Progress in Polymer Science 37 (2012) 1079 1104

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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: ferry.melchels@qut.edu.au (F.P.W. Melchels), marco.domingos@ipleiria.pt (M.A.N. Domingos), t2.klein@qut.edu.au (T.J. Klein),utrecht.nl (J. Malda), pbartolo@ipleiria.pt (P.J. Bartolo), dietmar.hutmacher@qut.edu.au (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