viscosity for geometricalring printing
[44]. Although the high viscos-
printing of a porous structure that can bequently by
photo-cross-linking, the diameter
of printed strands are considerably larger thanls. It is
expected that development of moreailored for specic AM techniques
will greatlye potential of AM.
ld-free tissue manufacture approachesely new trend in tissue
manufacturing is theo use cells or aggregates of cells as
buildingmanufacture tissue engineering constructs
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1089
Table 3Hydrogels used for additive manufacturing of cell-laden
tissue engineering constructs.
Hydrogel Technique Viability Proliferation Refs
NaturalCollagen Gelatin Matrigel Agarose Alginate
SyntheticPEGDA PEGPPOPEPPOPEGAPEGHPMAm
HybridHA-SH + PEGGelatin-MA Hyaluronan d)
a PEG-(PPO) ps.b PEG-(N-(2
without adaggregates interactionsdevelopmegeous to dirof
suspende(also referrshould be uof this appmolding tefrom cell aof
using highas also bSupercial after alginabiomaterialwith
differe
AnotherimplantableCells are cudish to forded in theiby a
reduchydrophiliccells can beby trypsin. evolved to types, and So
far, cell-cessfully fothe cornea,heart infarcis needed trobotics
couduction procell sheets, and stackindardizationa substantia
ities wiabilitcent rned ctegy tll sheeombinhieve gels ont
litelay ananufa
allen
onstru
digital to pring teetic rDispensing 86% Disp. + aldehyde
X-linking 98% Dispensing 99% Dispensing 93%Dispensing 94%Dispensing
91%
Stereolithography 65% G Dispensing 84%
la-MAma Disp. + UV X-linking 75% Lab Disp. + UV X-linking 94%
(1d)
85% (3d)
-4A Gel rod deposition 100%+ HA-MA Disp. + UV X-linking 100% +
Dextran-HEMA Dispensing + UV X-linking 94% (1d) 75% (3
2 blockcopolymer functionalized with alanine-methacrylamide
end-grou-hydroxypropyl)methacrylamide lactate)2 blockcopolymer.
ditional biomaterials. The rationale is thatof cells can fuse
through cellcell and cellECM
to form larger structures, similar to embryonicnt [106]. As
cellcell contact can be advanta-ect tissue formation, it is
believed that insteadd single cells, aggregates of thousands of
cellsed to as tissue spheroids or embryoid bodies)sed for tissue
manufacture. An elegant exampleroach (although still using agarose
rods as amplate) is the preparation of vascular graftsggregates
(Fig. 4D [46]). The benecial effecth densities of cells and their
associated ECM
een demonstrated for cartilage repair [107].and middle zone
chondrocytes recoveredte culture were layered without additionals,
resulting in continuous cell-derived tissuesnt properties in each
layer.
strategy that aims to engineer material-free tissue is the so
called cell-sheet technology.ltured on a thermo-responsive
polymer-coatedm a self-supporting sheet of cells embed-
denscell v
Repattea straof cethe cto achydrocurrewill psue m
4. Ch
4.1. C
A mentimagmagnr self-produced ECM, which can be harvestedtion in
temperature that renders the surface
and hence cell-repellent [108]. In this way, harvested without
destroying cellcell contactsOver the last decade, cell-sheet
technology hasengineer several tissues with one or more cellit has
recently seen clinical applications [109].sheet technology has only
been applied suc-r the regeneration of sheet-like tissues, such
as
and as cardiomyocyte patches to repair partialts. A next step in
technological developmento create thick 3D tissue structures.
Potentially,ld be employed to automate the cell-sheet pro-cess and
to assemble 3D structures by stackingas the handling steps for
cell-sheet harvestingg are fairly simple with high level of stan-.
Obviously, many sheets are needed to buildl 3D tissue volume and
the resulting high cell
anatomical[111,112] o3D laser scimages of bof tailored in breast
cametrical blheterogenecompositioMost AM ta constructTissues howent
ECM cothe osteochoped to modwith multipods will nenature of n30% in
24 h [38]None (3 months) [41]None (2 weeks) [42]None (2 weeks)
[42]N/A [37]None (2 weeks) [42]
N/A [34]>95% cell death in 3 days [42]None; after 3 days 60%
viable, up to 3 wks [104]N/A [36]
1050% in 4 days [105]Doubling in 7 days [91]N/A [44]
ill require sufcient vascularization to sustainy.technological
development includes micro-o-culture of broblasts and endothelial
cells aso generate pre-vascularized tissue from stacksts [110].
Other potential approaches includeation of cell sheets with
dispensing techniques,a third dimension by deposition of
structurednto and in-between cell sheets. Either way, therature
predicts that the cell-sheet technology
increasingly important role in the additive tis-cture in the
future.
ges and current developments
ct design
blueprint of an organ or tissue is a rst require-oduce an
anatomically accurate TEC. Medicalchniques such as computed
tomography andesonance imaging have been used to make
ly shaped implants using intermediate moldsr by direct
manufacturing [113]. More recently,anning was introduced to obtain
digital 3Dody contours, for example for the preparationbreast
prostheses implanted after mastectomyncer patients [114]. The
obtained digital geo-ueprint needs to be converted to a
buildable,ous model representation describing materialn,
distribution and geometrical information.echniques use only one
material for building, and only geometrical information is
needed.ever are heterogeneous, comprising of differ-
mponents, cell types and cell densities, such asondral tissue
(Fig. 6). Methods have been devel-el and design functionally graded
architecturesle biomaterials for AM [115117]. These meth-ed to be
applied to approximate the complexative heterogeneous tissues in
manufactured
-
1090 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 6. Examp erencesin the native t ial/hydrcell
type.Reproduced w ert [119
cell-materimajor advacess really baccording t
The stantion to AMTessellationtriangles thThis works(which is
uparts) that control sofporosity, fothat is usedtechniquesduced
that regions of regions wit
Howeveintegral paran impractiwith well-dangles, takirequiring
hlate. Howevnite volumeematical eqshapes, porfeatures sumore versaa
porosity fscopic shapufacturing table. Until tbe restrictesmall
struc
A new cal imagingExisting meanatomicalfor studyin[123]. In
thinterconnecsolid mode
iven dd by
of tetr(ST) arhese stertighus mode and/os to enlity. Thng
thehedron
thicknnstratanninan be roject
Hardw
itially,t and
proce3D strk (30 des thlso aper direately oilding lude thle of
functional graded construct design for osteochondral tissue. The
diffissue are reected in the design for the manufacturing process
by mater
ith permission from (2009) Future Medicine [118] (2009) Mary Ann
Lieb
al constructs. Only in this way can one of thentages of
including cells in the fabrication pro-e exploited, by deposition
of different cell typeso the tissue blueprint.dard le format to
feed geometrical informa-
control softwares is the STL format (Standard Language). The
format makes use of meshes ofat create watertight outer surfaces of
objects.
well for solid objects with limited complexitysually the case
for rapid prototyping of solidare to be built from a single
material. Some AMtwares give the user a degree of control overr
example by controlling the lament distance
to create the tool path for deposition-based. A novel modeling
approach was recently intro-automatically creates a tool path that
lls seta solid STL model, enabling to create distincth variable
porosity [121].r, if the internal pore architecture is to be ant of
the computer-aided design, the STL formatcal one. An STL mesh of a
few mm-sized scaffoldened porosity easily exceeds one million
tri-ng up hundreds of megabytes of disk space andeavy computation
power to design and manipu-er, the pore architecture of constructs
with in-s can be described using a single line of math-uation, with
freedom to design different poree sizes and porosity, and allowing
to includech as porosity and pore size gradients [69]. Atile le
format that would allow combining suchunction with a mesh that
describes the macro-
at a gnectemeshness and ta
waporotissustrutstabitrollitetrastrutdemo3D scties cthe p
4.2.
Ininkjea 2Dcate the inimpetion ato lasultimof buconce of an
organ would make designing and man-issue and organ constructs much
more achiev-hen, computer designs of porous structures willd to
either a coarse porosity for large models, ortures in the case of
ner, well-dened porosity.route to create porous models from
medi--derived data was recently developed [122].thods were adapted
that convert CT-derived
data into a volumetric mesh that can be used e.g.g biomechanics
using nite element modelingis case, the mesh is used to create a
completelyted strut-based porous model. In practice, thel obtained
by imaging is seeded with points
Existingfacilitate ticontrol of humidity, afeeders, etcOver the
lafor tissue mable, with a[124]. Dispmethod to speeds. Theogy
compoLiquid prec in tissue composition, mechanical properties and
cell typeogel composition, construct architecture and
encapsulated
] and (2009) Wiley [120].
istance (seeding distance SD), which are con-the nite element
software to result in a 3Dahedrons. Subsequently, struts of a given
thick-e designed around each edge of all tetrahedrons,truts are
joined at their intersections to createt model. Using this method,
one can generateels that have the overall shape of the scannedr
organ, built up from fully connected straight
sure manufacturability and optimal mechanicale pore size and
porosity can be tailored by con-
density of seeding points in the creation of the mesh, as well
as by choosing an appropriateess (Fig. 7). The example given in
this reviewes how from a solid breast model obtained byg, a range
of scaffold morphologies and porosi-designed and fabricated to the
requirements ofobjectives and aims.
are
tissue manufacture has focused on the use oflaser printers.
However, printing is inherentlyss. Inkjet printers are not designed
to fabri-uctures. The upper threshold for viscosity ofmPa s)
excludes the use of many hydrogels ande build-up of large 3D
structures. This limita-plies to biolaserprinting in its current
form, andct writing. To construct functional tissues andrgans,
techniques are required that are capable
structures at relevant scales and accuracies. Weat AM techniques
possess this capability. AM devices are currently being modied
tossue manufacturing [180]. This often entailsthe environmental
properties (temperature,nd sterility) and downscaling of
containers,., to reduce loss of costly biomaterials and cells.st
few years, AM devices designed particularlyanufacture have become
commercially avail-n emphasis on robotic dispensing
techniquesensing is a technologically straightforwardcreate
designed structures at relatively high
largest challenge for the dispensing technol-nent is to build
tissues with high accuracy.ursors need to be dispensed in thin
strands from
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1091
Fig. 7. Genera ta of somodels with v (SD) anmodels
manufReproduced w 14).
small-diamout initiallylayer (a videsupplemenpensed,
thiconcentratiIn this wayfrom lameencapsulatimer concenstructures
tor larger.
Light-barate than dand micromgeometry oprepared [1been
emplRGD-peptidtion with atechnique, scale-rangemicron featwith high
vcompared tto create hi(Fig. 8.), fab[127]. The w[128] wouldtoo
small tohigh level o
al tech at a r
were aolithogant sizting personalized scaffolds for breast
reconstruction. Top-row: CAD-daarying pore size and porosity as a
result of different seeding distancesactured by fused deposition
modeling.ith permission from (2011) IOP [103]
(doi:10.1088/1758-5082/3/3/0341
eter tips and solidify quickly before spreading on the platform
and later on the subsequento of dispensing hydrogels is available
online as
tary information). When only materials are dis-s can be achieved
by employing high polymer
opticparedthat stererelevons and a non-solvent for quick
coagulation., well-dened structures have been preparednts of only 1
m diameter [71]. However, forng cells non-solvents cannot be used
and poly-trations must be lower, so cell-laden hydrogelypically
have strands with diameters of 100 m
sed curing techniques are generally more accu-ispensing
techniques. With photolithographyolding, cell-laden microgels with
well-denedf up to several hundreds of m have been25,126].
Two-photon polymerization (2PP) hasoyed to locally functionalize
hydrogels withe sequences, leading to directed cell migra-ccuracy
below 100 m. As a light-directed AMstereolithography can be
performed at a large; from decimeter-sized objects down to sub-ures
can be built. Such high accuracy, combinedersatility and freedom of
design (particularlyo dispensing techniques) results in the
abilityghly detailed organic shapes, such as the alveoliricated by
2PP-based microstereolithographyoodpile structure in the bottom row
of Fig. 8
not be functional as a scaffold for the pores are facilitate
cell ingrowth, but does illustrate thef geometric control that can
be achieved with
at such resoIn the au
come for ligand gravitaOne of the hydrogel stphy
fabricathowever, cpeptides cothroughoutof channelsculturing
cecation of a sof PEGdiasulated celsettling to layer of celally
added phomogeneotiple gel compossible usicase the celcan easily
e
Anotherphoto-patteprinted on lid model and porous skeleton-mesh.
Middle row: CADd strut thicknesses (ST). Bottom row: physical
prototype
niques. Well-dened structures have been pre-esolution of several
tens of m from hydrogelslso used for cell encapsulation using the
sameraphy setup, although complex and clinicallyed hydrogel
structures with encapsulated cells
lutions still await to be reported.thors opinion, the largest
challenges to over-ht-based techniques are long fabrication
times,tional settling of cells in the precursor solution.rst
reports on stereolithographic fabrication of
ructures in 2005 argued that the stereolithogra-ion process was
too slow for cell encapsulation;ontrolled spatial distribution of
cell-adhesiveuld lead to control cell seeding and diffusivity
the scaffold, which in addition to the presence would be
superior to traditional seeding andlls on scaffolds [129]. More
recently, a modi-tereolithography apparatus for the fabrication
crylate-based hydrogel structures with encap-ls was reported
(Fig. 9) [70]. To prevent cellsthe bottom of the tank due to
gravity, eachl-containing prepolymer solution was manu-rior to
curing of that layer. Besides achieving aus cell distribution, this
also allows to use mul-positions and cell types, which is not
generally
ng the stereolithography technique [68]. In thisl suspension is
still dispensed manually, but onenvision automation of this
step.
approach for partially automated layeredrning of cell-laden
hydrogels uses masksa commercial high-resolution printer [130].
A
-
1092 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 8. Two eximage of a fabReproduced w
UV curing PEGDA cefor each sin-betweenlogical charsystem
(ampeptides) aactivity of hhydrogels a
Besides cross-linkinpossible. Fogravity can ating a micsettle.
Othe(chemical) rather thantern could one laser, samples of
structures prepared by two-photon polymerization (2PP)
techniquesricated alveolus. C, D. Woodpile structure resembling an
FDM-fabricated scaffoldith permission from (2007) Wiley [127] and
(2011) Springer [128].
unit was employed with the masks to cure all suspension in a
chamber that was replacedubsequent layer, with washes and relling.
The researchers performed an extensive bio-acterization including
optimization of the gelong which type and concentration of
adhesivend demonstration of the increased metabolicepatocytes
encapsulated in perfused patterneds compared to bulk
hydrogels.layer-by-layer deposition just prior to photo-g, other
solution paths to cell settling arer example, by continuous
tumbling of the setup,be counteracted by centrifugational forces,
cre-ro-gravity environment in which cells do notrwise, a physical
gel could be employed as thehydrogel precursor in which cells do
not settle,
using a liquid solution. In this case, a 3D pat-be cross-linked
by moving the focal volume ofeveral lasers creating an interference
pattern,
or by 2PP. Across-linkereversing thof gelatin-mbased gel)
astructured
Even if an importahigher resospecic volraphy. Howincrease
promination bthe surfacetus are equprojection increasing f
A currethat aims a. A. CAD image of a pulmonary alveolar
fragment. B. SEM, albeit at about 100 smaller scale.
fter cross-linking of the 3D structure, the non-d volume
including cells could be removed bye physical gelation (for
example, warming upethacrylate or ion exchange for an alginate-
nd recovered for later use, leaving a porous andhydrogel with
encapsulated cells.settling of cells is prevented, speed still isnt
processing parameter. When working atlution it generally takes
longer to build-up aume, and this is also the case for
stereolithog-ever, new technologies are being developed toduction
speed. For example, as opposed to illu-
y a computer-controlled laser tip drawing over in most
conventional SLAs, some new appara-ipped with a digital mirror
device that enablesof a whole layer at once, thereby
signicantlyabrication speed [35,68].nt development in
stereolithographic AMt high-throughput manufacturing of
accurate
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1093
Fig. 9. A schematic representation of the bottomup SLA
modication,in which the prepolymer solution is pipetted into the
container one layerat a time from the bottom to the top
[70].Reproduced by permission of The Royal Society of
Chemistry.
multi-material parts by a new process named
stereo-thermal-lithography [131]. It employs UV radiation
andthermal energy (produced by IR radiation) simultaneouslyto
initiate the cross-linking polymerization reaction in amedium
containing both photo- and thermal initiators.The amount of each
initiator is low enough not to startpolymerization by only one of
these two effects. However,at a point where the two effects
coincide, the amountof radicals generated is sufciently high to
initiate the
polymerization process. Temperature is used to bothproduce
radicals through the fragmentation of thermalinitiators and
simultaneously to increase the initiationand reaction rate of the
photo-initiated curing reaction.Added to this system is a rotating
multi-vat that enablesthe fabrication of multi-material structures
(Fig. 10).
4.3. Biomaterials
For application in additive tissue manufacture, bioma-terials
must meet more stringent requirements than formost other
applications such as in food, pharmaceutics orsensors.
Nevertheless, some innovations from other eldsmight possibly be
translated to AM techniques and cellencapsulation, using
alternative components and process-ing conditions. This section
gives an overview of suchdevelopments.
4.3.1. Degradation propertiesPolymer network chains give
hydrogels their mechani-
cal stability, but at the same time restrict the mobility
forcells to migrate and proliferate. Therefore, it is importantto
match the kinetics of degradation with rstly the cellmigration and
proliferation and subsequently tissue forma-tion, such that the
newly deposited ECM can take over theload to a certain extent from
the partially degraded poly-mer network. Moreover, the rate of
tissue formation and
Fig. 10. The sIR (heat radiamulti-materiaReproduced
wtereo-thermal-lithographic process with multi-vat system. Liquid
resins are soltion) source, both patterned using
computer-controlled digital mirror devices. Tl constructs.ith
permission from (2011) Springer [131].idied locally by
co-illumination from a UV (light) and anhe rotating multi-vat
system enables the construction of
-
1094 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 11. Schem olymer nization mecha
remodelingvarious tissthose in vit
By far mcell encapsbioinert poit is a non-are low enokidneys
areable macrofrom aqueoladen hydrgels can betration
andco-monomponent. Furlead to diffdation prostep-growtall allow
ceically functor tetheredrespects. Tlarly attracreaction
anstereolithogreactions atrol over thenabled theGPQG IWGby
cell-secrThese MMPlently bounmigrating aface of thegel that
waslinker. Latetion of such
can p of thing thhis sthallenormatiss and
far, techndiacryting idegradeeringforeme, tetherwill al
as thern in AMatic representation of the initial monomer
molecules and cross-linked pnism (B) end-linking mechanism and (C)
mixed-mode mechanism.
depends on many factors and is different forues. Hence, it is of
utmost importance to studyro and/or in vivo mimetics in great
detail [4].ost developments on degradable hydrogels forulation have
been based on the water-soluble,lymer poly(ethylene glycol) (PEG)
[93]. In itselfdegradable polymer, but PEG oligomers thatugh in
molecular weight to be secreted by the
often the basis for the synthesis of degrad-mers [132]. These
can be (photo)polymerizedus solutions with suspended cells, to form
cell-ogels [133]. The degradation kinetics of these
tuned by variation of the polymer concen- molecular weight, the
choice of degradableer and the ratio of PEG to the degradable
com-thermore, different cross-linking mechanismserent network
structures with varying degra-les (Fig. 11). Addition type chain
cross-linking,h end-linking and mixed-mode mechanisms
cells partsretaingel. Tthe csue fproce
SoAM PEGresulnon-enginthe alinkssites suchcatioll encapsulation
and the inclusion of biolog-ional entities such as cell-adhesive
peptides
growth factors [134], but differ in otherhe chain-cross-linking
mechanism is particu-tive for AM techniques because of the fastd
spatially directed initiation by light such as inraphy. However,
end-linking polymerizationre characterized by a particularly large
con-e network architecture. For example, it has
preparation of gels with the peptide link GCRD-Q-DRCG, which is
cleavable at the siteeted matrix metalloproteinases (MMPs)
[96].-cleavable gels (also supplemented with cova-d cell-adhesive
peptides) showed ingression ofnd proliferating broblasts seeded on
the sur-
gels, which was not observed in the control cross-linked with an
MMP-insensitive peptider studies have also shown the possible
applica-
gels for cell encapsulation [135]. In this way,
4.3.2. MechA speci
mechanicalbearing tissappropriatehydrogels isulation. Ththe main
limto larger sc
Hydrogetent of waactive chaious ways tincreasing sity;
howevfunction [9matrix comoutside of bgels with noetworks formed
through (A) chain-cross-linking polymer-
roliferate, migrate and form new tissue whilee gel that are free
of cells remain untouched,e overall shape and mechanical stability
of therategy of cell-mediated degradation mitigatesge of tuning
hydrolytic degradation with tis-on, which is a very site- and
condition-specic
therefore difcult to predict.fabrication of cell-laden PEG
structures byiques have mostly employed off-the-shelflates in
conjunction with RGDPEGacrylate,n gels that support cell viability
but areable and therefore of limited use in tissue. However, it can
be easily envisioned thatntioned strategies of introducing
hydrolysableed grow factors and enzyme-sensitive cleavageso be used
with alternative hydrogel platformsmosensitive PEGPPOPEG, allowing
the appli-
technologies.anical propertiesc disadvantage of hydrogels is
that their
strength is mostly far below that of load-ues, such as
cartilage. This fact has not beenly addressed by researchers
working withn tissue engineering, particularly for cell encap-e
lack of strong and tough hydrogels is one ofiting factors in
advancing tissue manufacture
ales and better quality of TECs.ls are intrinsically weak due to
the high con-ter, which dilutes the network of elasticallyns and
reduces physical entanglement. Obvi-o increase a gels modulus and
strength arethe polymer concentration and cross-link den-er this is
often detrimental for cell viability and5], and for the production
and distribution ofponents [100]. Recent developments in
areasiomedical engineering have resulted in hydro-vel chemical
structures that have considerably
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1095
improved mechanical properties due to the introductionof an
energy-dissipating mechanism, thereby increasingtoughness and
(tear) strength while still containing highwater volume fractions
[136]. These include slide-ring gels,double-netthe latter
twcompressivwater-contgels with re17 MPa at awhile the w
Relativein solutionfollowed bpenetratingdensity butAs a result
solution, thproduct whin much strinterpenetrunder inveswith
promidouble-netmanufacturweight monreinforcemstrategy is ized
macroor with othesuch as phytides [140].
Nanocomexhibiting tional hydrhydrophilicadsorption inorganic
(being consifunctionaliterties, incluat break anpression)
usynthesizedof N-isoproture with clreplacemeninitiation, gand
processtures. Poly(cell cultureof cell sheetion 3.3.2) [monomer
NHowever, mfully been pof 20 kg/motowards theadditive tis
Besides other appr
synthesize strong hydrogels for a variety of applications,some
of which may be translatable to tissue manufacture.The interested
reader is referred to the review by Calvert[144] for a more
comprehensive overview.
Hybrnce w
majolls intoach. H
of hyroteinanufac, ceramable med locasuccese cons
and canicaloved a
of natf multining , sincerial, a
and ceposititions fmatrixdiffereembr
solutptide uter-csuspeo a celg) imm
ascula
spiteional teering
as wite intolls exi
the nes and ajor c
centur of vieranspo
a tissgrafts , due
he hypth fact
a commigrature vwork gels and nanocomposite gels. Particularlyo
classes of gels show very high toughness and
e strengths, while retaining exibility and highent. Gong et al.
have prepared double-networkmarkably high compressive strengths of
up to
similarly remarkable strain at failure of 92%,ater-content was
as high as 90% [137].ly densely cross-linked networks were swollens
of a second monomer and cross-linker,y formation of the reinforcing
second, inter-
network, which has a relatively low cross-link higher
concentration than the rst network.of the high degree of swelling
in the monomere rst gel network is highly extended in the nalile
the second network is relaxed, which resultsonger reinforcing
effects than in conventionalating networks. These networks are
currentlytigation for use as articial articular cartilage
sing initial results [138]. In the translation of thework
strategy to cell encapsulation and tissueing, the major problem is
that low-molecular-omers are generally cytotoxic [139].
However,
ent of gels with encapsulated cells using thismore feasible
using double-bond functional-monomers of intermediate molecular
weights,r non-cytotoxic network-forming components
sically cross-linked gels or self-assembling pep-
posite gels are another class of hydrogelsmechanical properties
superior to conven-ogels. These are water-swollen networks of
polymers, physically cross-linked throughof the polymer chain
ends on nanometer-sizedclay) platelets. The nature of the
cross-linksderably large planar sheets with high junctiony somehow
yields unusual mechanical prop-ding very good toughness, high
elongationd ultimate stress (both in tension and com-p to several
MPa [141]. Generally, they are
through the in situ free-radical polymerizationpylacrylamide
(NIPAAm) in an aqueous mix-ay particles. Recent developments
included thet of the redox initiating system with photo-reatly
increasing the usefulness for patterninging with AM techniques to
form designed struc-NIPAAm) nanocomposite gels can be used for
on the gel surface and subsequent detachmentts without the use
of proteases (see also Sec-142], but the in situ polymerization of
the toxicIPAAm does not allow for cell encapsulation.ost recently
nanocomposite gels have success-repared from four-armed PEG
macromonomersl molecular weight [143], which is a big step
applicability of this gel reinforcing strategy insue
manufacture.double-networks and nanocomposite gels,oaches have been
followed attempting to
4.3.3.Si
and aof ceapproposedand pco-mmersfavordenwas
viablbersmechimprthoseuse ocontamorematetionsthe dcondnew
A the mwhenof peCompwith lead tlinkin
4.4. V
Infunctenginsues,diffusAs cefromptosithe m21st pointing
tFromthat tationand tgrowfromtion, immaid structuresater is a
crucial component of living systemsr component in most tissues, the
processing
designed hydrogel structures seems a logicalowever, most
organisms are not only com-drated cell-rich tissues, but also of
more dry-rich ECM such as bone and tendon. Therefore,turing of
solid biodegradable material (poly-ics) with cell-laden hydrogels
could combineechanical properties with cells positioned attions at
high densities. Recently, this approachsfully applied for the
generation of organizedtructs by alternate deposition of
thermoplasticell-laden hydrogels (Fig. 12) [145]. The resulting
properties of the constructs were signicantlynd could be
tailored within the same range asive tissues. Moreover, the
approach allows theiple hydrogels, and can thus build
constructsmultiple cell types or bioactive factors. Further-
the hydrogel is supported by the thermoplastic broader range of
hydrogel types, concentra-ross-link densities can be used compared
toion of hydrogels alone, thereby improving theor encapsulated
cells to proliferate and deposit.nt possible approach is by taking
advantage ofane-forming self-assembly process that occursions of
hyaluronic acid and particular typesamphiphiles are brought into
contact [146].ontrolled deposition of one of the componentsnded
cells into the second component wouldl suspension (potentially
gelled by photo-cross-obilized in membrane-surrounded strands.
rization
of considerable attempts in bioengineeringissues and organs,
most applications of tissue
have been restricted to avascular or thin tis-hout blood
vessels, nutrients and oxygen cannot
and out of TECs to retain cellular viability.sting more than a
few hundred microns awayarest capillaries would undergo hypoxia,
apo-ultimately cell death, vascularization is one ofhallenges
tissue engineers are faced with in they. Particularly with the
demand from a clinicalw to fabricate large TECs in which overcom-rt
limitations becomes increasingly difcult.ue transplant point of
view, it is well knowcan spontaneously vascularize after
implan-
to an inammatory wound-healing responseoxia-induced endogenous
release of angiogenicors [152]. The process of angiogenesis
followsplex cascade of events including ECs activa-
tion, and proliferation, their arrangement intoessels, addition
of mural cells (pericytes and
-
1096 F.P.W. Melchels et al. / Progress in Polymer Science 37
(2012) 1079 1104
Fig. 12. Co-m . Schemalternating ste ucts. B. connement o ent
LiveReproduced w 01).
SMCs), andThe molecuare being dfactors act theless, thiprovide
suftransplanteapeutic angmacrovascuoxygen in 3
Vasculartion (e.g., grfrom surrouformation fby a numbbiological
ptissue devewhere ungulogical effedeformed
vpatterning,photolithogform gradieco-deposit processes tAM
techniq
Several are being dof all, the mblood vessenough intscaffolds
getortuosity tniques suclevels of psmaller po
nels avesigno prelds fabrticulaead to
withop thntatioiquesgiogenuced ee pre
(endoation, anufacturing of solid biodegradable materials with
cell-laden hydrogels. Aps of printing polymer and cell-laden
hydrogel, yielding hybrid constrf the printed hydrogels. C. High
cell viability as demonstrated by uorescith permission from (2011)
IOP [145] (doi:10.1088/1758-5082/3/2/0210
matrix deposition as the vessels mature [147].lar mechanisms
regulating each of these stagesescribed, and it is obvious that
different growthat distinct steps of neovascularization. Never-s
induced vessel ingrowth is often too slow tocient nutrients to the
cells in the center of thed tissue. Conclusively, the limiting step
is ther-iogenesis, and both microvascularization andlarization are
required to provide nutrients andD.ization with or without
biochemical stimula-owth factor delivery), either through
ingrowthnding tissues or through de novo blood vessel
rom co-deposited cells is currently investigateder of research
groups [148151]. Mimicking
chanfold din vivscaffoor pahas lporesAV loimplatechnof anprodfor
thtypesmigratterning may be especially useful to controllopment
processes such as neovascularization,ided or uncontrolled growth
can lead to patho-cts including tumor growth, metastasis,
andessels. Techniques developed for microarray
microcontact printing, micromolding and laserraphy can be
translated to AM of tissues tonts of growth factors within the
scaolds or tocells. These are highly architecture-dependenthat can
benet from the specic advantages ofues.strategies for
vascularization at different levelseveloped [152], as illustrated
in Fig. 13. Firsticro-architecture of any scaffold must allow
el ingrowth, thus a pore network with largeerconnections is a
prerequisite. AM-producednerally have better interconnectivity and
lowerhan scaffolds fabricated by conventional tech-h as porogen
leaching. In addition, differentorosity can be designed to allow
cells to llres with new tissue while leaving large pore
likely impr(Fig. 13D).
4.5. Scale-u
An additlab-based aapplicationfrom the mto the centdecimeter
svolume, nuby a factor transport lquantities obtain
sufdifferentiatare mostly asks. Thenot only lhigh reproatic
overview of a hybrid bioprinting process encompassingLayering of
the dye-containing alginate results in specic/Dead assay. Scale
bars represent 2 mm.
ailable for vascularization (Fig. 13A). Such scaf-s would also
be benecial in combination withvascularization strategies; the use
of tortuousricated by thermally induced phase-separationte leaching
with arteriovenous loops in vivo
the formation of vascularized tissue but in small
interconnections too distant from thee tissue was prone to necrosis
in the longern time points [154] (Fig. 13B). Furthermore, AM
can aid vascularization by site-specic deliveryic factors,
possibly released on demand by cell-nzymes (Fig. 13C). Finally, AM
can be employedcise co-deposition of gels with relevant
cellsthelial cells, myoblasts, broblasts) to guidingproliferation
and network formation. This will
ove and accelerate in vitro prevascularization
p of the AM process
ional challenge in the development of currentttempts of tissue
manufacture towards clinical
is the gradual scale-up of the process. Goingillimeter scale
that current work is focused on,imeter scale of tissues or
eventually thecale of organs implies an increase in materialmbers
of cells and possibly construction timeof 103 or 106, respectively.
With such scale-up,imitations, as well as acquiring adequate
cellbecome increasingly difcult. Currently, tocient numbers of
cells, populations (eithered cells harvested from a patient or stem
cells)expanded in 2D monolayer using tissue culture
manual seeding, splitting and harvesting isabor-intensive and
expensive but also lacksducibility, and most importantly it is
also
-
F.P.W. Melchels et al. / Progress in Polymer Science 37 (2012)
1079 1104 1097
Fig. 13. The massociated vasa scaffold withadded macro-artery
and a ve8 weeks after the loop. Reprmembrane. Parelease, leadin(d)
In vitro prebroblasts (Mnetwork anastd-iii and captithe reader is
r
insufcientfor manufatissue manuthe concurrsystems [23riers [157]
ohowever aphas been eand commebioreactor-
Furthermwith cells, achieved minside hydof cells perost direct
approach to providing the necessary cues and allowing cells and
tissueculature is direct fabrication of functioning tissue.
Different strategies for improvin
meso-scale porosity (250 m) in which