REVIEW

------[14] [18] 1601118 Bio [18,20,21] Both of Both of [8–11] [21,24] (1 of 29) Therefore, in Therefore, in [9,19] When designing www.advhealthmat.de However, most 3D most 3D However, [4–7] [10,12,13] wileyonlinelibrary.com Presently, tissue engineering approaches tissue engineering Presently, Although significant successes have been Although significant successes have been achieved in engineered tissues, both in is research and clinical applications, it require organs complex 3D that obvious more precise multicellular structures with vascular and neural network integra mimetic design of the scaffolds, including scaffolds, mimetic design of the 3D structural characteristics and physical properties, can substantially enhance the physiological performance through cell–matrixappropriate cell–cell and further enhancing biolog interactions, ical functions. have been widely studied in cartilage, bone,have been widely studied and nerve regenera skin, vascular tissue tion, among others. scaffolds currently fabricated with tradi scaffolds tional techniques lack these qualities. a tissue engineered scaffold, the combi scaffold, a tissue engineered biological and engi nation of material, neering requirements must be consideredneering requirements manner. in an application-specific 3D printing of bioactive scaffolds contains contains 3D printing of bioactive scaffolds

[15–17] - [20,22,23] 3D printing is a rapid prototyping and additive manufac 3D printing is a rapid prototyping and the regeneration field, it can provide an excellent alternative the regeneration field, it can provide an excellent alternative by accurately positioning fabrication for biomimetic scaffold multiple cell types and biofactors simultaneously into complex that better represent the structural multi-scale architectures and biochemical complexity of living tissues or organs. This automated, additive process facilitates the manufac This automated, additive process facilitates architecture turing of 3D products having precisely controlled and interconnectivity) (external shape, internal pore geometry, with highly reproducibility and repeatability. In the past three decades, 3D bioprinting has been widely In the past three decades, 3D bioprinting has been widely developed to directly or indirectly fabricate 3D cell scaffolds medicine. of regenerative the field or medical implants for very precise spatiotemporal control on placement of It offers cells, proteins, DNA, drugs, growth factors, and other bioactive substances to better guide tissue formation for patient-specific therapy. tion. These requirements cannot be fulfilled using traditional traditional using fulfilled be cannot requirements These tion. methods. turing technique used to fabricate complex architecture with with architecture complex fabricate to used technique turing building process. high precision through a layer-by-layer two types of scaffold fabrication: acellular functional scaf two types of scaffold folds which incorporate biological components, and cell-laden constructs aiming to replicate native analogues. them aim to produce biocompatible, implantable constructs them aim to produce biocompatible, implantable constructs for tissue/organ regeneration, thus we refer to 3D printing in fabrication as “bioprinting”. the context of bioactive scaffold In this regard, the term bioprinting does not indicate whether

- [2] In the [3] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © Organ dysfunction or failure or dysfunction Organ [2–4] [1] , 1601118 6 , 2017

Adv. Healthcare Mater. Adv. www.advancedsciencenews.com DOI: 10.1002/adhm.201601118 is drastically increasing due to traumatic injury and disease. is drastically increasing due to traumatic Often, clinical treatments are limited by a paucity of avail clinical treatments Often, able donors and immune rejection of donated tissue. able donors and immune rejection of donated 1. Introduction by the organs are highly complex structures formed Human tissue types. The combined, functional organization of multiple together group and specialized highly are organs these in cells functions. distinctive perform to search for alternatives to conventional treatment strategies for the repair or replacement of missing or malfunctioning human tissues and organs, tissue engineering approaches are being explored as a promising solution. Regenerative medicine holds the promise of engineering functional tissues functional tissues holds the promise of engineering Regenerative medicine offering replace abnormal and necrotic tissues/organs, or organs to heal or needs. gap between organ shortage and transplantation hope for filling the bio (3D) bioprinting is evolving into an unparalleled Three-dimensional Haitao Cui, Margaret Nowicki, John P. Fisher, and Lijie Grace Zhang* and Lijie Grace P. Fisher, Nowicki, John Cui, Margaret Haitao 3D Bioprinting for Organ Regeneration for Organ 3D Bioprinting for patient- due to its high-integration potential manufacturing technology with high and rapid manufacturing capabilities specific designs, precise precise control over It enables versatility. resolution, and unprecedented and architectural accuracy/ multiple compositions, spatial distributions, microstructure, therefore achieving effective recapitulation of complexity, biological functions of target architecture, mechanical properties, and overview of recent advances in tissues and organs. Here we provide an as well as design concepts of bioinks suitable 3D bioprinting technology, applications of this technology focus on the for the bioprinting process. We more specifically on vasculature, for engineering living organs, focusing conclude with current challenges We neural networks, the heart and liver. development of 3D organ and the technical perspective for further bioprinting. Dr. H. Cui, M. Nowicki, Prof. L. G. Zhang Dr. Department of Mechanical and Aerospace Engineering Department of Biomedical Engineering Department of Medicine University The George Washington 3590 Science and Engineering Hall DC 20052, USA Washington, 800 22nd Street NW, E-mail: [email protected] Prof. J. P. Fisher Fischell Department of Bioengineering University of Maryland 3238 Jeong H. Kim Engineering Building Park, MD 20742, USA College REVIEW 1601118 www.advhealthmat.de printing techniques. raphy; thisprovedtobethepioneeringworkforfuture3D photopolymer-based manufacturingtechnologyofstereolithog specific functionsof3Dbioprintedorgananalogues. and neuralnetworkintegration,eventuallydevelopingthe egies forprintingmultiplelivingcells,includingvasculature 3D bioprintingfororganregenerationinvolvesadditionalstrat based ontraditional2Dinkjettechnologywasproposed. provided guidancefor3Dprintingtechniquesandproducts. Considerations forAdditive Manufactured Devices”, which Administration (FDA)issueddraft guidance,titled “Technical and architecturalinformation for tissues. are alsousedtocollectanddigitizethecomplextomographic aided manufacturing(CAM)toolsandmathematicalmodeling imaging (MRI). Computer-aided design (CAD) and computer- including computedtomography(CT)andmagneticresonance struct. organism levels,aidingthedesignofapatient-specificcon 3D structureandfunctionatthecellular, tissue,organand technology isanindispensabletooltoprovideinformationon zation oftheircomponents. comprehensive understandingofthecompositionandorgani heterogeneous architectureoffunctionaltissuesororgansisa tion process. cells aredirectlyprintedorinvolvedatanystageofthefabrica medical, analytical,andfoodapplications. $1.82 billionandwillincludeproducts andmaterialsfordental, By 2022,theglobal3Dbioprintingmarketisexpectedtoreach Vessel Bioprinter (Revotek,Sichuan, China)amongothers. Materials (SouthWindsor, CT, USA),andCommercialBlood (EnvisionTEC, Gladbeck,Germany), OxfordPerformance printer (Organovo, Inc., San Diego, CA, USA), 3D Bioplotter Hewlett-Packard (Palo Alto,CA,USA),NovogenMMXBio bioprinting market,suchas3DSystems(RockHill, SC,USA), and morebusinesseshavebeenestablishedintheexpanding Currently, with the increasing global interest and need, more cial 3Dbioprinters. 2009, OrganovoandInvetechcreatedoneofthefirstcommer organ shortageandsavinglives. engineering towardorganfabrication, ultimatelymitigating technology appearstoshowgreat promiseforadvancingtissue its infancyconsideringthecomplexity andfunctionality, this ties, andbiochemicalmicroenvironments. highly dynamicyetvariable3Dstructures,mechanicalproper or organ-specificmicroenvironmentbymimickingthenatural, addition, thistechniquehasthecapacitytobuilda3Dtissue- are crucialelementsofthewholeorganarchitecture. and incorporatemechanicalaswellbiochemicalcuesthat cation) canrecreatemorecomplex3Dorganlevelstructures and organs.Thisintegratedtechnique(imaging-design-fabri printing components drives the construction of the 3D tissues ical factorsarethenselected, and theconfiguration of these biomaterials (syntheticornatural),andsupportingbiochem techniques, the cell types (differentiated or undifferentiated), by-layer deposition.Consideringtheavailable3Dbioprinting that areimportedintoa3Dbioprintersystemforthelayer- tissue or organ model is divided into 2D horizontal slices Charles W. Hull, in1986receivedapatentfortheliquid, An essentialrequirementforreproducingthecomplex, (2 of29) [20,25] Itcommonlyoffers noninvasiveimagingmodality, [30,31] [28] wileyonlinelibrary.com In2003,acellularbioprintingtechnique Finally in2016,theFood andDrug [10,11] Therefore,medicalimaging [31] [25] [27] © 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim Althoughstillin The3Dimaged Inthismanner, [20,26] [29] In In [31] [32] ------

mental researchandtranslational medicine( technologies towardcreatingrealistic organsthatfurtherfunda rent challenges and exciting opportunities of 3D bioprinting printing fororganregeneration. Furthermore, wediscusscur plex tissue/organ, and alsopresent recent advances in3Dbio organs. We proposeastepwiseprocessofregenerating acom 3D bioprintingtechnologiesforgeneratingtissuesand and otheressentialelementspertainingtotheapplication of metastasis. stem cellengineering,drugdeliveryandbreastcancerbone bioprinting, complextissueengineering,nanobiomaterials, Engineering atGW. Herresearch interestsinclude3D/4D Bioengineering LaboratoryforNanomedicineandTissue In thisreview, wefocusongeneralprinciples,techniques, Adv. Healthcare Mater. She isthedirectorof and HarvardMedicalSchool. training atRiceUniversity and didherpostdoctoral Brown Universityin2009 Biomedical Engineeringat She obtainedherPh.D.in George Washington University. Aerospace Engineeringatthe Department ofMechanicaland associate professorinthe Lijie GraceZhang and nanobiomaterials. complex tissueengineering, include 3D/4Dbioprinting, Zhang. Herresearchinterests supervision ofDr. LijieGrace Applied Sciencesunderthe School ofEngineeringand George Washington University Mechanical EngineeringatThe completing herPhDin Margaret Nowicki regeneration. and complextissue/organ biomaterials, 3Dbioprinting, on thedevelopmentofnovel His researchcurrentlyfocuses Zhang’s groupinMarch2015. Sciences. HejoinedtheProf. Chemistry, ChineseAcademyof Changchun InstituteofApplied of polymersin2015from Ph.D. inchemistryandphysics Haitao Cui www.advancedsciencenews.com receivedhis Figure is an an is

iscurrently 2017

1 , ). 6

, 1601118 - - - - REVIEW

------[34] 1601118 Variations Variations (3 of 29) [20,33] www.advhealthmat.de Several additional manu Without the consideration Without [18] [35,36] Copyright 2015, the American Association Association American the 2015, Copyright wileyonlinelibrary.com [139] Finally, post-bioprinting, which involves the development post-bioprinting, Finally, 3D bioprinting for tissue engineering applications can be 3D bioprinting for tissue engineering of cell viability or bioactive components, several 3D printing techniques with higher temperatures, chemicals and other harsh environments can be utilized to manufacture implants. facturing techniques, including substrate supports and sacrifi cial templates, among others, are potentially required to create higher mechanical elasticity/strengths, more precise structures, functions due biological or multiple more complex structures Considering the specific requirements of the targeted tissues/ capa the account into take must design the properties, organs further modification before being imported into the printer. into the printer. further modification before being imported object into a stack of The slicing program can parse the solid 2D cross section is thin, axial cross sections; each respective as programmed. reproduced integrating various infill patterns, and deposits succes In this step, the printer reads the stl file build to materials other several or powder, liquid, of layers sive Several 3D the 3D model from a series of 2D cross-sections. multiple nozzles (mul printing techniques are capable of using multiple printing tiple materials), adjustable angles, and even combinations. incorporated living divided into two forms, with and without bioprinting Cellular cells printed directly into the constructs. viable cells to form techniques can directly deposit bioinks with strategies, they a 3D living structure. Based on the working droplet-based, can primarily be classified into three categories, bioprinting. extrusion-based and laser-assisted bilities and properties of the bioprinting systems (both bioinks and bioprinters), which we discuss next in detail. of biomimetic structures, mechanical supports and biological step to develop mature tissues/ is an essential functionality, organs for living applications. teristics of living tissue/organ constructs. Comparatively, acel teristics of living tissue/organ constructs. Comparatively, in the available bioprinting technologies also affect the charac in the available bioprinting technologies also affect for choices more extensive provide bioprinting techniques lular tissue regeneration applications.

- - - - - [20] The desired [25] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © , 1601118 6 , 2017

permission. with Reproduced review. this in covered information outlining Schematic diagram The 3D structure with patient-specific design is then printed Pre-bioprinting, also known as modeling, mainly includes Adv. Healthcare Mater. Adv. www.advancedsciencenews.com Figure 1. Figure for the Advancement of Science. structure of digital 3D models is precisely created using CAD and stored as a stereolithography (stl) files. Bioink or software biomaterial selection depends on the specific bioprinter type and the product properties required. deposition modeling process in the bio in layer-by-layer printing phase. According to the program design of different design of different to the program printing phase. According the loaded into files can be directly printers, the 3D design or must first be passed through a slicing program for printer, prehensive process requiring various design considerations, prehensive process requiring various design choice, bioink selec including imaging, modeling, printer development among tion, culture condition, and 3D construct manufacturing activities can be divided the others. Generally, into three steps: pre-bioprinting (modeling), bioprinting, and post-bioprinitng. 3D imaging acquisition, digital 3D design and bioink/bioma model. bioprinting 3D type of on the based selection terial 3D bioprinting is basically a rapid prototyping and additive 3D bioprinting is basically a rapid prototyping artificial implants manufacturing technique used to fabricate building through a layer-by-layer or complex tissue constructs shares three 3D bioprinting therapy. patient-specific process for with ordinary 2D printing – desktop printer basic concepts consisting (bioink ink model file), (3D printer), print file (3D and cells), and paper of biomaterials, bioactive components bioprinting is a com (print platform). Unlike 2D printing, 3D 2.1. Fundamental Principles 2.1. Fundamental 2. 3D Tissue/Organ Bioprinting and Related 2. 3D Tissue/Organ Bioprinting and Manufacturing Strategies Several imaging technologies, such as 3D scanner, CT, MRI CT, Several imaging technologies, such as 3D scanner, and others, are applied to collect and digitize the complex tomo graphic and architectural information of tissues. Giesel et al. graphic and architectural information of tissues. Giesel described and discussed the various methods of 3D imaging technology for 3D printing applications in detail. REVIEW 1601118 www.advhealthmat.de structs intofunctionaltissues/organs. and enhanceconstructmaturationtherebytransformingcon tion, oreveninsitubioprintingwillbeperformedtoinduce in vitroculture(preferenceabioreactor),vivoimplanta substitute, whilethecellularimplant often requiresanextrastep implant servesasanonliving implant deviceorartificialgraft forms, directimplantationand cellpost-seeding.Theacellular and printingmodality. Acellular bioprintingisdividedintotwo niques morespecificallyrefers toboththebioinkformulations to currentprintingtechniquelimitation. organ regeneration. Figure 2. osition. fusion, binderjetting,sheetlamination,anddirectedenergydep lymerization, materialjetting,extrusion,powderbed technologies consists of several parts including vat photopo Typically, ASTM(F2792) standardterminologyfor3Dprinting 2.2. AccustomedBioprintingTechniques simplified illustrationsoftypical3Dbioprintingtechniques. tree-diagram ofthevarious3Dbioprintingtechniqueswith (4 of29) [37] (a) A tree-diagram of the various 3D bioprinting techniques and (b) Simplified illustrations of typical 3D bioprinting techniques for tissue/ To someextent, theterminologyofbioprintingtech wileyonlinelibrary.com [26] [16]

© Moreimportantly, Figure 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim

2 showsa - - - - - and stereolithography. be categorizedintothreetypes: droplet-based,extrusion-based, nism), therepresentativetechniques ofcellularbioprintingcan Depending ontheprintingmodality (bioinkdepositionmecha logical materials,resolution,printingspeedandcellviability. limitations) intermsoftheavailableconditionssuchasbio logues, andeachofthemhasdifferent features(strengthsand have beendevelopedtocreate3Dlivingtissue/organana of 3Dprinting-basedrapidprototyping.Diversetechniques struct fabricationprocesstogetherwiththeinherentadvantages Cellular 3D bioprinting directly employs living cells in the con 2.2.1. Cellular Bioprinting the constructfabricationandcellularizationjointly. of generating a rapidprototypedtissueby accomplishing both In contrast,thedirectcellularbioprintingisaone-stepprocess depositing cellsontotheconstructsafter acellularbioprinting. (thermal-, electric-, laser beam-, acoustic- or pneumatic- mechanisms) (thermal-, electric-,laserbeam-,acoustic- orpneumatic-mechanisms) Droplet-based bioprinting relies on various energy sources Droplet-based bioprintingrelies onvariousenergysources [14,20,22,38] Adv. Healthcare Mater. www.advancedsciencenews.com

2017 , 6 , 1601118 - - - - REVIEW

------s) [42] [20] • [49] 1601118 [22] (5 of 29) [20] www.advhealthmat.de Overall, the inkjet bioprinting Overall, the inkjet bioprinting [45] s, and controls the droplet volume by • wileyonlinelibrary.com m). Moreover, inkjet bioprinting typi m). Moreover, µ Inkjet bioprinting dimensions are currently are dimensions bioprinting Inkjet [48] The size and distribution of these droplets can be droplets can these of and distribution size The Ultrasound parameters, including pulse, duration pulse, duration parameters, including Ultrasound [44] [39,46,47] However, for the inkjet and pneumatic pressure assisted for the inkjet and pneumatic However, The pneumatic pressure technique uses a set of electro The pneumatic pressure technique uses The electrohydrodynamic jetting (electrospraying or elec The electrohydrodynamic jetting (electrospraying limited by the diameter of the jetting needle. Typically, droplet droplet Typically, limited by the diameter of the jetting needle. size of the needle diameter is approximately two times the in the size as such, this technology has limitations diameter; to inkjet technology, range of tens of nanometers. In contrast from these limi electrohydrodynamic jetting does not suffer suspensions tations and can be used to process concentrated yet size in micrometers hundred few a are that needles from a few micrometers are capable of generating droplet deposits cell via on no adverse effects Furthermore, in size and smaller. cell-laden bioinks. bility have been observed when jetting the adjusting the pressure to the fluidic pathway and valve gating time. Although a higher liquid viscosity can be applied, there remain several concerns regarding droplet controls and cell via In order to obtain a favorable printing structure, the effect bility. of the printing conditions must be fully considered to include material preparation, droplet size, printing substrate stiffness, speed, surfactant usage, and agitation among others. mechanical micro-valves where the droplets are produced by opening the micro-valve under constant pneumatic pressure. cally exhibits over 80% cell viability after cellular bioprinting. after cell viability cally exhibits over 80% technique ensures rapid fabrication with highly repeatable pat technique ensures rapid terns; additionally, small volume droplets enable high printing small terns; additionally, 50 resolution (lower to trospinning) applies an electric potential difference between difference trospinning) applies an electric potential electrode to gen a positively charged needle and a grounded ejection occurs, in erate repulsive Coulombic force. Droplet when the charged the micrometer to nanometer size range, high-intensity electric medium exiting the needle enters the field. bioprinting, the viscosity of the available bioinks is too low to facilitate the rapid generation and sustainment of 3D struc perature or chemical reagents. The photocurable materials can be independently used, associated as the droplet bioinks, or deposited as supporting materials to assist in 3D molding. tures. Therefore, additional cross-linking methods are applied pH, tem light, to address this limitation, including UV/Vis controlled through the applied potential difference, the flow difference, controlled through the applied potential and the liquid solu rate to the needle, the distance of electrodes tion properties. tion and amplitude, can be adjusted to control the size of droplets to control the size can be adjusted and amplitude, of is capable radiation acoustic The of ejection. and the rate and ejection droplet size and controlling uniform generating used in these the acoustic frequencies However, directionality. to induce damage of the cell mem printers have the potential brane and cell lysis. Additionally, several modified inkjet tech several Additionally, brane and cell lysis. have been developed to build complex niques with multi-jets by arranging multiple cell types tissue and organ prototypes and other tissue components. This technique uses various types of liquid biomaterials with viscosities of up to 200 Pa Inkjet bioprinters do, however, still have limitations on material however, Inkjet bioprinters do, Devices are typi cell density and mechanical strength. viscosity, cally compatible with low solution viscosities (below 0.01 Pa cally compatible with and low cell concentrations (fewer than 10 million cells/mL), avoiding high shear stress and nozzle clogging. printer.

------[43] C in the ° 10 ∼ : The key fea 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © , 1601118 6 , 2017

[39–42] The necessary equipment is easily remolded from The necessary equipment is easily remolded C, the process only persists for a few microseconds ° [29] s) resulting in an overall temperature rise of 300 µ ∼ The inkjet bioprinting, which is granted with the earliest cel earliest the with is granted which bioprinting, inkjet The Stereolithography-based (vat-photopolymrization) bioprinting (vat-photopolymrization) Stereolithography-based Extrusion-based (dispensing, or direct writing) bioprinting Extrusion-based (dispensing, 2 ∼ Adv. Healthcare Mater. Adv. www.advancedsciencenews.com lular printing patent, is originated from commercial 2D inkjet lular printing patent, is originated from printing. ture of droplet-based bioprinting is that the droplets of cell- ture of droplet-based bioprinting is that and deposited laden bioink (hydrogels or slurries) are generated As a noncontact bio to pre-defined locations on the substrate. for method a high-throughput it provides technique, printing small droplets onto depositing multiple cells or biologicals in can be clas a targeted spatial position. Droplet techniques laser assisted- droplet ting, pneumatic pressure assisted-, and bioprinting. sified into four categories: inkjet, electrohydrodynamic jet sified into four categories: inkjet, electrohydrodynamic 2.2.1.1. Droplet-Based Cellular Bioprinting (DCB) 2.2.1.1. Droplet-Based Cellular which originates from fused deposition modeling (FDM) fused deposition modeling (FDM) which originates from mechanical- or electromagnetic- printing uses pneumatic-, cells based on a “needle-syringe” driven systems to deposit bioink dispensed by a deposition type. During bioprinting, cell-laden filaments forming desired 3D system precisely prints structures. a energy to deposit cell-laden bioink in mainly utilizes laser scanning or image projection modeling, reservoir via beam patterns. It offers allowing the molding of the high-precision of on deposition control precise to the advantages due greater biologicals and high resolution. to pattern the bioink micro-droplets of living cells and other of living cells the bioink micro-droplets to pattern greater It offers manner. in a high-throughput biologicals control precise with agility and simplicity to its due advantages factors, and cells, growth of biologicals including on deposition the most It has also been tissue/organ regeneration. genes for versatility, use due to its simplicity, common for pharmaceutical capability. and high-through put able and relatively inexpensive. In this technique, the bioink able and relatively inexpensive. In this factors, and cells is solution including biomaterial, bioactive and then transferred to the ink stored in a cartridge or reservoir, can be generated by chamber for droplet ejection. The droplets actuation, which can two mechanisms, thermal or piezoelectric be ejected from the inkjet-head nozzle to the print surface. 2D inkjet desktop printers making this technology widely avail 2D inkjet desktop printers making this technology ( They operate similar to the traditional “drop-on-demand” 2D They operate similar to the traditional “drop-on-demand” inkjet printers. The thermal actuation is based on a heating element, which can superheat the bioink to create vapor bub bles for ejecting the droplets. Although the temperature reaches 200 printer head. Many results have demonstrated that this increase this that demonstrated have results Many head. printer in temperature causes minimal damage to the viability of both printed cells and other integrated biologicals. The piezoelectric technique employs a voltage to induce a rapid shape change of the piezoelectric material, which generates a pressure pulse in the fluid forcing a droplet of ink from the nozzle. Droplet to voltage applied the by tuning adjusted be size can and shape the piezoelectric material. It allows a wider variety of inks than thermal inkjets as there is no requirement for a volatile com ponent, and no issue with coagulation. The acoustic radiation force associated with the ultrasound field is also utilized to eject on the piezoelectric interface the droplets from an air-liquid REVIEW 1601118 www.advhealthmat.de as highbioinkviscosity(1 control andanautomatedroboticsystemforbioprinting. technique consistingofafluid-dispensingsystemforextrusion based (or dispensing, direct writing) bioprinting is an integrated 2.2.1.2. Extrusion-BasedCellular Bioprinting(ECB) technique arestillmajorconcerns. efficiency, highcost and limitedavailability of bioinks for this free dropletmodel.Despitetheseadvantages,arelativelylow assisted dropletsystems have alsobeendeveloped. cells intoasacrificialmold. promise for “scaffold-free” bioprinting by depositing layers of up to10000droplets/s.Dropletbioprintingalsoshowsgreat deposition ratesrangingfrom1pLto300involumewith of this techniquehave reported controlling droplet sizes and ture by altering droplet densities or sizes. Recent developments of cells,materialsorgrowthfactorsthroughoutthe3Dstruc advantage isthepotentialtointroduceconcentration gradients low costbenefitsofthesebioprintingtechniques,another sidered andmanagedcarefullyduringfabrication. viability andbiologicalfunctionality; theserisksmustbecon crosslinking methodsmayresultinexcessivedamageofcell procedure mayslowdowntheprintingprocess,andseveral generation ofasolid,stable3Dstructure.Thecrosslinking print thebioinksandcross-linkersinturn,allowing Additionally, multi-jet bioprinting strategies can be used to co- pressure andpulsefrequency. configuration possessesahigher precisionduetoacontrolled Compared to thevalve-free configuration, the valve-based filaments usingavalve-free or avalve-basedconfiguration. microextrusion. pneumatic-, mechanical-(pistonorscrew),andsolenoid-based tion. Dispensingsystemscanbeclassifiedintothreetypes: tion providesbetterstructuralintegrityduringrapidfabrica deposited into the desired 3D structures. Continuous deposi filaments ordiscretevolumesofbioinksthatcanbeprecisely The bioinkisextrudedintothemannerofcell-ladencylindrical employing ahighcelldensities(upto10 cosity ofthebioinklayer. Moreover, thistechniqueiscapableof the donorlayerandsubstrate,thicknessvis tension, thewettabilityofsubstrate,airgapbetween many additionalfactors,includingthelaserfluence,surface higher printingresolution.Theresolutionisalsoinfluencedby viscosity andthicknessofthebioinklayercontributingtoa volume canbecontrolledfrom10to7000pLbyadjustingthe droplet production;thispreventsfromdirectlaser. Thedroplet strate. Theabsorbinglayerisusedtotransferheatforbioink pressure bubblethatpropelscell-ladendropletsontothesub focused laser to pulse on the absorbing layer generating a high- nium, andabioinklayer)receivingsubstrate.Itutilizes includes alaser-energy-absorbing layer, suchasgoldortita LIFT), consistsofapulsedlasersource,donorlayer(this bioprinting (also known as laser-induced forward transfer or bioprinting becauseofsomesimilarities.Laser-assisted droplet printing techniqueindividually, wecombineitintodroplet from severalotherreviewsthatlisteditasalaser-assisted bio Besides thethreeaforementioneddroplettechniques,laser- In additiontothehighresolution,simpleprocessing,and Pneumatic-based systemsutilize pressurizedairtoextrude (6 of29) [50] wileyonlinelibrary.com ∼ 300 mPa [42] Mechanical micro-extrusion • s) becauseofitsnozzle- 8 cells/mL)andaswell © 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim [40] : Extrusion- Differing [20,22] ------

coaxial nozzlesystems,amongothers. multiple direction-controllednozzleorstagesystems,and multiple independentlycontrollednozzlesorchambers, as: temperature-controlledcartridge(nozzle)orstagesystems, have beendevelopedforthecartridgesandstagessuch higher viscosities. of generatingalargerpressurefordispensingthebioinkswith fluid withlowviscosity, whereasthescrewsystem is capable commonly composedofsyringesandneedlesissuitabletoa (i.e., pre-crosslinkedbioink,bioplotting, coaxialcrosslinking, rials, self-healing materials),(2)crosslinkingagent integration processes include:(1)self-assembly (i.e.,shear-thinning mate be addressedintheexperimental design. very importantconsiderations for extrusionprintingandmust and thepropertiesofbioinks, as welltheirinteractions,are mechanical integrity of a 3D configuration, the molding process free of exogenousbiomaterials. In order to obtain appropriate cellular unitsenablingscaffold-free bioprinting,orprinting cate the3Dtissuestructure.Thistechniquedirectlyprintssolid After printing,themulticellularsystemsfusetogethertorepli cell-laden microcarriersaretwoexamplesofthistypebioink. without supportivebiomaterialsasabioink;cellspheroidsand using sphericalandcylindricalmulticellularsystemswith or limited byhighshearforcemanagement.Anothertypeinvolves the printingconditionsofcell-ladenhydrogelaresomewhat rapidly solidifiedintoa3Dconstructafter extrusion.However, solutions orlow-moduluscell-ladenhydrogels,whichneed be maintaining cellviabilityandfunction. lower-viscosity materialsprovidingasuitableenvironmentfor often providingstructuralsupportfortheprintedconstructand with microextrusionbioprinters,higher-viscosity materials in size, the displacement of the cartridge, and/or the stage motion can becontrolledbythedispensingprocedure,speed,nozzle dispensed usingamicroextrusionsystem.Theprintingprocess logicals forprinting.Thematerialsinsidethecartridgesmaybe pens) thatcanbeloadedwithcell-ladenbioinksorotherbio include astageandoneormorecartridges(i.e.,syringes from 30to tromagnetic drivensystems.Materials withviscositiesranging volume inpneumaticsystemsandthehighcomplexityofelec the material flow, because of the delay of the compressed gas dispensing systemsmightprovidemoredirectcontrolover microextusion systems. organ analogues.Therearetwomaintypesofbioinksusedin strength areoften employedtoreinforceprinted3Dtissue/ fibers. Syntheticpolymersthathaverelativelyhighmechanical micro-carriers, decellularizedmatricesandsyntheticpolymer tion ofbioinks,includingcellaggregates,cell-laden hydrogels, printing enablesrapidprinting,easyoperationandawideselec of controllingthebioinkprinting. (or direct writing) provides a simpler and more direct method netic plungerandaferro-magneticringmagnet. magnetic pullforcegeneratedbetweenafloatingferro-mag sion applieselectricalpulsestoopenavalvebycancelingthe laden cells.Solenoid(orelectromagneticdriven)microextru nozzle inmechanicalmicro-extrusioncanpotentiallyharmthe Compared to droplet-based bioprinting, extrusion-based bio In additiontodispensingsystems,theextrusionprinters x , y , and > 6 z × axes.Moreover, severaladvancedtechniques 10 [50] 7 mPa However, alargeshearingforcealongthe [50] • Thefirstishigh-viscosity, cell-laden s havebeenshowntobecompatible Adv. Healthcare Mater. www.advancedsciencenews.com [51–53] [20,50,54] [52] [50] Thepistonsystem Thetypicalmolding

2017 [42] Mechanical , 6 , 1601118 ------

REVIEW ------m µ 1601118 : Unlike the (7 of 29) 80% cell viability ∼ www.advhealthmat.de It is the most widely After removing the organic After [18,21,34,37] The DLP system uses a digital uses a system DLP The Therefore, it is important to apply [51,64] wileyonlinelibrary.com However, the photopolymerization is However, [22,57] [57] [22,37,57,58] SLA systems are capable of preparing structures SLA systems are capable of preparing The commonly photocurable bioinks include poly The commonly photocurable m features. µ µ [58] There is a limited choice of photopolymerizable bioinks, of photopolymerizable choice limited is a There Fused deposition modeling (FDM) or fused filament fabri deposition modeling (FDM) Fused face of the photocurable bioinks using a digital light procession using a digital light photocurable bioinks face of the patterned 2D entire an solidify can which (DLP), technique layer simultaneously. micromirror device (DMD) to project a set of 2D images from project a set of 2D images device (DMD) to micromirror beam- the to Compared structure. 3D sliced horizontally the be mask-image-projection printing can scanning technique, shape the form simultaneously to ability its to due faster much of an entire layer. can technically enable more however polymer modification options. cation (FFF) was developed in the early 1990s and is a major extrusion-based system. acellular, printing techniques can also employ acellular bioinks to fabricateprinting techniques can also employ acellular An additional cell seeding technique tissue engineered scaffolds. forcell-laden scaffolds can be employed to create artificial 3D a universal cell printing. Here, tissue/organ regeneration after seeding procedure can be used in a post-seeding process, or per tion to solid 3D structures. a cytocompatible photo-initiator. Additionally, the limited avail Additionally, a cytocompatible photo-initiator. high equipment costs ability of photocurable biomaterials and are major concerns with this technology. 2.2.2. Acellular Bioprinting acellular 3D bio Compared to cellular bioprinting techniques, for material selectionprinting provides more extensive choices cellular bio and manufacturing method. The aforementioned fusable cell seeding can be obtained using a bioreactor device. can be directly implanted without cells Also 3D printed grafts into injured patients for functional replacement or structural support during healing. The representative techniques of acel lular bioprinting fall into two categories: extrusions-based acel acellular bioprinting (LAB). lular bioprinting (EAB) or laser-based Bioprinting (EAB) 2.2.2.1. Extrusion-Based Acellular previously presented extrusion systems focusing on cellular bioprinting, acellular extrusion (or acellular direct writing) can utilize volatile or easily displaced organic solvents to dissolve polymers, followed by conversion from a highly viscous solu the cells can be seeded and grown on the solvent thoroughly, surface for tissue/organ regeneration. scaffold’s depending on the laser wavelength, power, exposure time and time exposure power, wavelength, laser the on depending toxicity of photo-intiator. driven by a radically induced chemical reaction, and the free driven by a radically induced chemical nucleic and proteins, membrane, cell the damage can radicals 40 acids. This technique can achieve up to ethylene glycol acrylate/methacrylate and its derivatives, meth ethylene glycol acrylate/methacrylate support material. tion and rapidly print constructs without with low to 50 Most commercial systems prepare structures acrylated/acrylated natural biomaterials (gelatin, hyaluronic biomaterials (gelatin, hyaluronic acrylated/acrylated natural and methacrylated/acrylated capped acid, dextran, and others), polymers. Overall, the main advantages among other synthetic bioprinting techniques are their of sterelithography-based complex designs with high resolu ability to simply fabricate features; with <5

------[50,55] The : Stere [22,24,58] [50] [20,22,57] 86%, control ∼ m in the z axis can 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag µ © Bioinks should possess Bioinks SLA) techniques, a resolu The types and concentra µ [20,50] The resolution is dependent is resolution The [60,61] [59] , 1601118 6 , m; nonbiological microextrusion printers 2017 µ m resolution.

µ m in the x/y plane and 10 µ [62,63] [56] The beam-scanning technique, or laser direct writing (LDW), The mask-image-projection printing system dynamically Overall, extrusion-based techniques are capable of greaterOverall, extrusion-based Adv. Healthcare Mater. Adv. www.advancedsciencenews.com tion of a 2D patterned layer. patterned 2D a of tion uses a laser beam to scan photocurable bioinks for solidifica generates a defined mask image that is projected onto the sur a defined mask image that is generates shear thinning ability to overcome surface tension to extrudeshear thinning ability to overcome surface nozzlethe at stress shear high resulting The form. filament in microextrusion viability after Cell may decrease the cell viability. with inkjet-based bio bioprinting is typically lower than that of 40 printing; cell survival rates are in the range lable by changing extrusion speed and nozzle gauge. lable by changing extrusion speed and nozzle erogeneous formulations, allowing physiologically relevant cellerogeneous formulations, scalability in a relatively short perioddensities, which facilitate extrusion-basedbenefits, great and versatility its Despite time. of involving lowerbioprinting still has several challenges mainly material selectionresolutions, higher shear stresses, and limited of the technology isamong others. The minimum feature size generally over 100 deposition and printing speed and have more tolerance for het deposition and printing are capable of 5 tion or scattering of the laser beam), and the selection of photo- initiator or any UV absorbers. on irradiant exposure conditions (laser spot size, wavelength, of absorp and the occurrence time/velocity exposure power, aerosol crosslinking or spraying crosslinking system), (3) UV/ crosslinking system), or spraying aerosol crosslinking deposition sensitive and (4) environmentally photocuring Vis bioprinting, In extrusion based and others). (pH, temperature, requiring a dispensing system refers to syringe bioplotting time. solidification over involving additional curing process lithography appearance (SLA) offers an additive manufacturing additive an offers (SLA) appearance lithography accuracy. technique with very high resolution and 2.2.1.3. Sterelithography-Based Cellular Bioprinting (SCB) Bioprinting 2.2.1.3. Sterelithography-Based Cellular be achieved. tion of bioinks, scanning speed and laser power contribute to contribute power laser and speed scanning bioinks, of tion the overall mechanical properties of the bioprinted structure. when printing multiple layers, early layers may Additionally, causing uneven mechanical be repeatedly exposed to the laser, the develop- strength or undesired 3D structures/patterns. With ment of micro-stereolithography ( tion of about 5 Therefore, we do not separately introduce it as a bioprinting separately introduce it as a bioprinting Therefore, we do not approach, cell-laden bioinks are technique. In the bioplotting a plotting medium (crosslinking pool) directly extruded into process. It requires the use of relatively to complete the curing into plotting medium that can sup viscous bioinks printed port the extruded structures temporarily until crosslinking is temporarily until crosslinking port the extruded structures complete. Sterelithography-based bioprinting technique (vat photopoly Sterelithography-based bioprinting technique irradiation of light merization) utilizes the spatially controlled layered through or laser to solidify a geometrically 2D pattern The 3D reservoir. selective photopolymerization in the bioink patterned layers in structure can be consecutively built on 2D fashion, and the uncured bioink can be easily a “layer-by-layer” of photo-polymerization The final product. from the removed bio in SLA-based 2D patterned layers is the most crucial step bioprinting techniques have two SLA-based printing. Traditional types: beaming-scanning and mask-image-projection. REVIEW 1601118 www.advhealthmat.de cross-linked aresuitableforFDMprinting. terials thatcanbemeltedandthenre-solidifiedorthermally with temperature controllers, an extrusion block and motors. construct fabrication.Theprinteriscomposedofheatingblocks different propertieswithinasinglestructure forcomplicated overhanging structuresormultiplematerialintegrationwith permit co-printingoftemporarysupportmaterialforcomplex are notused.Multiple printheadscanbeaccommodatedto to conventionalextrusionorinjectionmoldingexceptmolds tional crosslinking requirment. allowed ittosolidifyontheprintingstagewithoutanyaddi a semimoltenstate,passedthroughanextrusionnozzleand moplastic filamentsthatareheatedtotheirmeltingpointor it is low-cost and relatively fast. This technique employs ther used andgenerallywell-explored3Dprintingstrategybecause structures onthesurfaceofapowderbed. uses ahighpowerlaserforpowdersintering,formingsolid3D sintering (SLS)isanotherlaserbasedprintingtechniquethat it allows for more diverse scaffold properties. Selective laser cess. Theincreaseinmaterialselectionisbeneficialthat eliminate theconcernofcelldamageduringprintingpro crosslinking conditionsareavailablesinceacellularconstructs scaffolds. Inthesecases,however, morephotocuringresinsand raphy techniquescanalsobeappliedforfabricationofacellular 2.2.2.2. Laser-Based Acellular Bioprinting(LAB) for tissue/organregeneration. is requiredafter theFDMprintingtoseedcellsonconstructs the highmanufacturingtemperature.Therefore,anextrastep polymers anditisnotsuitableforprintingwithcellsdueto tages arethelimitedmaterialselectionrelatedtothermoplastic of with aleadscrew. Thesemodelsresultinanoverallresolution The extrusionforceisdrivenpneumaticallyormechanically it tothepreviouslyscannedunderlying layer. exposed particles together within the layer as well as connecting the ahigh-powerlaser, whichtracesthe2Dlayerdesignfusing ticles isachievedbythecontrolled additionalenergyinputof during thecoolingcycle.Localized thermalsinteringofthepar transition andthetemperature necessaryforrecrystallization laser. First, theparticlesarepreheatedbetweentheirmelting relies ontwoenergysources, a bed-heaterandhigh-power ionizing radiationornovelmaterialdesign. can beconductedviaanadditionalcrosslinkingreactionusing a conversionfromthermoplasticmaterialtothermoset offer a higher and more uniform strength between each layer, printing, preservingtheirfunctionality. Moreover, inorderto biologicals can be added though more mild processes after bio temperature thermoplastic biomaterials is preferable in that thermoplastic performanceandbiocompatibility. polyurethane, andtheirderivativeshavedemonstratedadequate terials suchaspoly(caprolactone) (PCL), poly(lacticacid)(PLA), objects positivelyimpactingscalability. Severalsyntheticbioma solvent submersionandhastheabilitytofabricatelarge-format tion applications.Thistechniquealsoeliminatestheneedfor mechanical propertiesmakeitsuitableforhardtissueregenera- bility, diverse synthetic biomaterialavailability, andfavorable applications areitssimpleemployment,rapidprintingcapa main advantagesoftheFDMmethodintissueengineering > 50 (8 of29) µ m inlayerheightandanaccuracyof wileyonlinelibrary.com [18,65] This method is analogous [21,31,68] © [66] 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim [37,68] [67] > Exploitinglow- 100 Thedisadvan Thetechnique [58] : Sterelithog Thisprocess Anybioma µ m. [18] The [58] ------

the liquid precursor where solidification cannot occur. to createanoxygen-containingzonebetweenthesolidpartand amorphous Teflon film)belowtheUVimageprojectionplane hand, employs an oxygen-permeable curing window (a thin, fication toinhibittheadhesionprocess.CLIP,onother bottom-up buildingapproachandthereforerequireslowsolidi The choiceofphotocuringresins isfairlybroadinCLIP;the size thatcouldcontainfeatures withresolutionsbelow100 ditional SLA,andgeneratesstructures tensofcentimetersin production inminutesinstead ofthehoursrequiredwithtra than layer-by-layer, fashion. Thisapproachallows3Dconstructs rate atwhichthepartcanbe formed inacontinuous,rather efficiency, and the resin reactivity all combine to determine the rate ofresinreplenishmentinthisdead-zone,theinitiation metals, polymersandtheircomposites. may beprintedusingseveralmaterialtypessuchasceramics, ered infabrication. properties affected bytheheatingprocessthatmustbeconsid shrinkage, andcrystallinitychangeareconcernsaboutmaterial Additionally, materialoxidation,thermaldegradation, bersome, andprovideslowresolutionsfortough,stiff grafts. other 3Dprintingtechniques,theSLSismoreexpensive,cum which are limited by filament prefabrication. Compared to this techniquearemorereadilyavailablethanFDMmaterials, to the UVwindow. sistent liquidinterface)preventingtheresinfromattaching through awell-controlledoxygeninhibiteddead-zone(per tinuous liquidinterfaceproduction(CLIP),whichisfacilitated A recentdevelopmentinSLA-based 3Dprintinginvolvescon 2.2.3. RecentDevelopmentsinBioprintingTechniques tural-supporting materials. suitable tothefabricationofhardbonereplacementsorstruc of availablebiomaterials.Specifically, ceramicsandmetalsare process fortissueengineeringapplicationsarethewiderange of metalorplasticcomponents.Themainadvantagesthis typing inmuchthesamefashionasindustrialfabrication dation byoverheating. spacing needstobecarefullycontrolledavoidpolymerdegra laser parametersofpower, beamsize,scanningspeedand recycled after bioprinting.For polymerpowdersintering,the 3D manufacturing, and unused powders may be removed or The un-sinteredpowderservesasthephysicalsupportduring resolution andallowingforadequatepowderdispensability. therefore weneedtoconsiderabalancebetweenobtainingfine achieved andmanipulatedthroughacarefulbalancebetween ferent SLSmachinesusuallyrangesfrom20to100 ical rolesinthefabricationprocess.Theresolutionofdif density, andthermodynamicvariationsofmaterialsplaycrit such asenergysource,particlesize,shape,freepacking layered structures. the surfaceofpowderedthermoplasticmaterialsintopatterned, (SHS) utilizesathermalprintheadratherthanlasertofuse SLS approach,atechnologyknownasselectiveheatsintering ance andpropertiesoftheprintedparts.Beinganalogousto be incorporatedintothepowdertofurthermodifyappear Selective lasersinteringisappliedtorapidscaffold proto [31] [21,58,70] [69] [68] Additionally, arangeoffillers can also Traditional SLA techniques use the [68] Adv. Healthcare Mater. Moreover, thepowdersusedin www.advancedsciencenews.com [68] Printingparameters,

2017 µ , m; thisis 6 , 1601118 [58] The µ m. [68] ------

REVIEW

------[77] 1601118 (9 of 29) (1) Biomate In tissue engi tissue In www.advhealthmat.de Strategies for bioink Strategies for bioink [10] [16,50,78–80] [22,38,50,52,75,81] ): 3

Figure wileyonlinelibrary.com In this section, we focus on the design strategies of bio on the design strategies In this section, we focus [20] Generally, biomaterials range from cell supportive soft soft supportive cell from range biomaterials Generally, cellular matrix (ECM) biomimetic structures that organize the cellular matrix (ECM) biomimetic structures for tissue functions, tissue regeneration, temporary substitutes or integration and guides for regenerating tissue ingrowth porosity, including elements basic Some tissue. host a within biodeg geometry, pore dimensions, internal interconnectivity, neering, the scaffolds fabricated by biomaterials serve as extra neering, the scaffolds and biocompatibility radation kinetics, mechanical properties, pro manufacturing the scaffold in account taken into also are play cru engineering science and/or Therefore, material cess. building block cial roles in programming an active and effective for tissue formation. 3.1.1. Design Principles into four major con The design principles can be combined siderations for selection ( particles and quantum dots for drug delivery and imaging, to dots for drug delivery and imaging, particles and quantum devices. medical functioning complex materials for functional scaffold bioprinting; scaffold-free bio bioprinting; scaffold-free scaffold materials for functional in detail in section 3.5. printing will be introduced nano and from metal or ceramic implants hydrogels, to stiff selection can be divided into two categories: functional scaf selection can be divided with/without cells as the printing fold bioprinting (biomaterials use cells as the printing bioprinting (only ink) or scaffold-free ink). The bioink material is crucial because it should provide the because it should material is crucial The bioink factors, chemokines, growth of biochemical (i.e., spectrum (i.e., inter and physical or signaling proteins) adhesion factors, stitial flow, mechanical and structural properties of extracellular properties of extracellular and structural mechanical stitial flow, promote a favorable environment for cell matrix) cues which and differentiation. survival, motility, tion mechanism and printing modality as discussed above. modality as discussed and printing tion mechanism rials should be affordable, abundant, and commercially available abundant, rials should be affordable, with appropriate regulations for clinical use. 3.1.2. Bioink Formulations 3D bioprinting technologies, including the deposition Acellular of metals, ceramics and thermoplastic polymers, generally rials must have suitable properties to meet specific bioprinterrials must have suitable properties to meet Printability refers todeposition requirements (printability). manufacturing andthe capability of the material to support interrelation of bioinksprintability and solidification, the rapid substrates must bein various bioprinters and on associated patterns. (2)evaluated carefully to produce accurate, high-quality Biomaterials must possess suitable physicochemical properties, char structure external and internal wetting/swelling, including acteristics range from nano- to macro-scale, degradation kinetics, (3) Biocompatibility mechanical strength and structural stability. and biological activity are necessary for tissue development and bioinklong-term in vivo implantation. The remodeling over with the endogenousmaterial should facilitate engraftment tissue without generating an immune response and provide a spectrum of biochemical cues (i.e., chemokines, growth factors, adhesion factors, or signaling proteins) that promote an environ (4) The mate and differentiation. ment for cell survival, motility, ------[31] The [76] Unlike [75,76] By exploiting By [22] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © [70] , 1601118 6 , This process is reasonably inexpensive This process is reasonably 2017

[31,71,74] This technology uses a binder solution, such assuch solution, binder a uses technology This [31,71–73] A nano-stereolithography technique, also called two-photon A nano-stereolithography technique, also 3D powder printing is a powder-based 3D bioprinting tech 3D bioprinting printing is a powder-based 3D powder Adv. Healthcare Mater. Adv. www.advancedsciencenews.com compared with other modalities, and provides more optionscompared with other and drug-delivery because it avoids thefor tissue engineering bioactive components. A major limi damage of incorporated tation of this system is the difficulty in removing unbound in is the difficulty tation of this system usage ofthe addition, desired hollow spaces. In from powder exhibits limited mechanical strengthaqueous binding agents This tech and resolution, and requires further post-processing. nique is also difficult for direct depositing or patterning living nique is also difficult the inkjet printer tocells. Several researchers are also using to the traditionaleject the binder droplet. This concept is closer acts like the paper. 2D inkjet technique where the powder bed polymerization printing (TPP), is used to photocure the liquid polymerization printing (TPP), is used to polymers by simultaneous two-photon absorption. photopolymerization that is triggered by nonlinear excitation photopolymerization that is triggered by not affected are but other regions at the focal point, happens 3D So it has the potential to print precise by the laser energy. 3D con enable and even resolution, very high with structure solution without struct printing inside the photocuring material other regions. This technique can achieve spatial affecting nm. solidification with a resolution of up to 100 nique that has been developed based on the principles of based on the principles has been developed nique that SLS. viscosity and reactivity of the monomers, however, are more monomers, however, reactivity of the viscosity and within the resin oxygen diffusion the they affect critical since permeable curing window. the affecting water, citric acid or phosphoric acid, among others, to selectivelyto others, among acid, phosphoric or acid citric water, biomaterial together in the designedbind the loose powdered for binder integration include biomaterials Available geometry. calcium phosphates, and hydroxyapatitestarch, dextran, gelatin, among others. the single photon polymerization process in SLA, two-photon process in SLA, polymerization the single photon over excited energy polymerization allows electron transitions when an atom levels; the polymerization process occurs a spe More specifically, absorbs two photons simultaneously. cific photoinitiator that reacts at low wavelengths simultane low wavelengths that reacts at photoinitiator cific their ener ously absorbs two photons with high wavelengths, low with photon one of energy the achieve to combine gies process. polymerization initiate the and thus wavelength sary for the successful construct fabrication. As printing and fabrication depend on the solidifying kinetics of the biomate or environmentally induced rials and the native, chemically, material properties, specific concerns arise based on the deposi In addition to the bioprinting techniques chosen for the tar 3. Material/Cells in 3D Bioprinting: From Bioink to 3D Bioprinting: From in 3. Material/Cells Modular Building Blocks 3.1. Design and Selection Principles of Bioink the high resolution of this technique, many researchers have researchers many technique, this of resolution high the focused on the realization of 3D environments for cell adhesion polymerization printing is an and proliferation. Two-photon limits often of materials cost and process the but improvement, products to a small scale. bioink selection, requirements, appropriate geted tissue including cells, biomaterials and biochemical signals, is neces REVIEW 1601118 www.advhealthmat.de for printingtogetherwithcells. Therefore,cellsareusually vents to facilitate printing, so that they are not appropriate materials typicallyrequirehigh temperaturesortoxicsol fabricate mechanicallyrobust anddurableconstructs.These ceramics, andcuring(thermoplastic) polymers.Theycan categories: gaining approval. byproducts needstobeperformedinvitroor/andvivobefore evaluation fornewlydevelopedmaterialsandtheirdegradation terials arepreferredintheseapplications;thebiocompatibility fore, Food and Drug Administration (FDA)-approved bioma ensuring cellviabilityanddevelopmentthroughout.There ible fabricationprocessesandbiomaterialsduringprinting, tion. other modificationisnecessaryforfurtherbiomedicalapplica an extrapost-processstepsuchaspurification,sterilization, or crosslinking agentsorothersevereprocessconditions.Assuch, involve theuseoforganicsolvents,hightemperatures, Figure 3. In general,theprintablebiomaterialsaredividedintotwo [21,78] (10 of29) Designofbioinksfor3Dbioprinting,includingdesignprinciples,formulations,solidification mechanismsandbio-functionalization. In contrast, cellular bioprinting requires biocompat [22,38,50] (1)Hard biomaterials,suchasmetals, wileyonlinelibrary.com © 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim - - - - - application. developed tomatchdesirable traits foravarietyofbiomedical specific andcomplexprintablematerialsaresteadilybeing properties andbioactivityofnativetissue.Moreover, themore been designedforcellprintingmimickingthemechanical erties of natural ECMs, many biomaterial combinations have like formduringprinting).Inordertobettermimictheprop liquid materialsorhydrogels(theyshouldbeinpaste- technologies currentlyavailableareonlycapableofdispensing a favorable environment for cells. The cellular bioprinting ural polymers, possess biomimetic characteristics, providing biomaterials suchashydrogels,comprisedofsyntheticornat nectivity ofporesallowsforuniformcelldistribution.(2)Soft is often utilizedtoimprovescaffold coverage.Theintercon conditions harmfultothecells.Adynamiccellseedingmethod seeded ontotheprintedconstructsafter fabrication,avoiding and SLA-based bioprinting areveryversatileindepositinga tion isalsoanimportantfactor. For example,extrusion-based In additiontothecomponents of bioink,theresultantforma Adv. Healthcare Mater. www.advancedsciencenews.com

2017 , 6 , 1601118 - - - - REVIEW

------[82] [8,11] 1601118 Moreover, Moreover, (11 of 29) The strategies The [6,86,88] [89] www.advhealthmat.de Based on the 3D printed [87] [66,84–86] wileyonlinelibrary.com Therefore, multiple printing techniques The bulk modification before or during the The bulk modification [7,20,65] [12,83] Herein, the surface modification involving physical Herein, [82,87] The inherent characteristics of these different printing mate The inherent characteristics of these different To date, the exploration of new biomaterials for tissue/organ date, To or material systems can be integrated into a 3D construct by appropriate with and materials the proper printer choosing Combining 3D bioprinting platforms and tech printability. alternative, especially niques has been proven to be an effective for complex organ manufacturing. rials, including solubility, viscosities, melting points, mechan viscosities, melting rials, including solubility, ical properties, and available chemistries for crosslinking and are responsible for the overall success of the functionalization the customized approaches described design. More importantly, above will provide potential strategies for creating versatile materials to support successful bioprinting. biomimetic spatial structure, incorporating bioactive compo biomimetic spatial structure, incorporating spatial distribution nents into constructs provides the proper and remod of biochemical cues for guiding tissue formation eling. adsorption or chemical conjunction has been widely utilized to adsorption or chemical conjunction has been regulate cell differ increase cell attachment, proliferation and entiation by means of interacting with cellular surface ligands surface entiation by means of interacting with cellular and/or modulating the signaling pathways. printing process may affect the physicochemical properties of the physicochemical properties affect printing process may modifica while post-processing surface the resultant scaffolds, between the interactions changes only tion on printed scaffolds cell/tissue and material surface. to be considered so that deformation of the final construct can be of the final construct so that deformation to be considered of bio-ink type. the proper selection prevented via 3.1.4. Bio-Functionalization excellent bioactivity for should possess An ideal scaffold Directly encapsulating growth factors regulating cell events. is a simple way of regulating cel or cytokines into bioinks approaches can also be used to modify Several conventional such as incorporation with bioac the printable biomaterials lular behaviors through diffuse release after bioprinting. release after diffuse lular behaviors through recognition sites, and adhesion factors tive factors, enzymatic among others. Other techniques such as microspheres or hydrogels can be Other techniques such as microspheres to achieve efficiently combined into 3D bioprinted scaffolds factors. bioactive various release of sustained the presence of nanoscale features also affects cell adhesion, affects the presence of nanoscale features also assembly. and cytoskeletal cell orientation, cell motility, to engineer biomaterials with specific physiological functions to engineer biomaterials with specific physiological of the complex bio requires a comprehensive understanding process, involving the logical mechanisms of the regeneration localization of bioac natural tissue-specific composition, the cascade of signaling tive components in ECM, and the complex pathways in normal physiological events. be given to Emphasis should bioprinting is still underway. role in cell sur that play a significant those printed scaffolds and during differentiation and migration proliferation, vival, prin the possess must also They processes. bioprinting after cipal means of mechanical support and biochemical signals of regeneration. Due to the limitations for the long term tissue material properties relative to specific printing techniques, 3D constructs with complex structures and characteristics were dif ficult to realize.

------2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © , 1601118 6 , 2017

The curing methods of hard biomaterials canbiomaterials hard The curing methods of [50] [38,50,52,81] Adv. Healthcare Mater. Adv. www.advancedsciencenews.com be easily understood. (1) The melt-deposition is based on phasebe easily understood. materials around their melting point. (2)transition of associated is mainly used in curable polymers. TheThe solution-deposition chosenbe need exchangeable) or (volatile organic solvents proper biomaterials, cell- soft polymers. For to dissolve the different bioinks.universal or typical most the are solutions hydrogel laden weight,as concentration, molecular properties such key Various important determi are stiffness gelation kinetics, and viscosity, As one of the typicalical crosslinking and covalent crosslinking. gelto transition from sol a phase methods, crosslinking physical environment change (var the printing can be controlled by state by these ions could be leached out or exchanged rions, however, thecompromising culture, term long in molecules ionic other physical interactionscontrol over the construct properties. Other bond, or inclusionsuch as hydrophobic, electrostatic, hydrogen 3D structures, how complex can employ the solidification of their weak mechanical strength limits their application. ever, preferred in order toTherefore, covalent network formation is In general, the radical based enhance the mechanical stability. and temperature; thecross-linking can be induced by light, redox, addition, enzy non-radical crosslinking methods involve Michael genipin among others,matic, glutaraldehyde, carbodiimide, and should be considered.so the toxicity of the crosslinking agents strategies, thelight-induced deposition or solidification In the photoinitiators or photosensitizers are commonly applied to ini tiate the radical crosslinking reaction of monomers and/or pre nants. The solidification (or gelation) mechanisms include phys The solidification (or gelation) mechanisms nants. pH, or others. Theious external stimuli) such as temperatures, via multivalent counte ionically cross-linked network is formed polymer solutions, such as D-p-chromophore (known for its high sensitivity in 2PP processes), Irgacure 2959 (I2959; high biocom nate salt (LAP, biocompatible initiator and visible light working (high biocompatible initiator and visible light range), VA-086 initiator with(CQ; an range), and camphorquinone working amongrange) working light visible and applications dental many others. Some chemical crosslinking reactions are too slow to support 3D structures during rapid printing, thus multiple-step the printing tem a better choice. Normally, crosslinking offers aroundbe should hydrogels the in encapsulated cells of peratures the physiological temperature in order to avoid ice nucleation or overheating, which are harmful to cells. In vivo stabilities, perme hydro polymer of rates degradation and compatibilities, ability, patible initiator and UV working range), lithium acylphosphi lithium range), UV working initiator and patible gels should be seriously considered before the 3D constructs can tissues or organs. In addition,be implanted, particularly for soft the swelling and contraction characteristics of the bio-inks have Some general types of curing approaches are described in of curing approaches are described Some general types wide array of bioink types, including hydrogels, microcarriers, hydrogels, microcarriers, of bioink types, including wide array strands and decellularized cell pellet, tissue tissue spheroids, is capable bioprinting also Extrusion-based matrix components. delivery in a fugitive liquid small building blocks of depositing has the nozzle tip design, and remains flexible in medium, in near solid state, due to larger nozzle ability to extrude bioink diameter ranges. 3.1.3. Solidification Mechanisms Figure 2. Figure REVIEW 1601118 www.advhealthmat.de metal matrixcompositeshavebeenreported. posites, whichconsistofthepuremetalsthemselves,alloys,or based, zinc-based,orotherbiodegradablemetal-basedcom clinical trials. studied in3Dprintingaswell,andsomehaveprogressedto such asstainlesssteel,titanium,and certain alloyshavebeen tone (PCL)andpoly lactide (PLA)arethemostwidelyused mineralization abilities. materials fordental,jointandboneimplantationduetotheir cations canprogress. comprehensive researchisneededbeforefurtherclinicalappli vivo. Astheavailabilityofbiodegradablemetalsincrease,more metabolism canbeobservedinstudieseithervitro,or biocompatibility, suitabledegradationrates,andcompleted metallic components,polymers,ceramicsorcomposites. either fromnaturalorsynthesizedmaterials,containingin In thefieldoftissueengineering,hardbiomaterialsarederived Thermoplastic Polymers) 3.2.1. HardBiomaterials(Metals,Ceramics, and printable biomaterials. bioprinting, andwillfurtherdiscussthedevelopmentofnew briefly coverthetraditionalanduniversalbiomaterialsusedin ties ofbioinksaswe discussed above. In thissection, we will printing techniques have specific requirements for the proper able biomaterialsremainsamajorchallenge.Thedifferent functionalization. Therefore,thelackofvarietyinideal,print structs insteadofexploringnewprintablematerialsandtheir lution, speedorothers)thebio-applicationofprintedcon printing techniques, the update of printing parameters (reso Currently, mostresearchfocusesonthedevelopment ofnew 3.2. Common BioinksandRecentDevelopments ated tissue). body) to resorbable materials (eventually replaced by regener bioprinting fieldrangingfromceramicoxides(inertinthe both metallicandnonmetallicelementsareappliedinthe3D mechanical strengthandinvivosafety. repair inthebiomedicalcommunitybecauseoftheirhigh used inFDMandSLSbioprinting. degradable polymersandtheir copolymersareextensively at lowcostwithoutimmunogenicity. SomeFDA-approved engineering outcomes,andthese materialscanbeproduced thetic polymerscanbeeasily modifiedforenhancingtissue osteogenesis. adequate mechanical strength, andtheabilitytopromote been printed into bone scaffolds with biomimetic structures, calcium phosphate(TCP)and(CaP), has in humanteethandbones.It,alongwithitsanaloguestri concept hasbeenproposed. ferred fortissueregeneration,thusa“biodegradablemetal” for longtermimplantation.Biodegradableimplantsarepre metal ions are serious considerations being further evaluated niques, metalcorrosionandageing,potentialtoxicityof Ceramics andglassesarewidelyusedasbiocompatible Metals havebeenusedclinicallyforbonereplacement or The physicochemical and mechanical properties of syn (12 of29) [92] [93] [68,69] Hydroxyapatite(HA)isaprimarycomponent However, limited3Dmetalprintingtech wileyonlinelibrary.com [72,91] Therefore,bioceramicscontaining [90] Somemagnesium-based,iron- [22,31,81] [21] © Commonmetals 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim [90]

Polycaprolac Thefavorable [21,31,78] ------bility allowingeasyprintingandprocessing. because oftheirpropermeltingtemperaturesandgoodsolu biocompatible andbiodegradablepolymersusedwithFDM sites) tosuitparticularapplications. tive anchoredsites(i.e.,adhesionmotiforenzymedegradation composition andfunctionalgroupchemistryaswellbioac response suchasspecificmolecularweight,chemicalstructure, be tailoredwithspecificphysicalpropertiesintermsoftissue weight. Theadvantageofsyntheticpolymersisthattheycan ical strengthsandalackofcontrolincomposition/molecular however, theyarelimitedbyimmunogenicity, weakmechan their similaritytohumanECM,andinherentbioactivity, of mostsoft tissuesinthebody. and polymerconcentration,thusmimickingtheelasticmoduli values throughmanipulationofchemistry, crosslinkingdensity, teeth, hydrogelscanrecapitulatearangeofelasticmodulus With theexceptionofstiffest tissuetypessuchasboneand 3.2.2. SoftBiomaterials(Hydrogels) the tailoringofmechanicalpropertiesforscaffolds. in thecopolymerstructureandpolymerconcentrationenable it showslimitedpromiseforbiomedicalapplication.Variations applied toengineerhydrophilicityandenablebiocompatibility, native biocompatibility. Althoughsurfacemodificationhasbeen ison tobiomaterialssuch as PCLandPLAwhichoffer greater ymer (ABS) is notwidely used in medical devices in compar printing applications.Acrylonitrile-Butadiene-Styrene copol microorganisms thathasalsoattractedattentionfor3Dbio tyrate) (PHB)isanaturalthermoplasticpolyesterproducedby another favorablefeatureofthismaterial.Poly(3-hydroxybu polymerization ratiobetweenthelactideandglycolidegroups, sion bioprinting;degradationcanbecontrolledbyadjustingthe co-glycolide) (PLGA)isanothermaterialthatidealforextru hydrophobicity, andlowwaterabsorptioncapacity. Poly(lactic- very slowdegradation,duetoitssemicrystallinestructure, materials intheprintedconstructs.PCL,however, exhibits can also be used asa 3D structuralsupport for cell-laden soft to being used for fabricating tissue engineering scaffolds, they scaffolds. physical or chemicalnetworks when using as tissue engineered ronic F127shouldbechemically modifiedpriortoforming 16 F127 cantransformfromaliquid under4 gelation dependentonthesolutionconcentration.Pluronic because itpossessesthecharacteristicofthermo-reversible or others). and their derivatecopolymers, includes polyesters, polypeptides synthetic materials(polyethyleneglycol(PEG),PluronicF127 hyaluronic acid, often isolated from animalor human tissue) or tives (including alginate, gelatin, collagen, chitosan, fibrin and based oneithernaturallyoccurringpolymersandtheirderiva used inthecellularbioprintingtechniquesarepredominantly acrylation ormethacrylation, where thechemicallymodified plex 3Dconstructs. used asarepresentativesacrificialmaterialforfabricatingcom Pluronic F127,arewater-soluble polymers,theyareintensively As thesyntheticpolymers,bothpolyethyleneglycolPEGand ° C whenabove20%w/wconcentration. BothPEGandPlu [95] [21,65,79,81] Thetypicalmethodforachieving gelformationis Theadvantagesofnaturalpolymersare [50] PluronicF127isofparticularinterest Adv. Healthcare Mater. [14,50,94] www.advancedsciencenews.com [50] Soft biomaterialsmainly ° C toagelatover [78,79]

2017 Inaddition , 6 , 1601118 ------REVIEW ------[67] 1601118 For For The com Alder reac [65,77,94] − [21] Engineering Decellularized Decellularized (13 of 29) [7,94] [104] www.advhealthmat.de Challenges in tissue decel tissue in Challenges Additionally, some toxicity has Additionally, [22,38,50,65,79,80] [107] [100,105,106] wileyonlinelibrary.com Other natural polymers such as starch, cel such as starch, Other natural polymers [105] Moreover, the strongly desired characteristics of advanced Moreover, Currently, composites of polymers and bioactive materials composites of polymers and bioactive materials Currently, in high concentration, or cell scaffolds in low concentration, in low concentration, scaffolds or cell in high concentration, hydrogel the fibrin However, rapid gelation property. due to its 3D shape. to maintain a and fragile is too soft a tissues contain from different matrices (dECM) extracellular of native and glycoproteins proteins, proteoglycans variety of so it has been used as bioink capable tissue ECM components, microenvironment in printed of recapitulating a tissue-specific analogues. tissue/organ 3D logical activity. Generally, the stiffness of the cured composites the stiffness Generally, logical activity. or will increase with raising the concentration of nanoparticles be combined with other materials. Synthetic polymers can often bioactive materials, naturally derived materials or other func posite scaffolds combining a variety of biodegradable polymers posite scaffolds and bioactive ceramics are fabricated by 3D printing and are capable of achieving high mechanical strength and good bio tionalized materials to create more complex hybrid structures. both biomimetic properties in struc involve tissue scaffolds ture and the ability to regulate cell behavior. example, the photocrosslinkable macromers or prepolymers example, the photocrosslinkable macromers multi-armed acrylated/methacrylated by prepared be easily can Photocrosslinked oligomers/polymers or branched polymers. a high degree networks have a high gel content, which indicates solvents used in these sys due to the of crosslinking. However, drying may occur after tems, shrinkage or swelling of scaffolds and mechanical or soaking, resulting in changes in structural above the melting properties. Some macromers can be heated cases, in such the suitable viscosity; temperature to obtain no obvious material no solvent is needed for bioprinting and other covalent cooling. Additionally, shrinkage is observed after in the 3D printable crosslinking systems have been developed Diels inks. A thermally reversible dynamic covalent tion was used to synthesize a printable PLA blend for dramati tion was used to synthesize a printable PLA of the scaffolds. cally improving both strength and toughness are being developed with the aim of increasing the mechanical are being developed with the aim of increasing stability or improving tissue interaction. scaffold 3.2.3. Latest Development of Bioinks may provide a prom The chemical modification of biomaterials bioinks. 3D printable for extending approach ising lularization are with ensuring the complete removal of cel lularization are with maintaining of the fine vascularture lular components while and other tissue structures. cells are grown on decellularized tissue been observed when decellulariza potentially due to the retention of the scaffolds, tion detergent. techniques that mimic the critical aspects of natural healing and growth cascade are preferred to augment the proliferation this is cells; implanted or the recruited of differentiation and achieved through the integration of growth factors and often cytokines that provide suitable biochemical and physicochem ical factors for tissue regeneration. Engineering these dynamic further control ECM mechanisms into biomaterials offers lulose, and dextran, among others, have been developed for 3D among others, have been developed for 3D lulose, and dextran, or engineering tissue in use potential with scaffolds printing other biomedical applications.

------[94] Hyaluronic [96] Additionally, the Additionally, Acrylate or meth Acrylate 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © [102] [98] They can also pro The crosslinking pro The reversible gelation [50] C and a random coil struc [96,97] ° [20,50,81] Limitations of HA as a bio [38] Both Col and Gel have abun Both Col and Gel It is widely used as surgical glue as surgical used is widely It ) instantaneously, making it attrac ) instantaneously, + [101] 2 However, the high cost and weak and cost high the However, , 1601118 [103] 6 , , Zn + 2 [99,100] 2017 This material is composed of two repeating This material is composed of two repeating

[96] C. Gelatin methacrylate (or gelatin methacryla C. Gelatin ° C limiting its direct application as the cell scaffold at physi C limiting its direct application as the cell scaffold ° Natural polymers and their chemical modifications are the Natural polymers and Alginate (Alg) is an anionic polysaccharide derived from Alginate (Alg) is an anionic polysaccharide Adv. Healthcare Mater. Adv. www.advancedsciencenews.com monosaccharides (i.e., L-guluronic and D-mannuronic acids), monosaccharides (i.e., L-guluronic and multi using be formed can hydrogel ionic the typical therefore, valent cations (i.e., Ca ture above 40 mechanism in aqueous conditions is based on the formation of an alpha helix structure below 30 mide, GelMA) has been widely used to fabricate scaffolds via has been widely used to fabricate scaffolds mide, GelMA) the various light-based printing platforms. acid (HA), or hyaluronan is a linear polysaccharide compo acid (HA), or hyaluronan is a linear polysaccharide which has nent of the ECM (non-sulfated glycosaminoglycans), treatments such as been used clinically for several decades for therapy for damaged joints and arthritis. mechanical strength limit its application. Gelatin (Gel) derived (Gel) Gelatin mechanical strength limit its application. possesses also and inexpensive, is Col hydrolyzed partially from thermosensitive properties. acrylate modified HA can be crosslinked to form a hydrogel a to form crosslinked be HA can modified acrylate of HA can via light based 3D bioprinting. Thiol-modification reactions with form a hydrogel through Michael-type addition active vinyl based crosslinkers. aforementioned thiol-ene crosslinking method has also applied materials. thiol-based adding system when the GelMA to sure. Additionally, their derivative copolymers including poly including copolymers derivative their Additionally, sure. PEG or Pluronic F127 is generally crosslinked under UV expo crosslinked under F127 is generally PEG or Pluronic esters and polypeptides, among others, have been widely syn others, have been polypeptides, among esters and cell for used and hydrogels chemical or physical as thesized printing applied in 3D cellular which can also be encapsulation, and manufacturing. printable biomaterials and encapsulating most widely used as the to components their of similarity the to due cells living native tissue microenvironment. cesses are reversible, however, so the printed structures cannot so the printed structures cesses are reversible, however, applications. culture longterm for maintained be ological temperature, thus it commonly serves as the sacrificial material in 3D printed structures. vide tissue-specific biochemical and physical stimuli to guide and physical stimuli to guide vide tissue-specific biochemical migration, proliferation, differen cellular behaviors including chemical crosslinking approaches. tional groups to induce algae or seaweed. tiation, and maturation. For use in bioprinting, natural poly use in For tiation, and maturation. of in several ways, to include the use mers have been employed or ionic interaction characteristics the temperature sensitivity and the use of covalent addition of func to facilitate extrusion, dant proteins including fibronectin, vimentin, vitronectin, and arginineglycine-aspartic acid (RGD) peptides, which promote around lies Gel of temperature transition The adhesion. cell 30 Fibrin is comprised of fibrinogen monomers that are cleaved Fibrin with thrombin by a blood coagulation crosslinking mechanism, thus fibrin plays an important role in the blood clotting and healing processes. wound tive for 3D tissue/organ printing. tage in 3D printing. 3D in tage material for bioprinting are that HA hydrogels are typically too material for bioprinting are that HA hydrogels swelling significant show and structures, robust form to soft Collagen (Col) is a main structural protein of ECMs; behavior. gelation it responds to simple crosslinking via thermosensitive be a major advan under physiological conditions, which can REVIEW 1601118 www.advhealthmat.de ments. Cells usedfor3Dprintingshouldtakeintoaccountseveralele 3.3.1. PrinciplesofCell Selection tissues/organs. cells forcontributingtocompletefunctionalityofcomplex printed construct,butalsohaveacloserelationshipwithother only involvethearrangementofprimarycelltypesin3D and integratewithhosttissueororgan. homeostasis, self-renew, respond totissuedamageorinjury, the bioprintedconstructmustbeabletomaintaincellular In ordertomaintainlong-term function after implantation, 3.3.2. Cell Sources development involvesbiologicalsignalpaths. different cell types; (6)Interactionof multiple cellsfor tissue (5) Physiologicalspecificitybothstructuralandfunctionalon ronment, includingphysicalforcesandbiologicalstressors; oped after implantationbystimulationwiththein vivoenvi in vitrotocloselymimicthetruephysiologicalstate,anddevel in vivoimplantation;(4)Cellular functionscanbemaintained 3D printedscaffolds arerequiredforeitherinvitrocultureor priate cellproliferation and controllabledifferentiation inthe to surviveduringorafter thebioprintingprocess;(3)Appro in vitrocultureforbioprinting;(2)Cells mustberobustenough clinical application. functionality of the fabricated construct, especially for future The choiceofcellsfortissueororganprintingiscrucial 3.3. Cell SourcesandSelection effects beforeadvancingtoinvivotestingandapplication. understood andcontrollablestructuralfunctionalbiological applications. Also, itisessentialthatthese materials havewell- sion mustbebetterunderstoodforprogressiontowardclinical degradation characteristicssuchastimeandbyproductemis structs usingbioprintingtechnology. Material printabilityand to incorporatethesebiologicallyinspiredmaterialsintocon over cellbehavior. Onechallengeisindevelopingmethods bioprinted constructsfortissue regeneration.Examplesofthese some limitationsmakeitdifficult toapplyautologouscellsin approaches, to avoid negative immuneresponses. However, tiation of autologous stem cells or through reprogramming the patientsthemselvesthrough thegenerationanddifferen cells isthepreference,autologous cellsmaybeobtainedfrom tion ofexogenouscells.Therefore,theautologoussource of primary cells. rounding forfunctionalmaintenanceanddevelopmentofthe or areinvolvedinvascularizationprovideanessentialsur types thatprovidesupportive,structuralorotherfunctions, primary functionalcelltypes,mosttissuescontainvarious be recapitulatedintheregeneratedtissue.Inadditionto multiple celltypeswithspecificbiologicalfunctionsthatmust Host immuneresponsemaybetriggeredbytheimplanta (14 of29) [16,20,81] (1)Sufficient numbersofcellscanbeexpanded [20] Therefore,theoptionsforprintingcellsnot [108] wileyonlinelibrary.com Tissues andorgansarecomprisedof [16,20] © 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim ------stimulation invivo. composition of the ECM as well as the native biomechanical various celltypes,gradientsandarrangementofbiologicals, environment, suchasspecificorganizationandhierarchy of functions, thusacompleteunderstandingofthetissuemicro cellular components,needstoreproducespecific Regeneration ofatissueororganincludingcellularandextra 3.4. ModularFabrication ofMini-tissue regenerating complextissue/organ. cell printing can providea simple andeffective approach for bination oflocationorspatialarrangement.Therefore,3Dstem ferentiated into target cell types by bioactive factors in the com for complextissue/organregeneration.Stemcellscanbedif factors, mayreducethecomplicationoffabricationprocess seeding stemcellsontheconstructwithregionalbioactive Printing stemcellswiththeregionalbioactivefactors,orpost- specific cellsonthedesiredregionsofcomplex3Dstructure. step. Additionally, acellularprintingisdifficult topost-seed allel, requiringcomplicatedandprecisecontroloftheprinting bioinks withdifferent cellsneedtobepreparedprintinpar spontaneous self-organization happensduringdevelopment gation alongwiththe3Dprinted scaffold degradation. through directassemblyinscaffold-free conditions oraggre tissue/organ developmentrelies oncellularself-organization them toassembleinto3Dliving structures.Theprocessof niques canbeusedtoprintthese buildingblocksandguide easily beassembledtocompletetissues.3Dbioprintingtech proliferation anddifferentiation abilities. the printingprocesseshavenoadverseeffects onthestemcell obtained throughoptimizingtheprintingparameters;overall cations. According topreviousstudies,highcellviabilitycanbe for clinicalusesandshowgreatpromisebioprintingappli multipotent differentiation potential, they are considered safer biomedical applications.Althoughtheyhaveamorelimited cells, hepatocytes,andneuralcellscanbeusedinmany cytes, adipocytes,cardiaccells,endothelialsmoothmuscle ical orothersourcescandifferentiate intoosteoblasts,chondro Adult mesenchymal stem cells from bone marrow, fat, umbil ficulty andlimitationsassociatedwiththecurrentcellsources. requires reprogrammingthecelltypethusovercomingdif further differentiation. mary cellswithsupportingorprogenitors/stemfor regeneration need to be fabricated with either functional pri notypes. bility togeneratemultiplefunctionaltissue-specificcellphe ferentiated butmultipotentstate(self-renewal) andtheircapa ising celltypesduetotheirabilityproliferateinanundif cells (ES)andinducedpluripotentstem(iPS)areprom sourced cells.Pluripotentstemcellsincludingembryonic of manyprimarycell types, andthe effectiveness of patient- vitro cultureofcells,finiteexpansionorregenerationcapacity challenges are:techniquerestrictionsontheisolationandin blocks. tional components,whichcanbedefinedasfunctionalbuilding an aggregatestructureconsistingofsmallstructuralandfunc The 3Dprintedconstructsforthecomplextissueororgan [23,36,50] [20,38] Especially, iPSderiveddirectlyfromadulttissues Modularfabricationofthesebuildingblockscan [17] Tissues ororganscanbeconsideredas [16,23,26] Adv. Healthcare Mater. Incellularprinting,multiple www.advancedsciencenews.com [50]

2017 , 6 , 1601118 [20] This ------REVIEW

------[65] [87] 1601118 [4,17,26] Prior to reca (15 of 29) [16] www.advhealthmat.de wileyonlinelibrary.com In complex tissue/organ bioprinting, the In complex tissue/organ bioprinting, the [20,38] [38,65] Compared with slow permeation in static culture, the use of a in vivo, could be more ben or direct implantation bioreactor, mass trans eficial for ensuring homogenous and efficient port. printed constructs facilitates efficient transfer of nutrients printed constructs facilitates efficient and oxygen. This perfusion system can be used to mimic haemodynamic forces and pressures that occur naturally in the human body thus improving ECM production and mechanical properties of the artificial tissues/organs. out any supplement of extra nutrition, the cell viability will out any supplement of extra nutrition, the Therefore, the bioprinting time, including decrease severely. incorporation of preparation time, should be shortened; the improve culture media during printing can also significantly cell viability. printing process directly encapsulated in the bioinks in the it is essential or are post-seeded onto the printed constructs; to supplement them with nutrients and oxygen during the fabrication, the constructs with viable culture periods. After cells must not only remain viable for the long culture period, but also must be able to function as intended. Bioreactors, load or compressive perfusion, tensile typically employing ing, rotation or other conditioning, may provide a dynamic surrounding and mechanical stimulation for cell culture in vitro, mimicking the native fluid environment and physical forces in vivo. procedure and the sensitivity of cells may impact the cell procedure and the sensitivity of cells may If the time is long and the cells are fragile, then with viability. The requirements for 3D printing biological tissues and The requirements for organs can be summarized in three elements: (1) Biomimetic in three elements: (1) Biomimetic organs can be summarized and resolution); (2) Biocompatible and bio structure (modeling or Bio-microenvironment, either in vitro active components; (3) The first two and biochemistry. in vivo, including biomechanics parts of this bioink and the technique discussed in have been bioprinting techniques and bioinks manuscript. The various of multiple cell types can facilitate the hierarchical fabrication into desirable tissue types. and direct them to differentiate provide an adequate The post-bioprinting process is crucial to chemical or bio bio-microenvironment including mechanical, growth. remodeling and tissue to regulate signals logical by medium flow through the 3D Shear forces created The development of new bioreactor technologies enables the The development of new bioreactor technologies for sur rapid maturation of tissues, multiscale vascularization vivability of tissues, and mechanical integrity and innervation vivability of tissues, and mechanical integrity for implantation. structures compared to all traditional tissue engineering tech tissue traditional all compared to structures niques. Although avascular or thin tissue bioprinting has shown shown has bioprinting tissue thin or avascular Although niques. organ bio complex tissue or in current research, great promise a challenge. implantation remains printing for tissue is creating functional organ-level complexity, pitulating of contains a hierarchical arrangement an essential stage that includes This microenvironment. 3D a in types cell multiple along type, tissue on depending cells functional primary the of vasculature in stroma and paren with a multi-scale network chyma, as well as lymphatic vessels and neural networks. chyma, as well as lymphatic post-bioprinting process or the tissues/organ regenerated pro post-bioprinting process or the tissues/organ The cells are transport and mechanical stimulation. Mass (2) cess involves three phases: the time of printing cellular bioprinting, For viability. Cell (1)

------[110] The After After [20] [109] It allows the [35] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © m can be printed as building [23] µ , 1601118 This technique enables scaffold-free scaffold-free This technique enables 6 , [23] Tissue spheroids are thought to possess Tissue [17,23] 2017 [23]

[15] Moreover, the multiple cell types can also be organ be also can types cell multiple the Moreover, [20,23] One advantage of this technique is potentially accelerated One advantage of this technique is potentially It is noteworthy that in either biomaterial based bioprinting It is noteworthy that in either biomaterial As previous mentioned, bioprinting techniques have the In addition to the traditional “cell-scaffold” approach, cell approach, “cell-scaffold” the traditional to addition In Adv. Healthcare Mater. Adv. www.advancedsciencenews.com obtaining sufficient mechanical integrity, aggregates with diam with aggregates integrity, mechanical sufficient obtaining eters ranging from 200 to 500 blocks to form 3D structures. However, the directly printed con blocks to form 3D structures. However, structs are fragile and lack cohesive tensile strength. Therefore, structs are fragile and lack cohesive tensile formation the for process important very a is fusion successful ability of multicellular of 3D structures that rely on the cohesive of ECM, aggregates and additive properties. The accumulation enhancing tissue associated restriction of cell motility and kinetics or impede the cohesion in tissue spheroids can change fusion process. tissue spheroids’ precise simultaneous 3D positioning of multiple cell types with high density to mimic their natural counterparts. tissue organization and the ability to direct the formation of tissue organization and the ability to direct complex structures. or scaffold-free bioprinting, cell-laden bioprinting techniques bioprinting, or scaffold-free for regenerated and oxygen transport require suitable nutrient vasculature or architectures pore proper through tissues/organs analogues. ultimate goal is industrial, scalable, biofabrication of patient- specific functional 3D living human organs suitable for clinical implantation. most tremendous potential on manufacturing the complex ized during cell aggregation, especially endothelia cells can be can cells endothelia especially aggregation, cell during ized vascularized cellular aggregates. co-cultured to form 3D bioprinting of organs is a comprehensive or integrative a pathway for scalable and reproducible approach that offers mass production of engineered living organs. 4. 3D Bioprinting of Organs 4.1. Definition, Elements and Procedure aggregation is a typical technique of tissue fabrication, in of tissue fabrication, is a typical technique aggregation blocks directly used as building cellular units are which solid to engineer the tissues. in vivo, but has also been recapitulated in numerous in vitro in numerous has also been recapitulated in vivo, but applications. bioprinting, even free of exogenous biomaterials. The con bioprinting, even free the knowledge of developmental biology cept originates from and organs are formed without any and the fact is that, tissues cellular Generally, development. during embryonic scaffolds have some typical procedures: (1) Cell aggregation techniques pellet Cellular (3) aggregation; cell of Initiation (2) expansion; as cylinder or sphe molding, such collection; (4) Geometric roid. material properties that can replicate the mechanical and func material properties that can replicate the tional properties of the tissue ECM. Moreover, by manipulating by manipulating tional properties of the tissue ECM. Moreover, can be con the host bioactive composition, self-organization in assisting the ECM trolled. Unlike the critical role of bioinks bioprinted self-assem production in traditional 3D bioprinting, bling cellular spheroids may produce a suitable ECM environ bling cellular spheroids may produce a suitable ment by themselves. More importantly, it enables the creation of functional com it enables More importantly, networks. and neural vasculature with plex tissues REVIEW 1601118 www.advhealthmat.de Furthermore, themechanicalcues,presentaton pancreatic, orlivertissue.Vasculature withinthetissuesor high volumetricoxygen-consumption rate,suchascardiac, organs mayresult,especially when scaling-uptissueswitha these, lowcellviabilityandmalfunction ofartificialtissues/ (3) Construct maturation.Inthisstage,thecellsmustproliferate necessary forengineeredtissue maturation. exchange, andmetabolicwaste removal,allofwhicharealso require thesupplementationofadequatenutrients,gas To maintainmetabolicfunctions,thenativetissuesororgans 4.2.1. Vasculature 4.2. 3DBioprintingApplicationsinOrganRegeneration future perspectives. toward clinical translation and provide potential solutions and and organconstructs.We willalsodiscuss majorroadblocks including vasculature/vascularizedtissue,neuralregeneration, of complextissueandorganconstructsforimplantation, will presentrecentdevelopmentsonbioprintingscale-ups neering technologiesarefacing.Inthefollowingsection,we structs, whichisalsoaproblemthemajorityoftissueengi cular networkandaneuralinthe3Dprintedcon reality. Themostcriticalchallengeistheintegrationofavas be progressivelyaddressedtomakeorganprintingbecomea post-implantation. efficacy, andmonitoringofconstructintegrityfunction manufactured constructimmuneacceptance,invivosafetyand in vivointegration.Thisphasewillinvolvetheissueofbio neered tissueneedstobeimplantedintothepatients’ bodyfor After in vitro maturation of 3D printed constructs, the engi Therefore, asmentionedabove,severalchallengesmust tissue formationandfurtherintegration. cal conditions,havebeenknowntopositivelyinfluence set of several signaling pathways in normal physiologi populations tospontaneouslyreorganizeinto3Dtissue. actions betweencellsandcell–matrixoffer theabilityofcell such astightjunctionsandadherensjunctions.Theseinter matrix adhesions,suchasintegrins,andcell–cell time. Overtime,cellsreachequilibriumstatesbetweencell– can alsobereplacedbydepositednativeECMwithincreasing cell development,bothbiomechanicallyandbiologically, and The constructsmayprovideanappropriateenvironmentfor celerated usingtechniquessuchasmechanicalconditioning. involved inmaturationthatwasdiscussedabovecanbeac further integratingintothehosttissue.Thephenomenon matrix componentsandperformnaturalbiologicalfunctions, ing witheachother; theymustbeabletosecretetheirown to formtheappropriate cell-cell connections for communicat of theconstruct. ity, aswellincreasematrixreorganizationandmaturation organs experienceinvivocanincreasestrengthandflexibil that mimicsthephysicalforcesitscorrespondingtissues or composition, periodic stretching,pulsing,orcompression to the intrinsic mechanical stimulation from biomaterial (16 of29) [16,20,50] [38] wileyonlinelibrary.com © 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim [111] [112–114] Inaddition Without [38] ------basement membrane(40 boresistent confluentmonolayerofECsandisattachedtoa blasts andperivascularnerves. outermost layer, isacollagenousECMcontainingmainlyfibro SMCs withbandsorfibersofelastictissues,andadventitia,the is comprisedofadensepopulationconcentricallyorganized construct. sufficient nutrientsandoxygentothecellsinsideoftissue inosculation withmicrocirculationinvivoistooslowtoprovide angiogenic growthfactors),theprocessofangiogenesisand healing responseandhypoxia-inducedendogenousreleaseof cularize after implantation(involvinganinflammatorywound- only consistofEClayer. Althoughgrafts canspontaneouslyvas (<6 mm)vasculaturecurrentlyremainsaformidabletask. large-diameter vasculargrafts, manufacturingsmall-diameter in vivo.Comparedtothecommercialandclinicalsuccesses in The rateofsproutingangiogenesisisaround1.0mmperweek activation, migration,andproliferationaswellarrangement). ogenesis process (a complex cascadeof events including ECs sprout fromthelargebloodvesselsbasedonnativeangi to considerthefabricationofcapillaries,becausetheycan ally, fromatissueengineeringstandpoint,itisnotnecessary prone tonecrosisafter longer implantationtimes.Addition from thearteriovenous(AV) loopandthetissue, invivo,was to theformationofvascularizedtissue,capillariesaretoofar only capillariescanbecreated.Althoughsuchdesignslead organize intobloodvesselsautonomously(angiogenesis),thus blood vesselsinartificialtissuereliesontheabilityofECsto complex tissuesandorgans. network, whichisalsoamajorproblemin3Dbioprinting of tissue andorganregenerationistheintegrationofavascular titia respectively. from the vessel lumen: intima, tunica media and tunica adven muscular arteriesandveinshavethreedistinctlayersstarting printing strategies,isabsolutelyessential. adequate vesselgeometriesanddimensionsthrough3Dbio of interconnectedvasculature. without prefusablechannelshasdevelopedforself-assembly maintaining tissuefunctions. organs iscrucialfortransportingoxygenandnutrients eter ofbloodvesselsrangesfrommicroscopicsize,5 multi-scale and multilayer arrangements. The inner diam cular anatomy. laries arerequiredtobemanufacturedmimicnaturalvas where vasculartreesspanningarteriesandveinsdowntocapil fabricating hierarchicalandcomplexstructuralvasculature, (aorta). for thesmallestcapillaries,to30mm,largestartery orders. units willformahierarchicalstructure withdifferent branching branching unit(abifurcatedpattern); therepeatofmulti-scale a challenge.Themainunitof thevasculartreeisa‘Y’shape toward functional,mechanically integratedvasculatureisstill ability. apoptosis andultimatelycelldeath,duetolimiteddiffusion 200 Encapsulating ECsorSMCsintobioprintedconstructs Native bloodvesselshavecomplexuniquestructuresin 3D bioprintingtechnologyiscurrentlyunsuccessfulin µ m awayfromthenearestcapillarieswillundergohypoxia, [112] [109,115] [116] Therefore, the most critical challenge for complex [120] Walls ofthelargevessels,namelyelasticarteries, Theconstructsatsubmicrometer scalearedifficult Obtaining a functional vasculature, consisting of [112,114] [117,118] Moreimportantly, successfulmaturation Intima,theinnermostlayerisathrom ∼ 120 nm).Media, themiddlelayer, [26,109,110,115] Adv. Healthcare Mater. [122] [113] [119] Thismethodofgenerating Cells existingmorethan Incontrast,thecapillaries www.advancedsciencenews.com [112,118,121]

2017 , 6 , 1601118 ∼ 10 [123] µ m ------REVIEW

- - - - [85] [125] 1601118 a). After After 4

After a After [125] 1cm thick 1cm > Copyright 2012, Copyright Figure (17 of 29) [95,124,126] [125] In other studies, www.advhealthmat.de Copyright 2014, Wiley- Copyright [126] [124] Copyright 2015, Wiley-VCH. 2015, Copyright [136] 1 mm in the GelMA hydrogel 1 mm in the GelMA m. Reproduced with permission. ∼ µ m Copyright 2011, Wiley-VCH. (c) Confocal Copyright µ [95] wileyonlinelibrary.com The 3D printed constructs were incubated at The 3D printed constructs were incubated In a thick osteogenic tissue model ( osteogenic tissue a thick In [127] [124] in volume), the embedded vascular network ensured in volume), the embedded 3 crosslinking the ECM gels, the glass filaments were dissolved glass filaments were the ECM gels, the crosslinking intervessel junctions. In co-cultures with to form vessels with space, endothelial cells lining the 10T1/2 cells in the interstitial surrounded by the 10T1/2 cells and vascular lumen became the extending from sprouts multicellular single and formed into the bulk hydrogel ( patterned vasculature and 10 cm a straight EC-laden gelatin line was printed inside of the col a straight EC-laden gelatin line was printed lagen layers. Thermosensitive fugitive ink, Pluronic F127 was also explored explored was also ink, Pluronic F127 fugitive Thermosensitive 4b). networks (Figure print microvascular to significantly increased cell viability near the channel regions near the channel increased cell viability significantly In an with other deeper regions. construct compared inside the was used as a cytocompatible carbohydrate glass earlier study, 3D filament lattices. template to print rigid sacrificial cooling process, the perfusable channels possessed final diam possessed channels perfusable process, the cooling 100 eters ranging from ca. uniform and long-term perfusion culture throughout the con the throughout culture perfusion and long-term uniform of encapsulated struct, promoting the osteogenic differentiation human mesenchymal stem cells (hMSCs). (Figure 4c). (Figure - - 10 mm) Reproduced with permission. = 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag Copyright 2016, Wiley-VCH. (g) Confocal microscopy images show interconnected structures of the 2016, Wiley-VCH. (g) Confocal Copyright © [87,95,124] [87] : In indirect bioprinting, cell-laden: In indirect bioprinting, , 1601118 6 , 2017

m. Reproduced with permission. µ red) were perfusable vascularized tissue constructs. A confocal z-stack montage demonstrating HUVECs (expressing mCherry, (a) 3D printed Copyright 2016, Wiley-VCH. (f) Copyright fluorescence images of hMSCs and HUVECs co-cultured in designed vascular channel regions for Confocal [130] Here, we focus on the manufacturing of interconnected chan we focus on the manufacturing of interconnected Here, Adv. Healthcare Mater. Adv. www.advancedsciencenews.com Nature Publishing Group. (b) Fluorescent image of a 3D microvascular network fabricated via omnidirectional printing of a fugitive ink (dyed red) Nature Publishing Group. (b) Fluorescent image of a 3D microvascular network fabricated via within a photopolymerized Pluronic F127-diacrylate matrix. (Scale bar VCH. (d) Confocal fluorescence images of hMSCs and HUVECs co-cultured on various scaffolds in a static culture condition for 5 days. hMSCs fluorescence images of hMSCs and HUVECs co-cultured on various scaffolds VCH. (d) Confocal bars indicate 200 were labeled with cell tracker green, and HUVECs were stained with cell tracker red. The scale 1 week. HUVECs encapsulated in the hydrogel were inclined to aggregate and migrate to form annular ring patterns along the channel. The scale bars scale The channel. the along patterns ring annular form to migrate and aggregate to inclined were hydrogel the in encapsulated HUVECs week. 1 indicate 200 4.2.1.1. Sacrificial Templates nels or free-standing tubular structures, allowing native blood tubular structures, allowing native blood nels or free-standing anastomosis. Over all, three approaches vessel ingrowth and (1) indirect bioprinting through utilizing have been developed: (a fugitive ink) that is removed to create sacrificial templates a bulk construct; (2) directly bioprinting hollow channels in of in a construct; (3) direct bioprinting interconnected channels or blood vessel in a tubular shape. a vasculature network the bulk matrix to fabricate vascularized hydrogels serve as are printed with tissue constructs; here the vascular networks is generated inside fugitive inks. The endothelium lumen the perfusion culture of endothelial tubular channels after network shows cells (ECs). The integration of the vascular sion. Copyright 2016, Wiley-VCH. (e) 3D-printed PPF scaffolds as venous interposition grafts at the time of in vivo implantation. Reproduced with permis 2016, Wiley-VCH. (e) 3D-printed Copyright Figure 4. throughout a bulk fibrin gel, after one day in culture. residing in the vascular space with 10T1/2 cells (expressing EGFP, green) uniformly distributed 1 mm. A partial z-stack of two intersecting channels demonstrated endothelialization of channel walls and across the intervessel junction, Scale bar, Reproduced with permission. while in the surrounding bulk gel 10T1/2 cells are seen beginning to spread out in three dimensions. to be printed using the current techniques. Therefore, instead techniques. Therefore, using the current to be printed some researchers tree, biomimetic vascular a of fabricating chan (bifurcated or branched) print perfusable alternatively nels at micrometer or higher scale to mimic a vascular network, to mimic a vascular or higher scale nels at micrometer for cell oxygen supplementation medium flow and facilitating and formation. tissue maturation viability, image of live HUVEC cells lining the microchannel walls using the same fugitive ink method. Reproduced with permission. image of live HUVEC cells lining the microchannel walls using the same fugitive ink method. encapsulated HUVECs after migrating to outer regions of the bioprinted fibers at day10. Reproduced with permission. with Reproduced day10. at fibers bioprinted the of regions to outer migrating HUVECs after encapsulated REVIEW 1601118 www.advhealthmat.de system ofmice. functionality for6monthsafter implantation inthevenous and wallthicknessesof150 enabled the printing of scaffolds with inner diameters of 1 mm and invivostudiesshowedthatthisgraft-fabrication strategy using a DLP based SLA technique (Figure 4e). graft wasfabricatedwithpoly(propylenefumarate) (PPF) into complexspatialstructures. tiple bioprinting platform and also facilitates patterning them tubular constructsallowsthemtobeintegratedwithinamul other fabricationmethods,directprintingoftheself-supporting tubes thatareprintedinsideamold pattern. Compared to lature; and(2)bioprintingofscaffold-free branchedvascular a tubularshapevia:(1)bioprintingofgrafts orvascu is directbioprintingofbloodvesselsorvascularnetworks in 4.2.1.3. DirectPrintingofTubular Constructs vessels underpulsatilearterialflow. structs canprovidesimilarflowcharacteristicstonativeblood 4.2.1.2. DirectPrintingofInterconnectedChannels tural designandthebiomimeticdistributionofmultiplecells. external constructsislimited,suchasthehierarchicalstruc on thebulkhydrogelconstructs,thusstructuralfabricationof explored. Finally, thestructureofvascularnetworkstotallyrely lation with microcirculation in vivo has yet to be systemically the traditionalscaffold design.Thefunctionality and inoscu constructs, which is similar with the interconnected pores in could providesufficient nutrientsforcellviabilityinsideof ally, thecurrentstudiesonly suggestthe3Dprintedchannel the multilayercellstructureofnativebloodvessels.Addition vasculature onlypossessesendotheliumandisunabletorepeat this techniquestillfacessomechallenges.First, thefabricated tion hasexhibitedfeasibility, flexibilityandangiogenicability, the bioprintedsacrificialtemplatemethodforvascularfabrica by SLA. in poly(ethyleneglycol)dimethacrylate(PEGDMA)hydrogels millimeter-sized branchedchannelsweredesignedandprinted approach tofabricationofvascularizedtissue.For example,the printing ofaconstructwithinterconnectedchannelsisanother 37 sprouts were generated with fully covered ECs. gelatin liquefaction.Thefunctionalvascularchannelsandthe for angiogeniccell-encapsulatingpatches. lution, as well as open fluidic channels with 100 utilized toprint3Dcell/biomaterialpatternswith<5 printer (Figure 4d). of fullyinterconnectedmicrovascularnetworksusingaFDM printed vascularizedtissueconstructwithauniqueintegration connected channels.Inarecentstudy, wedevelopeda3Dbio have alsobeenusedtoproducethemultidirectionalandinter subsequent layers.Therefore,other3Dbioprintingtechniques nels would be crosslinked by light exposure after printing the is stilllargelyrestrictedtouniaxialchannels.Theopenchan lution featuresandfluidicchannels,architecturalcomplexity based methodsarecapableofproducingextremelyhigh-reso technique basedontheFDM and SLAbioprintersystems. plex vascularizedtissueconstruct usingadual3Dbioprinting Recently, abiodegradable,3D-printed,acellularvascular ° C for30mintocompletethecollagengelationand (18 of29) [128] Inaddition,ahigh-resolution [130] Moreover, ourgroupalsodevelopedacom [85,129] wileyonlinelibrary.com Themicrovasculardesignofthecon µ m, whichsustainedpatencyand µ : Thethirdapproach © [63] SLA techniquewas 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim Althoughlaser [130] [127] Both in vitro µ : Directbio m diameter Although µ m reso [87] ------

with PVA (6%w/v)solution. tinuous extrusionofhighlyconcentratedalginate(16.7%w/w) fibers areprintedusingashell/corenozzlethroughthecon in 3Dprintedbloodvessels. tube structurewasprintedviaaninkjet-printingprocess. CaCl due to a lack of natural ECM receptors. Finally, the alginate/ layer. Second,theencapsulatedcellshavelimitedproliferation grow aroundthetubularconstructtoformendothelium adhesion duetoitsstronglyhydrophilicnature,thusECscannot erated bythedissolutionofPVA. diameter of the microgel beads. (Figure 4g). an innernozzleandcalciumchloridethroughouter GelMA fibersbyextrudinganalginate/GelMA mixturethrough coaxial extrusionwasusedtocreateagridofsolidalginate/ of, or along with, alginate gel is required. In a different design, ions. Therefore,theuseofmorebiomimeticmaterialsinstead its ionicinteraction,owingtotheexchangereactionwithother transferred toaCaCl tion ofalginateandcalciumchloride(CaCl along withthecrosslinkingprocess.Typically, thecombina crosslinker. Thefreestandingtubularconstructsaregenerated biopolymer, whiletheinnernozzlecontainscorresponding fluidic channels.Theouternozzlecontainsanuncrosslinked coaxial extrusionnozzlesareemployedtoproducefree-standing from 35 to 40 eters ofthetubularstructurescouldbeadjustedrespectively of endothelialcells(Figure 4f). generated inourengineeredconstructafter theencapsulation A biomimetic vascular lumen with capillaries was successfully tissue construction. of bloodvessels,however, itisnotanidealmaterialforliving concentrated, cell-ladensodiumalginate(2 CaCl outer tubeofacoaxialnozzleisimmediatelycrosslinkedby this design,thesodiumalginatesolutionflowingthrough lized tofabricatehollowtubeswithoutanysupports. height of5mm. height of 10mm,anoverhang angle of 63 droplets weredepositedtoformafreestandingtubewith By refining the operating conditions, 210 layers of Ca-alginate tubular structures. into alginateinkdropletsbylaminatingprintingtofabricate printing. currently achievebranchedstructures. extrusion oftubularstructures,thisprintingmethodcannot nozzle systemoffers averystraightforwardapproachfordirect but itdidnotprovideabiomimetic, hollow, vascularstructure. questions oftheinstabilityand weakbioactivityofalginategels, fibers forendotheliumformation. Thistechniqueaddressedthe ECs encapsulatedinthefibers could migratetotheedgesof nected fluidicchannelsinthe constructs.Theresultsshowed crosslinked alginatedisintegrated leavinghollow, intercon ther reinforcethefibers.Overcultureperiod,ionically crosslinking, GelMA was covalentlyphotocrosslinkedtofur In oneparadigmfordirectextrusionoftubularstructures, Alginate hasshownpromisingadvantagesforfabrication The alginate/CaCl 2 2 solutionthroughtheinnertube. hydrogelisunstableforalongculturetimebecauseof [45] Microgelbeadswereformedbydiffusion ofCa [136] µ After a temporary solidification by physical m and from 30 to 200 [135] [47] [50,96,97,136] 2 Thewallthicknessandtheinnerdiam 2 systemhasalsobeenappliedtoinkjet solutionandthehollowfibersaregen Adv. Healthcare Mater. [131,132] [133] First, itdoesnotpromotecell [87] After printing, constructsare [47] www.advancedsciencenews.com Thestacksofself-standing In addition, a 3D zigzag [133] [134] µ m through varying the Furthermore, thelow Although,thecoaxial ∼ ° 2 6%) hasbeenuti andan overhang ) arewidelyused

2017 , 6 [131,134] , 1601118 [135] In 2 + ------

REVIEW

- - - - - [145] 1601118 [150,151] It consists of (19 of 29) [145] www.advhealthmat.de On the other hand, On the other [85,87] In our previous review and Although the PNS has a greater [146,150] Misdirection towards the wrong At the cellular level, neurons are the are neurons level, the cellular At wileyonlinelibrary.com Following the PNS injuries, Wallerian the PNS injuries, Wallerian Following [149,150] Therefore, tissue engineering techniques engineering Therefore, tissue [149–151] [147,152,154] The CNS consists of the brain and spinal The CNS consists of the brain and spinal [152] [152,153] The PNS consists of nerve fiber (Schwann cells The PNS consists of nerve fiber (Schwann [145] [146–148] Prior to creating neural networks, the repair of nervous Prior to creating neural sure, sufficient interconnectedness of endothelium to prevent interconnectedness sure, sufficient patency rate to support occlusion-free thrombosis, and a high with indirect bioprinting of a vascular circulation. Compared be bioprinting of tubular construct can network, the direct anastomosis to the host. more convenient for 4.2.2. Neural Networks system is widely distributed throughout The human nervous vasculature. which is similar with the aforementioned the body, that processes biological the all controlling for responsible is It as well as transmit coordinate voluntary and involuntary actions parts of the body. signals among the different ticity and tensile strength) to satisfy retention and burst pres satisfy retention and tensile strength) to ticity and the design and bioprinting of vascular networks should be vascular networks and bioprinting of the design possess vessels, thus it should to native blood easily connected (elas mechanical properties such as proper certain properties, book, we have discussed the self-repair process of the nervous the of process self-repair the discussed have we book, system in detail. core components of both the CNS and the PNS, which connect the CNS and the core components of both neural networks. to each other to form neural circuits or capacity for axonal regeneration after injury, spontaneous injury, capacity for axonal regeneration after peripheral nerve repair is nearly always incomplete with poor functional recovery. nological rejection. To overcome these disadvantages, current disadvantages, these overcome To rejection. nological research is focused on the development of novel tissue engi neering alternatives to repair peripheral nerve gaps. target reduces functional outcome, thus grafts between the target reduces functional outcome, thus grafts nerve stumps are required to bridge the gap and support axonal ability in the PNS shows poor self-repair regrowth. Additionally, in clinical treatments large defects. Utilization of an autograft inevitably creates additional nerve injury and loss of function induce immu techniques often near the donor site; allografts degeneration commonly occurs, and SCs are activated to con degeneration commonly occurs, and SCs enabling guided tribute to forming the bands of Büngner, the reconstruction process axonal regeneration. However, cannot take place in CNS injuries because the CNS lacks SCs. growth axon both impeding forms, tissue scar glial Instead, and myelination. cord and is responsible for processing the information from cord and is responsible for processing the PNS. the central nervous system (CNS) and the peripheral nervous the central nervous system (CNS) and the system (PNS). Based on biological viewpoints, the primary function of the Based on biological viewpoints, the primary via connecting the nervous system is to control the whole body fabrication of neural brain to all tissues/organs. Therefore, the tissue and organ networks is an essential process for complex regeneration. and disorders system injuries caused by disease, trauma remains a formidable task. (SCs) wrap around the axons) bundles and connective tissues (SCs) wrap around the axons) bundles and the CNS and limbs/ that detect and transmit signals between and sensory efferent) peripheral organs through motor (or nerves. afferent) (or effect on angiogenesis in our works. on angiogenesis effect combining cells and scaffolds is the most feasible approach for combining cells and scaffolds CNS regeneration.

------In [135,142] [140] Concentri [138] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag m luminal diameter © [139] µ In another study, gelatin In another study, The presented artificial [138] [140] After removing the template, the removing After [141] m, using a polytetrahydrofuranet µ , 1601118 6 Various vascular cell types, including Various [143,144] , [143] 2017

[143,144] The printed networks showed the reduced cross- reduced showed the networks printed The [137] [141] Combination of high-resolution TPP with SLA enables the Combination of high-resolution Scaffold-free bioprinting provides an alternative method bioprinting provides an alternative Scaffold-free Combining direct extrusion and a supporting slurry of and a supporting direct extrusion Combining Although numerous 3D bioprinting approaches have Adv. Healthcare Mater. Adv. www.advancedsciencenews.com eter. Then they were printed by molding a sacrificial template sacrificial a molding by printed were they Then eter. or agarose. collagen of SMCs and fibroblasts, were aggregated into discrete units of either multicellular spheroids or cylinders of controllable diam fabrication of refined and complex geometries for printing and complex geometries for printing fabrication of refined structures or bifurcated tubular The blood vessels. tubular polymers and were created using photo-crosslinkable synthetic enabled the fabrica biopolymers. The high resolution of TPP tion of branched tube structures with 18 and wall thicknesses <5 herdiacrylate (PTHF-diacrylate). ibility, degradation behavior or potential immunogenicity. degradation behavior or potential ibility, based on cells and the genuine matrix they secrete while based on cells and the genuine matrix as biocompat such of biomaterials the complexity avoiding tial for this technique to produce biological structures with to produce biological structures with tial for this technique structure. heterogeneous internal hydrogel microparticles was also developed. This approach can developed. This approach was also hydrogel microparticles tubular networks. Photoreactive print hierarchically branching printing and was crosslinked after poly(vinyl alcohol) (PVA) of were recovered from the slurry fully crosslinked structures in stirred water. particles by immersion Carbopol were also printed, highlighting the poten cally nested objects emerged to fabricate the vasculature, generating vasculature with multi-scale, multilayer structures that replicate the geom etry, complexity, and longevity of human vascularized tissues and longevity complexity, etry, remains a challenge. In addition to fabricating the vasculature of in within the construct, the functionality and anastomosis vivo microcirculation should be taken into account for implan 3D printing and site- tation. On one hand, a strategy combining specific delivery of angiogenic factors should be developed to promote vascularization. Several studies have shown a positive erating the biomimetic mechanical properties in the range of erating the biomimetic mechanical properties native porcine carotid arteries. linking density and high contents of reversible H-bonds, gen linking density and high contents of reversible able hollow tubes using a blended bioink and alginate removal bioink and alginate tubes using a blended able hollow process. Subsequently, they modified the design to fabricate perfus to fabricate the design modified they Subsequently, rials. order to obtain a proper mechanical strength, dithiol-mediated order to obtain a proper mechanical strength, mate chain transfer was also applied to photo-crosslinkable cular reconstructions. The extrusion of cell aggregates consisting of either one cell The extrusion of cell aggregates consisting for the fabrication of type or several cell types was developed approach tubular structures. A fully biological self-assembly vas small diameter, was proposed to fabricate the scaffold-free, vascular constructs possessed 3D microstructured wall architec vascular constructs possessed 3D microstructured tures, including high porosity and high interconnectivity. the fusion of the discrete units resulted in single- and double- layered hollow vascular tubes. Unique advantages of this is capable of engineering it method are speed and scalability; blood vessels with distinct shapes/diameters and hierarchical structure. hydrogel microparticles were used with freeform reversible were used with freeform reversible hydrogel microparticles hydrogels (FRESH) as a support bath embedding of suspended arterial tree. to print embedded branching REVIEW 1601118 www.advhealthmat.de neural connectivityinthreedimensions. gested thepatternedneurons showed neuriteoutgrowthand could beusefulinprintednerveguidanceconduits. during printinganditwashypothesizedthatsuchgradients ents ofmoleculeswerepatternedalongthelengthchannels complicated nervousnetwork. of theconduitsandevenhavegreatpotentialforreplicating cision andcontroloftheinternalarchitectureoutershape for theorientedfeature.3Dprintingtechniquesoffer greatpre control overgeometryandinternalmicrostructure,especially tional scaffold or conduitfabricationtechniquesoffer limited ture andspatiotemporalchemicalphysicalcues.Conven neural cellsresidewithina3DECMwithmicro/nanoarchitec conduits (NGCs) with poly(ethylene glycol)resinwas printed using and the branched scaffolds can mimic a nerveplexus. allel tothelongaxisoflumencouldmimicnervefascicles successful fabricationofconduitswithmultiplechannelspar different numbersofchannels. geometries, includinghexagonalandcircularpatternswith luronic acid(GMHA)wasprintedasscaffolds withdifferent outgrowth of neurites. Glycidyl methacrylate modified hya micro structuresthatmimicnaturalECMandfacilitatethe structs withhighresolution,creatinghighlyalignednano/ most typicaltechniquetocreategeometricallypatternedcon utilized toprintconstructsforneuraltissue engineering. onic dorsalrootganglia(DRG). (Puramatrix or agarose) was made to encapsulate the embry supplying structureandacell-permissive,self-assembling gel conduit, where PEG hydrogel served as a cell-restrictive region over, adualhydrogel approachwasdevelopedforapatterned neurons werefirstfabricatedusingathermalinkjetprinter. structures ofprimaryembryonichippocampalandcortical neural tissue. In an earlier study, the controlled patterns and shown greatpotentialforfabricatingconduitsengineered 4.2.2.1. 3DPrintingforPNSRegeneration The photocurableformofPEG waspermissiveforneuronal differentiate well after printing. Schwann cellsandastroglialcansurvive,proliferate immunostaining studies. and promoting cellular differentiation which was confirmed by The bioinkperformedverywellinpreventingcellaggregation ology could be retained after bioprinting. of neurons,includingneuronalphenotypes,andelectrophysi The results showed cellular properties and functional fidelity and forminimalscarformation. motes cellsecretionofinductivefactorsforaxonalelongation provides adirectframeworkforneuronstoproliferateandpro at thetargetedsite.Thescaffold orconduitbridgesthelesion, implantation ofaneuralscaffold orconduitfabricatedinvitro different commercially available inkjet bioprinting systems. medium wasusedtoprintneuron-likePC-12cellsusing two low-acyl gellangum)inasurfactant-containingtissueculture novel bioinkbasedonamicrogelsuspension(endotoxin-free printer with4-channeldispenser. printed intosingle-layerandmultilayerconstructsusingabio 3D collagenconstructionwithastrocytesandneuronswas Overall, nervoussystemregenerationinvolvesthesurgical (20 of29) wileyonlinelibrary.com ∼ 50 mm resolution from photocurable [157] TheLIFTtechniquehasalsobeen [34,152,153,155] [160] [159] [158] [161] [145,152] Amultilayered, on-demand Theauthorsproposedthat In 3D printing, SLA is the Theimmunostainingsug : Inkjetbioprintinghas Inthehumanbody, [161] [156] © µ 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim Nerveguidance SL technique. Furthermore, a [159] [159] More Gradi [162] [157] [158] [156] ------

tion, andinductionoftheendogenicrecoverysystems. beneficial effects suchasanti-inflammation,neuralcellprotec various cytokines and growthfactorsthatgenerateavariety of printing (Figure 5c). was utilized tocreate neural constructs via extrusion-based 3D of graphenewithsmallamountspolylactide-co-glycolide, printable graphene (3DG) composite liquid ink consisting most lation andmoreeffectively guideneuraltissuerepair. A3D stimulate andcontrolneuronactivitiesunderelectricalstimu highly elongated morphologies with features similar to axons neurogenic differentiation. ThiscoincideswithhMSCs adopting genic stimuli,revealthat3DG supports MSCproliferationand than 800S/m.Invitroexperiments, intheabsenceofneuro cally robust and flexible with electrical conductivities greater growth wasobserved. sensory functionsexhibitedrecoveryeventhoughlimitedaxon parable levelonthesciaticnervedefectmodel.Bothmotorand collagen conduitgraft, the bioprintedgraft performedatacom especially for CNS regeneration. a promisingtherapeuticoptionforneuraltissueregeneration, ricate scaffold-free conduitsusingMSCandESCcylinders. lineage. embryonic stem cells(ESCs) and MSCs towards a neurogenic were made by SLA to guide the growth and differentiation of The micro-well/channelarraysastopographicnetworkpatterns in eachrepairgroupbeingsimilar( after 21days,withthenumberofuniqueaxonsatdistalend rescently labelled peripheral axons) 3 mm gap injury model YFP tively withanautograft controlinathy-1-YFP-Hmouse(the had acceptablehandlingpropertiesandperformedcompara growth andexperimentaldifferentiation invitro.Theconduits stitutes andtargetinjurednervetissue. axon outgrowthandenhanceconnectionbetweenartificialsub tological assessment. results closetothatofautografts intermsoffunctionalandhis porting re-innervationacrossa10mmsciaticnervegap,with implantation inaratmodel,thebio-conduitwascapableofsup up-regulated theexpressionoftheirneurotrophicfactors.After attachment, proliferationandsurvivaloftheseededASCs, The invitroresultshowedcryoGelMA scaffolds supportedthe channel orbifurcatingmodelsandpersonalizedstructures. andkey” molds,suchasthe designedmulti- 3D-printed “lock The conduitswerefabricatedwithdifferent geometriesusing was preparedforPNSregeneration3Dprinting(Figure 5b). laden cryopolymerizedgelatinmethacryloyl(cryoGelMA) gel A bio-conduitconsistingofadipose-derivedstemcell(ASC) has beendevelopedtofabricatecustomizedconduitmolds. duit graft. resulted inthreehollowchannelsformingafully cellular con After the removal of the agarose rods, the fused construct pared andextrudedintomoldedwellsmadebyagaroserods. The multicellularcylindricalunitsofMSCsandESCswerepre important approach. organizational complexity of the tissuehasbeenregarded as an 3D printedscaffolds thatmimicthebiologicallyfunctionaland ical function of stem cells in the implanted tissue site, the use of The stemcellreplacementhasattractedmuchattentionas Conductive nanobiomaterials have been shown to improve Conductive nanobiomaterialshavebeenshowntoimprove + transgenicmousestrainpossessesapopulationoffluo [164] [165] Extrusion-basedbioprintingwasemployedtofab Comparedwithautologousgraft andcommercial [167] [152,163] [166] [165] Theresulting3DGmaterialis mechani Theindirect3Dprintingtechnique Inaddition, stem cellscansecrete Adv. Healthcare Mater. [163] www.advancedsciencenews.com Figure To support the physiolog [152,153]

5 a). They can assist to Theycanassistto [162]

2017 , 6 , 1601118 [153,163] [166] [165] ------

REVIEW

- - - [174] 1601118 (21 of 29) www.advhealthmat.de The custom scaffolds The custom scaffolds reference angle) stained for stained angle) reference [169] ° Compared to the control m over 3 days due to the µ [168] m). Reproduced with permission. µ 76.1 ± wileyonlinelibrary.com Recently, an imaging-coupled 3D printing an imaging-coupled 3D printing Recently, [168] Copyright 2015, Elsevier. (b) A patient’s sciatic nerve 2015, Elsevier. Copyright [162] reference angle) stained for GFAP (green) and laminin (red). ° Copyright 2015, American Chemical Society. (d) SEM image of 2015, American Chemical Society. Copyright Copyright 2016, Nature Publishing Group. (c) 3D printed graphene 2016, Nature Publishing Group. Copyright [167] [166] approach was developed, facilitating customized neuroregener ation in previously inaccessible ways. samples (fibrin without the VEGF or VEGF printed directly in collagen), the printed C17.2 cells in the collagen hydrogel and migrated toward the fibrin gel with showed high viability, a total distance of 102.4 VEGF induction. growth factor (VEGF)-releasing fibrin gel were printed to con struct an artificial neural tissue. SLA. The PEG nerve guide was implanted in to a Thy-1-YFP-H common SLA. The PEG nerve guide was implanted in to µ - - [167] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © They have been used in [151–153] Copyright 2015, Wiley-VCH. (e) Printed gel scaffold comprising optimal 5% w/v alginate, 5% w/v carboxymethyl 2015, Wiley-VCH. (e) Printed gel scaffold comprising Copyright , 1601118 [169] 6 , 2017

guide made with a wall thickness of 50 mm by (a) A PEG nerve Neurotrophic factors are endogenous molecules critical to Adv. Healthcare Mater. Adv. www.advancedsciencenews.com was reconstructed based on MR neurography, and then a personalized nerve guidance conduit (NGC) was fabricated. The images show an intraop show images The fabricated. was (NGC) conduit guidance nerve personalized a and then neurography, MR on based reconstructed was of the in a rat sciatic nerve transection model with 10 mm gap, and the general observations erative photograph of the NGCs for nerve regeneration Reproduced with permission. regenerated sciatic nerve at 16 weeks post-surgery. chitosan, and 1.5% w/v agarose. NSCs (31 d post-printing, including 21 d differentiation) stained with DAPI (blue) and expressed TUJ1 (red), with cell chitosan, and 1.5% w/v agarose. NSCs (31 d post-printing, including 21 d differentiation) stained the Z-axis (0–59 clusters interconnected by neurites. The lower right panel shows depth coding of cells along a 3D printed hollow nerve pathway displaying an axially oriented physical cue on the luminal surface. Photograph of an implanted 3D printed nerve printed implanted 3D of an Photograph luminal surface. physical cue on the axially oriented displaying an nerve pathway a 3D printed hollow (90 cue physical oriented horizontally printed, the 3D on neurons embryonic primary Cultured suturing. to prior guide tau (green), while cultured Schwann cells on the horizontally oriented physical cue (90 Reproduced with permission. Figure 5. Copyright 2016, Wiley-VCH. Copyright nerve graft conduit at various sizes. Photograph of tubular nerve conduit that was implanted into a human cadaver via longitudinal transection and of tubular nerve conduit that was implanted into a human cadaver via longitudinal transection nerve graft conduit at various sizes. Photograph along the previously described longitudinal transection wrapped around the ulnar nerve (white arrows). The nerve conduit was then sutured closed Excess 3DG nerve conduit length was then cut with (white dotted line) as well as to the surrounding epinerium and nerve tissue (inset, yellow circle). surgical shears to expose additional nerve tissue. Reproduced with permission. fibular mouse, small gap, 3 mm injury model. The nerve graft repair image illustrated intervals marked with sample axon tracing from 4.0 mm interval nerve graft repair image illustrated intervals marked with sample axon tracing from 4.0 mm fibular mouse, small gap, 3 mm injury model. The from of axons at each interval was counted to obtain a sprouting index value; axons were traced position back to 0.0 mm (start) interval. The number calculate with a previously traced axon (as highlighted in expanded sections with green circles), to distal intervals back to 0.0 mm, or a branch point each interval. Reproduced with permission. percentage of unique start axons represented at the maintenance, survival, proliferation and differentiation of the maintenance, survival, proliferation and differentiation various neuronal populations. and presynaptic terminals. In vivo experiments using a human cadaver nerve model illustrate that 3DG has exceptional han dling characteristics and can be intraoperatively manipulated. neural tissue engineering to promote axonal regeneration, neu murine neural ronal plasticity and neurogenesis. In a study, stem cells (C17.2), collagen hydrogel, and vascular endothelial REVIEW 1601118 www.advhealthmat.de rons 3Dprinting. improved theaverageneuritelengthofprimarycorticalneu fiber. Inaddition,the3Dprintednanocompositescaffolds also rites anddirectedneuriteextensionofPC-12cellsalongthe factor (NGF)nanoparticlesgreatlyincreasedthelengthofneu bicuculline-induced increasedcalcium response. Moreover, the neurons are spontaneously active and show a cells expressingastrocyteandoligodendrocytelineagemarkers. of hNSCsresultedinGABAergic neurons,togetherwithglial printing (Figure 5d). in enhancedfunctionalreturnoftheregeneratednerve3D successful regenerationofcomplexnerveinjuries,resulting nerve gapinratsshowedthatthe3Dprintedscaffolds achieved the regenerationofbifurcatedinjuriesacrossa10mmcomplex tractant/chemokinetic functionality. Invivostudiesexamining and biochemicalcuesprovideaxonalguidancechemo GDNF). factor, NGFandglialcellline-derivedneurotrophicfactor, physical cuesandpath-specificbiochemical(nervegrowth cating pathwayswereaugmentedwith3Dprintedbiomimetic were fabricatedviaamicroextrusionprintingsystem.Thebifur cells, retinalganglioncell(RGC)neuronsandglia. tric inkjetprintingwasusedtoprinttwotypesofadultratCNS 4.2.2.2. 3DPrintingforCNSRegeneration most research currently focuses on neural regeneration with its huge potential for neural tissue regeneration. Although interior microarchitectures. Therefore, 3D printing has shown idly fabricatesubtleexterior geometries aswellcomplex the functionofimpairednervoussystems. construct promoted the repair of damaged CNS and rescued injury modelofadultzebrafish.Theresultsshowedthe3D stem cell (NSC)-ladenconstruct was implanted into a brain on self-developed FDMequipment. gradable polyurethanedispersionsweresynthesizedforuse cortical neurons3Dprinting. aspartic acid)-gellan gum (RGD-GG), combined with primary of anovelpeptide-modifiedbiopolymer, (arginine-glycine- 3D brain-likestructureswereprintedusingabio-inkconsisting retain theirgrowthpromotingproperties. neurite outgrowth itself. Moreover, the printed glial cells could The printingprocessdidnoteffect RGC/glial survivalandRGC encapsulation. a gelby chemical cross-linking following extrusion with hNSC nate, carboxymethylchitosan(CMC),andagarose,whichform and supportingneuroglia.Thebioinkwascomprisedofalgi were usedforinsitudifferentiation towardfunctionalneurons by microextrusionbioprinting. novel 3Dneuralmini-tissueconstruct(nMTC)wasfabricated spraying techniques. factor (NGF)delivery, usingSLAprintingandco-axialelectro embedded core-shellnanoparticleswithsustainedneurogenic patterned scaffold, whichhastunableporousstructureand the RGDpeptideinRGD-coupledGGthantopurifiedGG. hydrogels, indicatingthatcorticalneuronsrespondedbetterto tical neuronsandglialcellsin3DprintedRGD-GGmodified cessful encapsulation,survivalandnetworkingofprimarycor As wediscussedabove,3Dprinting hasthecapacitytorap In arecentstudy, twothermoresponsivewater-based biode (22 of29) [169] Invitrostudiesrevealedthat3Dprintedphysical [174] [170] The results showed that the differentiation [170] [169] wileyonlinelibrary.com Theprintedscaffold withnervegrowth Ourgroupalsodevelopedanovel3D [172] [174] Theresultsdemonstratedsuc Frontal corticalhumanNSCs [173] The3Dprintedneural : Inastudy, piezoelec [171] [173] © 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim Inanotherstudy, Mostrecently, a [174] [172] [171] ------

tural andfunctionalcardiactissues. been anefficient approach toreproducethecomplexityofstruc or diseasedcardiovasculartissues. 3Dbioprintinghasrecently creating engineeredtissuestorepaircongenitaldefectsand/ engineering techniques have shown a promising approach for rejection, anticoagulationtherapy, andlimiteddurability. Tissue several disadvantages,suchasdonortissueshortage,immune autografts, allografts, xenografts, andartificialprostheses,have cardiovascular disease(CVD),traditionalapproaches,including fibroblasts, andendothelialcells.Inthetreatmentofserious includes threemaincellularcomponents,cardiomyocytes, venting itfromoverfillingwithblood. tect theheart,anchoringittosurroundingwalls,andpre pericardium isadouble-wallfibroseroussacthatactstopro responsible forcontractionandrelaxationoftheheart.The dium consistingofcardiomyocytesisthethickmuscularlayer rier toprotectthevalvesandheartchambers.Themyocar made upofendothelialcells,whichactasablood–heartbar organ, andothers. serve acommonfunction,includingsensoryorgan,visceral organ isacollectionoftissuesjoinedinstructuralunitto vascularized tissueandtheirfunctionalization.Inbiology, an printed organfieldfocusesonthebiomimeticfabricationof (aortic andpulmonary). valves (mitralandtricuspid)thesemilunar/outflow ensure unidirectionalflowofblood:theatrioventricular/inflow four chambers,valvesandaheartwall.The nopleuric mesenchymecelllayer. Inanatomy, thehearthas the firstfunctionalorgantobedevelopedfromsplanch epicardium/pericardium. layers: theinnerendocardium,middlemyocardiumandouter off aseriesofcomplicatedandirreversibleprocesses, involving the blockageofcoronaryarteries. Myocardialischemiasets plexities. nents hasremainedelusivelargelyduetoorgan-levelcom whole-organ bioprinting,incorporatingallofthesecompo bioprinting holds great promise forachieving all goals, but full-sized organsusingtissueengineeringtechnology. 3D researchers arenowexploringdevelopmentoffunctional, Due totheshortageoforganssuitablefortransplantation, 4.2.3. Organs for engineeredcomplextissues/organs. cess wouldalsopavethewayforregeneratingneuralnetworks the purpose of repairing the nervous system, we believe its suc covering onthesurface. cells (SMC)respectively, withvalvular endothelialcells(VEC) mainly containvalveinterstitialcells(VIC)andsmoothmuscle and afibrousannuluswall(rootwall).Leafletsrootwalls system. which pumpsbloodthroughoutthebodyincirculatory 4.2.3.1. Heart and functions. unique challengesinreplicatingtheirhighlycomplexstructures organs (internalorgans),suchastheheartandliver, duetothe The mostfatalCVDismyocardial infarction(MI),causedby [110,176] [20,23,26,35,110,175] Duringembryonicdevelopment,theheartis : Theheartisamuscularorganinhumans, [1–3] Herein, wemainlyfocusonvisceral Therefore, current research in the 3D [176] [176] [176] Eachvalveiscomposedofleaflets Theheartwallismadeupofthree Theendocardiumisprimarily Adv. Healthcare Mater. www.advancedsciencenews.com [110] [176] Overall,theheart

2017 , 6 , 1601118 ------REVIEW

------In [183] [184] [185] [187] 1601118 [186] Between [176] (23 of 29) 85% of the liver ∼ The engineered The engineered www.advhealthmat.de [186] The laminated hepato laminated The [184] [110] In another study, a liver-on-a- a In another study, In anatomy, the liver is divided In anatomy, [185] wileyonlinelibrary.com [1,176] [186] Histology shows two major types of liver shows two major Histology [176] : In the human body, the liver is composed of the liver body, : In the human A multi-nozzle extrusion system was applied to applied to A multi-nozzle extrusion system was [176] [110] The liver has extensive regeneration capacity due to high pro The liver has extensive regeneration capacity In an earlier work, a 3D hepatocyte/gelatin construct was construct was In an earlier work, a 3D hepatocyte/gelatin Recently, metabolically active, anatomical, 3D hepatic metabolically active, anatomical, 3D hepatic Recently, the hepatocyte plates are liver sinusoids, which connect the are liver sinusoids, which connect the the hepatocyte plates portal triads for mixing of the oxygen-rich central vein with the artery and the nutrient-rich blood from blood from the hepatic the portal vein. volume. The non-parenchymal cells are sinusoidal endothelial cells are sinusoidal endothelial volume. The non-parenchymal and hepatic stel cell (KCs), Kupffer cells (SECs), phagocytic late cells (HSCs), among others. Owing to its location and late cells (HSCs), among others. Owing also prone to many multidimensional functions, the liver is diseases. cell: parenchymal cells and non-parenchymal cells. The paren cell: parenchymal cells chymal cells are hepatocytes that constitute 70 chymal cells are hepatocytes bioreactor could be interfaced with a bioprinter to fabricate fabricate to bioprinter a with interfaced be could bioreactor hydrogel. 3D hepatic spheroids encapsulated within GelMA The engineered hepatic constructs remained functional for 30 alpha-1 albumin, of rates secretion the monitoring while days immu as well as transferrin, and ceruloplasmin, antitrypsin, nostaining for the hepatocyte markers, cytokeratin 18, MRP2 bile canalicular protein and tight junction protein ZO-1. cytes remained viable and performed biological functions in functions in cytes remained viable and performed biological showed This technique 2 months. than more construct for the reconstitute the struc a promising and stepwise approach to of human livers. ture of the in vivo microenvironment liferation ability of hepatocytes, even if it is subjected to vast liferation ability of hepatocytes, even if techniques damages. Therefore, various tissue engineering liver tissues. How have been developed to fabricate biomimetic ever, the traditional methods have a limited achievement on the on achievement limited a have methods traditional the ever, adhesion. volumetric liver tissues with highly intercellular printed from a 38 layer assembly. 38 layer a from printed chip platform of 3D human HepG2/C3A spheroids was also spheroids was also chip platform of 3D human HepG2/C3A developed for drug toxicity assessment. tissues have also been developed. For example, a double- tissues have also been developed. For nozzle bioprinting technique was used to fabricate an anatomical liver structure with a vascular-like network. anatomical liver structure with a vascular-like fabricate human liver hepatocellular carcinoma cell (HepG2) cell (HepG2) fabricate human liver hepatocellular carcinoma 3D architecture. laden alginate hydrogel in an organized tures with higher cell densities. 3D bioprinting facilitates the fabrication of complex liver struc 3D bioprinting facilitates the fabrication into four lobes, each of which is microscopically made up of of which is microscopically made up into four lobes, each lobules are roughly hexagonal, and consist hepatic lobules. The radiating from a central vein. of hepatocyte plates tion, and detoxification. 4.2.3.2. Liver It of mostly hepatocytes. tissue consisting highly specialized functions, with numerous role in metabolism plays a major of storage, decomposition regulation of glycogen including produc synthesis, hormone cells, plasma protein red blood Some researchers also focus on fabricating micro-organs micro-organs fabricating on focus also researchers Some for drug metabolic or organs-on-a-chip using 3D printing studies. mimetic drug metabolic process in the micro-organ under mimetic drug metabolic process in the micro-organ under continuous perfusion flow. The biofabricated micro-organ device was performed as an performed as an The biofabricated micro-organ device was in vitro drug metabolism model. The results showed a bio addition, an acetaminophen-induced toxic response test in this this in acetaminophen-induced toxic response test addition, an similar to platform was performed to verify the effectiveness that of animal studies.

------[177] [180] 4.0% for ± -SMA) in the α They also used Human aortic Human 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag [181] [181] © Direct encapsulation of [182] a). Compared with traditional with Compared In addition, a 3D simplified 6

[110] [180] 3.4% for SMCs and 83.2 , 1601118 The printed construct retained the The printed construct 6 Figure ± , [182] [179] Although it is still not a functional heart not a functional Although it is still 2017

[179] [139] Compared with the random patch, the patterned Compared with the Human cardiac derived cardiomyocyte progenitor cardiac Human [178] [178] Dysfunctional valves caused by stenosis or regurgitation Dysfunctional valves Some researchers have developed bioprinted cardiac and cardiac bioprinted developed have researchers Some Adv. Healthcare Mater. Adv. www.advancedsciencenews.com VICs) over 7 days in culture. Moreover, the encapsulated SMCs VICs) over 7 days in culture. Moreover, expressed higher alpha-smooth muscle actin ( aortic root sinus SMCs in the valve root and aortic VICs in the aortic root sinus SMCs in the valve root leaflet were viable (81.4 ture enhanced gene expression of the early cardiac transcrip ture enhanced gene as well as the sarcomeric Mef-2c and tion factors Nkx2.5, Gata-4 protein TroponinT. cardiac phenotype with high cell viability. Moreover, the 3D cul Moreover, high cell viability. cardiac phenotype with alginate/gelatin hydrogel to bioprint a living 3D heart valve with alginate/gelatin hydrogel to bioprint a living anatomical architecture ( cally accurate, living, engineered valves with heterogeneous with valves engineered living, accurate, cally multiple cells. mechanical properties and well -distributed native anatomic and axisymmetric aortic In an earlier study, with 12–22 mm wall and trileaflets) valve geometries (root printer. inner diameters were 3D printed with a dual-nozzle hydrogel, which VICs were encapsulated into the bioprinted by and remodeled the initial matrix maintained high viability, depositing collagen and glyosaminoglycans. impair the proper opening and closing of the valves, affecting the valves, affecting impair the proper opening and closing of performance. heart efficient approaches, tissue engineering has great potential to address approaches, tissue engineering has great by providing living current limitations of non-living prosthetics remodel and integrate in patients. For constructs that can grow, anatomi create can bioprinting 3D valve, heart engineered the valve constructs for cardiac and valve tissue engineering, but a functional 3D heart construct has yet to be explored. The whole heart organ not only has complicated structure, but is also comprised of multiple cell types with spatial distribution, associating with specific and integrated functions comparable also been has FRESH technique the Recently, native tissue. to developed to create a 3D heart construct of a 5-day-old chick embryo, demonstrating the capability of recapitulating the com plex trabecular structures of a whole heart through CAD mod 6b). eling (Figure with a vascular network, it offers a potential approach to gen with a vascular network, it offers erate a large-scale, complex 3D internal and external anatomical architecture. tation. alginate hydrogel was used to print the cell (hCMPC)-laden 3D cardiac construct. patch showed increased vessel formation and found significant found significant vessel formation and showed increased patch implan following hearts infarcted of improvement functional cell death, scar formation, and ventricular dysfunction. scar formation, and cell death, heart valve construct with root and trileaflets was printed from heart valve construct with root and trileaflets based hybrid hydrogel. a HAMA/GelMA Therefore, engineered myocardial tissue has been explored to tissue has been engineered myocardial Therefore, LIFT- techniques. printing 3D using functions restore cardiac to prepare a were first applied printing techniques based cell seeded with cardiac patch urethane urea (PEUU) polyester in a defined pattern for cardiac regen HUVECs and MSCs eration. ricate the heterogeneous aortic valve constructs with different with different ricate the heterogeneous aortic valve constructs maintained near mechanical properties. VIC seeded scaffolds 100% viability over 21 days. PEG-DA hydrogels and alginate hydrogels were utilized to fab PEG-DA hydrogels and alginate hydrogels printed stiff matrix, while the soft matrix elevated vimentin the soft matrix, while printed stiff expression of the VICs. REVIEW 1601118 www.advhealthmat.de wall viaFRESHtechnique.Reproducedwithpermission. internal trabecularstructurefromtheCADmodel.Adarkfieldimageof3Dprintedheartwithvisiblethroughtranslucent heart trabeculation basedontheconfocalimagingdata.Acrosssectionof3Dprintedheartinfluorescentalginate(green)showingrecreation ofthe (blue), andF-actin(red)imagedwithaconfocalmicroscope.Acrosssectionofthe3DCADmodelembryonicheartcomplex internal (b) A dark field image of an explanted embryonic chick heart. A 3D image of the 5-day-old embryonic chick heart stained for fibronectin (green), nuclei intrinsic/extrinsic structures. erful toolforfabricatingcomplexliverconstructswithspecial that thisdouble-nozzleassemblytechniquecouldbeapow decreased after anincreasingprofile.Theseresultsindicate ture, whilethelevelsofureaandalaninetransaminasewere the embeddedhepatocytesincreasedduring2weekcul endothelial growthfactor. Thealbuminsecretionlevelof were inducedtodifferentiate intoendothelial-likecellswith nate/chitosan hydrogelwasplacedaroundit.TheADSCs vascular-like network,andhepatocytesladengelatin/algi within agelatin/alginate/fibrinogenhydrogeltoform Adipose-derived stromal cells (ADSCs) were encapsulated cell nuclei(blue);StainingforVIC(ii)intheleaflet,andstainingSMC(iv)root.Reproducedwithpermission. root after7dayculture. Representative imageof immunohistochemical staining for aSMA(green) and vimentin (red), andDraq 5 counterstaining for cell trackergreenandVICforvalveleafletwerelabeledbyred.Live/deadassayencapsulated(i)intheSMC(iii) invalve Figure 6. hepatic celllinesandprimary hepatocytes. architecturally andphysiologically relevantfeaturesfortwo CA, USA),3Dliverconstructswerefabricatedcontaining printing technology(OrganovoHoldings,Inc.,SanDiego, sent, andtightjunctionprotein expressionwasobserved demonstrated thatseveralcritical liverfunctionswerepre with the addition of ECs and HSCs. Biochemical studies 3D hepaticneotissueswerefurther enhancedincomplexity in fluorescentimages.Reproducedwithpermission. (red) in2.5%GelMAwith1%GMHAonday0.Scalebars,500 taken underfluorescentandbrightfieldchannelsshowingpatternsoffluorescentlylabeledhiPSC-HPCs(green)in5%GelMAsupporting cells albumin (Alb),E-cadherin(E-Cad), andnucleus(Dapi)stainingofhiPSC-HPCsin3Dtricultureconstructs.Scalebars,500 (24 of29) (a)3Dprintedaorticvalveconduit.Fluorescentimageoffirsttwolayersaconduit;SMCforrootwerelabeledby wileyonlinelibrary.com [187] UsingNovoGenTM bio- © [188,189] 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim [190] [139] Copyright 2016,NationalAcademyofSciences. Copyright 2015,theAmericanAssociationfor AdvancementofScience.(c)Images(5 Bioprinted µ m. Grayscaleimages(5 - - - - technique (Figure 6c). microscale hexagonalarchitecturewasdevelopedusingaDLP ical veinendothelialcellsandadipose-derivedstemin a hepatic progenitorcells(hiPSC-HPCs)withhumanumbil hydrogel-based triculturemodelthatembedshiPSC-derived and enhancedcytochromeP450 induction. gene expression levels, increased metabolicproduct secretion, improved morphologicalorganization, higherliver-specific the hiPSC-HPCsoverweeksofinvitroculture,especiallyfor showed both phenotypicandfunctionalenhancementsin culture anda3DHPC-onlymodel,thetriculturemodel promising resultstowardthe generation of‘mini-organs’ that organs currentlyremainsanarduous challenge,ithasshown in drugdiscoveryanddevelopment. the potentialutilityofhuman3Dbioprintedlivertissues clinically relevantinjuryresponse.Theseresultsdemonstrate In additiontotheliver-specific functions,italsoexhibiteda sues alsounderwentotherbiochemicalstudiesforsixweeks. throughout the3Dtissue.Moreover, the3Dprintedlivertis Overall, although3Dprinting ofcomplexandlarge3D × ) andconfocalimmunofluorescenceimages(40 [190] Incomparisonwith2Dmonolayer Adv. Healthcare Mater. www.advancedsciencenews.com [182] µ [189] Copyright 2012,Wiley-VCH. m inbrightfieldand100 Mostrecently, a3D [190]

2017 , × 6 ) showing , 1601118 µ × m ) - - REVIEW

------[193] 1601118 A unique A unique (25 of 29) [82,192] www.advhealthmat.de It can enable growth of thick It can enable growth and our lab has developed and our lab has developed [27] [26,50] wileyonlinelibrary.com [26] Furthermore, 3D bioprinting techniques have 3D bioprinting techniques have Furthermore, (levetiracetam), which was the first FDA-approved (levetiracetam), which was the first FDA-approved [36,195] ® [61,194] Finally, in order to ensure effective industrial translation and industrial translation in order to ensure effective Finally, New niches for technological advancements on instrumen New niches for technological advancements some 4D bioprinted constructs for regulating cell/scaffold some 4D bioprinted constructs for regulating cell/scaffold behavior. shown the potential to facilitate the development of real istic tissue/organ models, therefore this technology is also istic tissue/organ models, therefore this technology is also expected to translate advancing the needs of other specific applications such as models for pharmaceutical/toxicological screening. turers who are producing devices through 3D printing tech turers who are producing devices through Pharmaceuticals’ the FDA approved Aprecia niques. In March, SPRITAM facturing along with its governing software, product testing and software, with its governing facturing along guidance issued draft finally the implantation process. The FDA Considerations for Additive 10, 2016, titled “Technical on May guidance for manufac which provides Devices”, Manufactured commercialization of organ printing technology, the key issue organ printing technology, commercialization of and regulation of bioinks, bioprinters and is quality assurance 3D product requires bioprinted products. This customizable control in every a comprehensive regulation to assure quality equipment and raw step of the process: production of printing factors and cells), biomaterials, biological materials (bioprinter, of the manu design control of the 3D printed model, validation aspect of this technology is its ability to achieve a personalized aspect of this technology is its ability to achieve a personalized therapeutic schedule to address individual patient needs. tion, ethical concerns will be considered for future attempts. tion, ethical concerns will be considered made on animals, Although the majority of the trials have been tissues or organs ethical concerns will be raised when printing for implantation in humans. drug manufactured using 3D printing. In addition to regula to addition In printing. 3D using manufactured drug tation, with improved spatial and temporal resolutions as well resolutions as well tation, with improved spatial and temporal for specific organs, as optimized bioinks and cell sources eventually become provide promise that 3D bioprinting will reliable, and convenient methods one of the most efficient, near future. The high to biofabricate tissue constructs in the flexibility and controllability of 3D bioprinting enables com ceuticals with spatiotemporal gradients for regulating cellular functions during tissue/organ regeneration. plex and tailored release profiles of multiple active pharma niques have been proposed, turing biologically dynamic variations will further allow tem poral evolution of bioprinted tissue constructs that potentially meet the requirements of dynamic tissue remodeling during instance, 4D bioprinting tech developmental processes. For Moreover, advanced materials engineering approaches fea Moreover, technology is the rapid or accelerated tissue maturation pro tissue maturation is the rapid or accelerated technology rapid undergo should constructs organ printed where cess, a solid and maturation toward remodeling, matrix deposition, integ ensuring structural with bioink degradation, living tissue implan for functionality biological and rigidity mechanical rity, in situ bioprinting is an advanced tation. As an alternative, where living cells can be printed trend in organ regeneration, instead of the post- during an operation in the human body in vitro. bioprinting process tissues in critical defects with the help of vascularization driven with the help of vascularization driven tissues in critical defects Although in situ bioprinted in the body. by natural processes tested, the manufacturing of complex skin and bone have been in view of the limited success of their organs is still uncertain fabrication in vitro.

------[191] 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag © , 1601118 6 , 2017

Considering the pathway from 3D organ printing to implanta Considering the pathway from 3D organ printing Adv. Healthcare Mater. Adv. www.advancedsciencenews.com posite tissue/organ structures at clinically relevant sizes. Sec posite tissue/organ structures at clinically considered. The 3D construct bioinks and cells must be ondly, generated by bioprinting serves as a biomimetic construct with desired composition and cellular contribution to support func any single material and it is unlikely that Moreover, tionality. single cell possess all the properties required to recapitulate tissue function. Therefore, developing appropriate bioink formu supply are lations associating with cell sources with sufficient strategy. fabrication is consideration third The important. very Organs have highly complex architectures and properties; as such they may require a combination of several bioprinting techniques along with specifically designed bioinks to introduce repeatable and Highly structural heterogeneity and functionality. straightforward technologies and protocols should be developed complex. Thefrom simple to steps, in logical the organs print to manufacturing process. Considering the is consideration fourth the micro-scale resolution in cellular bioprinting, the process requires enough nutrients and oxygen supplementation for printing sterile stable, a in constructs living scalable sustaining system over long printing times. The post-bioprinting process consideration. Another challenge for organ printing is the fifth operative care are essential for customized functional organoperative care are essential for customized current considering printing techniques, fabrication. First, many technical chal 3D tissue/organ printing has to address speed and flexibilitylenges to increase the resolution, printing complex and com with relevant biomaterials for creating more niques, maturation processes, surgical operations and post niques, maturation processes, surgical tionamount of time, standardizedinto a human in a reasonable fabrication tech protocols involving patient-specific design, tional organ fabrication. Many of the challenges facing the 3Dthe facing challenges the of Many fabrication. organ tional bioprinting field relate to specific technical, material, and cel lular aspects. 5. Current Challenges and Future Perspectives and Future 5. Current Challenges technology includingOrgan printing is a multidisciplinary and medi computer science, materialogy, engineering, biology, cine, which requires a multidisciplinary team and long-term sus cine, which requires a multidisciplinary team few years, researcherstainable financial support. Over the past examples of artifi not only have demonstrated proof-of-concept technologies, bioprinting cial tissue fabrication using different 3D bioprinting offers but also have shown possibilities that method of recapitulating structural and functional an effective especially for func complexity for engineered tissue fabrication, contain the same functional components of large organs. of large same functional components contain the Mini-organs can be considered a future trend in organ in organ a future trend can be considered Mini-organs organs. to fully functional might be a gateway printing and coun scale than their natural be built in smaller They can function of the most vital closely performing terparts while the current studies, the simulta From the associated organ. of materials could allow fabrication neous printing of multiple heterogeneous internal organization engineered tissues with could more accurately model native of cells and ECM, which cell multiple organ-specific Additionally, tissue organization. functional to form spatially organized required to be types are the integration of vasculature within the organs, especially for constructs. REVIEW 1601118 www.advhealthmat.de Discovery andTranslational ResearchGrant. program grant#1642186andMarchofDimesFoundation’s Gene 1DP2EB020549-01, NSFBMEprogramgrant#1510561,MME This workissupportedbyNIHDirector’sNewInnovatorAward Acknowledgements bioprinting. rent challengesandrealizetheemergingpotentialof3Dorgan bioink materialsandengineeringdesignscanaddressthecur ciplinary researchtoadvanceprintingtechniques,printable challenges still remain in this research field, further multidis the cells,andapplicationsfororganregeneration.Although relative components,includingthetechniques,bioinks, review presentstherecentadvancesinbioprintingandtheir in vitro tissue/organ models for various research studies. This versatility hasalsobeenexpandedtootherapplications,suchas future developmentand3Dscale-upoffunctionalorgans;its nology hasalreadydemonstrateditsremarkablepotentialfor and isintheinitialstagesofdevelopment.However, thistech encompasses specifictechnical,materialandcellularaspects, 3D bioprintingfororganregenerationisanemergingfieldthat 6. Conclusion [13] [14] [10] [12] [11] [2] [3] [4] [5] [8] [9] [6] [7] [1] Y. Wei, 15 G. Zan,Q.Wu, P. Dubruel, T. Billiet,M.Vandenhaute, J.Schelfhout,S.Van Vlierberghe, P. Zhang,Y. Huang,Y. Wei, X.Chen, Z. Zhang,P.B.X.S.Chen,Y. Wei, G. JuliusVancso, L.Moroni, S. F.Badylak,D.J.Weiss, A.Caplan, P.Macchiarini, McGraw-Hill Education,Boston,NY, USA Organogenesis K. C.Rustad,M.Sorkin,B.Levi,T. Longaker, G.C.Gurtner, 379 Publishing, Stanford, Regeneration: AdvancesinMicroandNanotechnology L. G.Zhang, A. Khademhosseini, T. Webster, J. L. G.Griffith,M.A.Swartz, a) N.J.Castro, C.M.O’Brien,L.G.Zhang, Acta Biomater 2015 L. J.Zhang,T. J.Webster, B. Derby, T. Dvir, B.P.Timko,D.S.Kohane,R.Langer, 2011 E. M. Benetti, M. K. Gunnewiek, C. A. van Blitterswijk, 432; b)W. Zhu,F.Masood,J.O’Brien,L.G.Zhang, macromolecules H. Cui,Y. Wang, L.Cui,P.Zhang,X.Wang, Y. Wei, X. Chen, 440. E. S.Place,N.D.Evans, M.Stevens, E. P. Widmaier, H. Raff,K. T. Strang, (26 of29) , 1115; e) H. T. Cui, J. Shao, Y. Wang, P. B. Zhang, X. S. Chen, , 943. , , 6 11 , 13. Biomacromolecules , 693;c)H.Cui,X.Zhuang,C.He,Y. Wei, X.Chen, Science Biomaterials

2010 2015

Adv. Mater. 2014

2012 wileyonlinelibrary.com , , 6 , 11 , 151. 15 , 2014 338 , 183;d)H.T. Cui,Y. D.Liu,Y. L.Cheng,

, 3146. 2012 Nano Today

, 921. .

2016 Nat. Rev. Mol.Cell Biol. 2013 J. Mater. Chem.B Published online:December20,2016 , 33 , , 6020. , 28 14 , 2099. Received:

2009 , 1904; f) H.Cui,L. , 1904;f) Vander’s Human Physiology Macromol. Biosci. Revised: Nat. Mater. Biomacromolecules 2010 , © 4 , 66. 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim

September 30,2016 2016 . AIChE J. Nat. Nanotechnol. Tissue and Organ October 26,2016

, 2006

, PanStanford 2009 Nanomedicine 4 Lancet , 4244.

, 2014 2014 , 7 8 , 211. , 457.

2014 2012 , , Bio- 60 14 - - - , , , , ,

[35] [34] [33] [36] [32] [29] [27] [28] [26] [15] [31] [16] [40] [39] [37] [38] [42] [20] [24] [23] [22] [43] [18] [19] [17] [21] [47] [41] [44] [45] [46] [25] [30] J. Li, L. He, C. Zhou, Y. Zhou, Y. Bai, F. Y. Lee, J. J. Mao, V. Mironov, V. Kasyanov, R.Markwald, Elsevier Ltd,SanDiego, Nanotechnology inTissueEngineeringandRegenerativeMedicine L. G.Zhang,J.P.Fisher, K.W. Leong, X. Zhang,Y. Zhang, 2011 F. Pati,J.Gantelius, H. Svahn, A. C. Leigh-Lancaster, R.J.Law, K. Murphy, S.Dorman,N.Smith,L.Bauwens,I.Sohn,T. McDonald, ACS Biomater. Sci.Eng. R. Jose,M.J.Rodriguez,T. A.Dixon,F.Omenetto,D.L.Kaplan, Manufactured Devices Food andDrugAdministration, 2003 W. C. Wilson Jr., T. Boland, 2016 B. Gao,Q.Yang, X.Zhao,G.Jin,Y. Ma,F.Xu, C. W. Hull, I. T. Ozbolat, Assist. Radiol.Surg. 2015 C. Mandrycky, Z. Wang, Kim, K. Kim, D. H. Part B C. M.O’Brien,B.Holmes,S.Faucett, L.G.Zhang, a) R.D.Farahani, M.Dube,D.Therriault, C. Mezel,A.Souquet,L.Hallo,F.Guillemot, 5794; b)U.Kalsoom,P.N.Nesterenko,B.Paull, I. T. Ozbolat,Y. Yu, 34 C L. Gasperini,D.Maniglio,A.Motta,C.Migliaresi, Biomater. Res. B. K.Gu,D.J.Choi,S.Park,M.Kim,C.Kang,H. 55 A. Skardal,Atala, Technol. K. Akita,S.Mochizuki,Takiura, M.Nakamura, Y. Nishiyama,C.Henmi,S.Iwanaga,H.Nakagawa,K.Yamaguchi, S. Khalil,J.Nam,W. Sun, S. V. Murphy, A.Atala, Adv. Mater. J. W. Nichol,A.Manbachi,H.Bae,S.Chen,Khademhosseini, P. Zorlutuna,N.Annabi,G.Camci-Unal, M.Nikkhah,J.Cha, R. Unterhinninghofen, H. U. Kauczor, F. Giesel, L. F. Rengier, A.Mehndiratta,H.vonTengg-Kobligk, C.M.Zechmann, R. Markwald, V. Mironov, V. Richard, K. P. Visconti, G. Forgacs, C. Drake, J. doi: 10.1007/s10439-016-1611-9. Mater. A. V. Do,B.Khorsand,S.M.Geary, A.K.Salem, J. H.Park,Jang,S.Lee,D.W. Cho, 60355; c)A.Ambrosi,M.Pumera, Drug DeliveryFormul. a) X.Cui,T. Boland,D.D’Lima,M.K.Lotz, 2 Technol. K. Yamaguchi, K.Akita, S.Mochizuki,K.Takiura, M. Nakamura,Y. Nishiyama,C.Henmi,S.Iwanaga, H.Nakagawa, 394 U. Demirci,G.Montesano, Biomaterials T. Xu, W. Zhao,J.M.Zhu,Z.Albanna,Yoo, A.Atala, P. A. Eagles, A. Qureshi, N. S. N. Jayasinghe, U. S.Schubert, , 014103.

2015 , 422. , 4650. , 375. , , , , 22 272 34 40

2015 , 2015

21 2008 2008 , 667. , 746. , 145. , 491. , 123.

US 4575330A , 2012 ,

2013 21 4 , , Trends Biotechnol.

, 1742. 2016 52 52 , 103. Soft Matter Biomater. Sci. , , 60201. , 060201. , 24 34

, 2010 IEEE Trans. Biomed.Eng. , 1782. Cell Biochem.Biophys. Ann. Biomed.Eng. 20 , 130. ,

Nat. Biotechnol. , 12.

2016 2016 2012 , 2015 5 , Rapid PrototypingJ. , 335. Adv. Healthcare Mater.

Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 2008 1986 Lab Chip . , , US 20120116568A1

2 . 2009

6 , 1662. 2015 Technical Considerations forAdditive , 149;b)E.Tekin, P.J.Smith, , . 4 Angew. Chem. Int. Ed. Engl. www.advancedsciencenews.com , 703. Chem. Soc.Rev. , ,

30 2007 33

2164. 2015 2014 , 395. Ann. Biomed. Eng. ,

2015 7 Curr. Opin.Biotechnol. ,

, , 1139. 2013 Adv. Mater. Biotechnol. Adv.

43 32 2005 3D Bioprintingand Biofabrication , , 730. , 773. 72 Biochem. J.

, Trends Biotechnol 2017 Rsc Adv. ,

, Tissue Eng.,Part 60 2016 Adv. Healthcare , 777. 2012 11 J. ImagingSci. Int. J. Comput. J. ImagingSci. , 691. , 9. , Recent Pat. Tissue Eng., 6 , .

MRS Bull. , 1601118 45 2016

2016 , 2740.

2016 2016 2016 2010 2006 , , 28 6 , , , , , , , ,

REVIEW

, , , 24 18 , 1. , 1; Acta Acta , , 1601118 2016 77

, 333. , 6136; , , 3453; , 1343; , 322 42 2006 24 43 2012 , 182. ,

, 1600058. J. Biomed. ,

, Tissue Eng. Tissue Eng. Tissue 104 , , 767. Chem. Rev. Curr. Opin. Curr. 3 40 2014 , H178. , 351. , ,

, 11 2014 , 23 , 68. , 86; b) J. Jang, 2016 Mater. Sci. Eng. Mater. 2014 Adv. Healthcare Adv. 2014

, 245

(27 of 29) , , 106. 2016 86 2016 53 2016 Biomaterials

, 12531.

, , 2014 37

, 782; b) S. R.S. b) Shin, 782; , 42 2011 , Adv. Mater. Adv. www.advhealthmat.de , 2016 , 2139.

46 , 2174. , 164. 2016 2016 Chem. Mater.

,

5 37 , 4. 62 , , 539. 2012 , 9 Adv. Sci. Adv.

2016 9 ,

, , 2013 , 5415. , 20.

Exp. Cell Res. Exp. Cell 2016 2015 , 539. 3 8

, 92. 2015 2015 ,

Adv. Mater. Adv. , Chem. Soc. Rev. , 800; c) J. H. Shim, S. E. Kim,

Acta Biomater. 2012 21

Adv. Funct. Mater. Funct. Adv. , Mater. Sci. Eng. R-Rep. Mater. 95 119 , 1210; b) A. Kumar, K. C. Nune, K. C. , 1210; b) A. Kumar, Biomaterials , , Dev. Dynamics Dev. 2015 2016

, 1984. Ceram. Int. Ceram. , 766. 2016 104 , 394.

Semin. Thromb. Hemost. 26 67 Prog. Polym. Sci. , 2016 2016 , ,

34

Prog. Polym. Sci. wileyonlinelibrary.com , J. Biol. Eng. Biomaterials , 025003; b) K. Legemate, S. Tarafder, Y. Jun, Y. , 025003; b) K. Legemate, S. Tarafder, Expert Opin. Drug Delivery Acc. Chem. Res. Chem. Acc. 2016 8 Polymers 2015 , 697. , 103. 2016 Biotechnol. Lett.

, 1; b) S. Ahadian, R. B. Sadeghian, S. Salehi, , 1; b) S. Ahadian, R. B.

, 88; c) F. Pati, D. H. Ha, J. Jang, H. H. Han, , 2016 20 40

J. Biomed. Mater. Res. Part A J. Biomed. Mater. Molecules 30 33 , , , 1980. , , Curr. Drug Delivery Curr. , 1701. 2016

Adv. Healthcare Mater. Adv. 4 20 Technol. Rev. Technol. , , J. Dent. Res. J. Mater. Chem. B J. Mater. 2014 2016

2016 2016 , 1496.

2015 2014

116 , , 156. I. T. Swinehart, S. F. Badylak, Swinehart, I. T. J. W. Rhie, D. W. Cho, Rhie, D. W. J. W. T. G. Kim, B. S. Kim, S. W. Kim, S. M. Kwon, D. W. Cho, D. W. Kim, S. M. Kwon, G. Kim, B. S. Kim, S. W. T. Biomater. a) P. de la Puente, D. Ludena, b) H. T. Shiu, B. Goss, C. Lutton, R. Crawford, Y. Xiao, Y. Shiu, B. Goss, C. Lutton, R. Crawford, b) H. T. Part B-Rev. B. J. Klotz, D. Gawlitta, A. Rosenberg, J. Malda, F. P. W. Melchels, A.D. Gawlitta, Rosenberg, J. Malda, F. P. W. B. J. Klotz, Biotechnol. Trends K. H. Hussein, K. M. Park, K. S. Kang, H. M. Woo, K. H. Hussein, K. M. Park, K. S. Kang, H. M. Woo, Biol. Appl. C-Mater. R. I. Litvinov, J. W. Weisel, Weisel, J. W. R. I. Litvinov, K. Su, C. M. Wang, K. Su, C. M. Wang, W. Wu, A. DeConinck, J. A. Lewis, A. DeConinck, Wu, W. J. M. Aamodt, D. W. Grainger, Grainger, J. M. Aamodt, D. W. a) F. M. Chen, X. H. Liu, 2016 C. J. Dong, Y. G. Lv, G. Lv, C. J. Dong, Y. T. Jungst, W. Smolan, K. Schacht, T. Scheibel, J. Groll, Smolan, K. Schacht, T. Jungst, W. T. c) Q. Wang, Q. Q. Xia, Y. Wu, X. L. Zhang, F. Q. Wen, X. W. Chen, X. W. X. L. Zhang, F. Q. Wen, Wu, Q. Q. Xia, Y. c) Q. Wang, Ouyang, He, H. W. S. F. Zhang, B. C. Heng, Y. Mater. K. Y. Lee, D. J. Mooney, Lee, D. J. Mooney, K. Y. 854; d) R. R. Costa, J. F. Mano, 854; d) R. R. Costa, T. Garg, A. K. Goyal, T. X. M. Li, M. J. Gao, Y. Jiang, X. M. Li, M. J. Gao, Y. e) K. C. Hung, C. S. Tseng, L. G. Dai, S. H. Hsu, e) K. C. Hung, C. S. Tseng, D. K. Tripathi, D. K. Tripathi, T. K. Giri, D. Thakur, A. Alexander, Ajazuddin, H.Badwaik, Ajazuddin, A. Alexander, Thakur, K. Giri, D. T. R. D. K. Misra, K. Bourzac, a) E. M. Goncalves, F. J. Oliveira, R. F. Silva, M. A. Neto, a) E. M. Goncalves, F. J. Oliveira, R. F. Silva, M. Vila, M. Amaral, M. Vallet-Regi, M. H. Fernandes, Mater. Res. Part B Mater. C. H. Lee, 83 Lee, C. H. Jun, C. Chou, M. R. Awadallah, A. Koch, Y. a) S. Tarafder, Biofabrication Y. F. Zheng, X. N. Gu, F. Witte, Y. Part A J. Y. Park, J. Kundu, S. W. Kim, S. S. Kang, D. W. Cho, D. W. S. Kang, Kim, S. S. W. J. Kundu, Park, J. Y. S. J. Lee, D. Lee, T. R. Yoon, H. K. Kim, H. H. Jo, J. S. Park, J. H. Lee, H. R. Yoon, S. J. Lee, D. Lee, T. A. Park, S. D. Kim, I. K. Kwon, W. L. G. Zhang, H. Cui, W. Zhu, M. Nowicki, X. Zhou, A. Khademhosseini, M.X. Zhou, A. Nowicki, Zhu, W. H. Cui, a) Y. L. Wu, N. Putcha, K. W. Ng, D. T. Leong, C. T. Lim, C. T. Leong, Ng, D. T. N. Putcha, K. W. L. Wu, a) Y. Chen, C.Loo, X. S. 3203. B. Holmes, L. G. Zhang, Zhu, H. Cui, W. B. Aghaei-Ghareh-Bolagh, X. Gao, M. Nikkhah, S. M. Jung, B. Aghaei-Ghareh-Bolagh, X. Gao, M. Nikkhah, M. R. Dokmeci, A. Dolatshahi-Pirouz, S. B. Kim, S. M. Kim, A. Khademhosseini, Tang, S. X. c) V. Gribova, R. Auzely Velty, C. Picart, Gribova, R. Auzely Velty, c) V. J. Y. Park, J.-H. Shim, S.-A. Choi, J. Jang, M. Kim, S. H. Lee, Jang, M. Kim, S. H. Shim, S.-A. Choi, J. Park, J.-H. J. Y. Cho, D.-W. Z. Tang, Y. Wang, P. Podsiadlo, N. A. Kotov, Podsiadlo, N. A. Kotov, P. Wang, Y. Z. Tang, a) A. C. Mitchell, P. S. Briquez, J. A. Hubbell, J. R. Cochran, Briquez, J. A. Hubbell, J. R. Cochran, a) A. C. Mitchell, P. S. Biomater. A. Khademhosseini, H. Bae, M. Ramalingam, S. Ostrovidov, Bioconjugate Chem. M. Lei, X. Wang, M. Lei, X. Wang, W. Zhu, X. Ma, M. Gou, D. Mei, K. Zhang, S. Chen, D. Mei, K. Zhang, S. Zhu, X. Ma, M. Gou, W. Biotechnol. H. N. Chia, B. M. Wu, B. M. Wu, H. N. Chia, [86] [91] [95] [99] [94] [96] [98] [97] [92] [93] [89] [90] [87] [88] [81] [85] [82] [84] [83] [80] [79] [107] [101] [105] [103] [100] [106] [102] [104]

, , , , , , , 5 62 44 , Adv. Adv. ACS 347 Acta , , 2016 2014 2016 ,

, 312;

2016 67 , 740. , 624.

2015 2015 , 1079. J. Biomed.

2015 ,

34 42

37 Biomaterials , 54. , , , 32 , B67. 2000 , 16961. , 4273. ACS Macro Lett. Macro ACS , , 321. 8

2016 2004 10 ,

2012 Tissue Eng., Part A Part Tissue Eng., 27 Science 76

, 32. , , , 7 , 12. 2016 ,

2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag Mater. Technol. Mater. 6 Biomaterials 2016

, © 2008 2015

2016

J. Mater. Sci. Mater. Med. Sci. Mater. J. Mater.

, 27226. 2004 6 Chem. Soc. Rev. Soc. Chem.

, 685. , 2014 , 1.

, 160. 21 , 98 , 1. Adv. Healthcare Mater. Adv. , 2016 , 041101. 2014 Biomed. Eng.-Biomed. Tech. Biotechnol. Adv.

Dent. Mater. 2 , 56. , Prog. Polym. Sci. , 2 2016 Biotechnol. Bioeng. Adv. Mater. Adv.

Robot. Comput. Integr. Manuf. Integr. Robot. Comput. 2015 , , 32876. Biomaterials 2011 ,

6 2014 Mater. Today Mater. ,

2015 Adv. Eng. Mater. Adv. , 1679; b) M. Hofmann, b) 1679; ,

2015 2 Sci. Rep. Biofabrication

, 1601118 2015 ,

6 2016 ,

Molecules 2016 , 164.

2017 ACS Appl. Mater. Interfaces ACS Appl. Mater.

14 , 8192. , J. Polym. Sci., Part A: Polym. Chem. J. Polym. Sci., Part A: Polym. Sci. Rep. 26 , Appl. Phys. Rev. Int. J. Polym. Sci. 2015 J. Nanomater.

, 403. , 4437. , 845. , 382. 2014

21 23 25 3 , , , , , 295. , 66. , 267. 2015 31 J. R. Tumbleston, D. Shirvanyants, N. Ermoshkin, R. Janusziewicz, J. R. Tumbleston, K. Chen, R. Pinschmidt, J. P. Rolland, A. R. Johnson, D. Kelly, Samulski, J. M. DeSimone, A. Ermoshkin, E. T. C. Y. Yap, C. K. Chua, Z. L. Dong, Z. H. Liu, D. Q. Zhang, L. E. Loh, Yap, C. Y. S. L. Sing, R. A. Smaldone, J. R. Davidson, G. A. Appuhamillage, C. M. Thompson, W. Voit, Voit, W. J. R. Davidson, G. A. Appuhamillage, C. M. Thompson, L. C. Zhang, H. Attar, M. Calin, J. Eckert, Calin, M. Attar, Zhang, H. L. C. S. A. Skoog, P. L. Goering, R. J. Narayan, S. A. Skoog, P. L. Goering, Mater. Res. B Appl. Biomater. Mater. C. C. Chang, E. D. Boland, S. K. Williams, J. B. Hoying, C. C. Chang, E. D. Boland, 2002 I. T. Ozbolat, H. Chen, Y. Yu, Yu, Ozbolat, H. Chen, Y. I. T. R. Landers, U. Hubner, R. Schmelzeisen, R. Mulhaupt, R. Schmelzeisen, R. Landers, U. Hubner, 30 R. Schmelzeisen, U. Hubner, A. Laib, R. Landers, Pfister, A. R. Mulhaupt, a) D. J. Odde, M. J. Renn, A. Panwar, L. P. Tan, L. P. Tan, A. Panwar, J. W. Stansbury, M. J. Idacavage, Stansbury, J. W. 2014 I. T. Ozbolat, M. Hospodiuk, Ozbolat, M. Hospodiuk, I. T. C. Jia, D. Yu, M. Lamarre, P. L. Leopold, Y. D. Teng, H. Wang, Wang, H. D. Teng, L. Leopold, Y. M. Lamarre, P. C. Jia, D. Yu, Mater. H. Tao, B. Marelli, M. Yang, B. An, M. S. Onses, J. A. Rogers, B. An, M. S. Onses, B. Marelli, M. Yang, H. Tao, D. L. Kaplan, F. G. Omenetto, D. L. Kaplan, J. A. Lewis, G. M. Gratson, J. A. Lewis, G. S. J. Lechner, A. Pich, H. Fischer, A. Pich, H. Fischer, S. J. Lechner, M. N. Birkholz, G. Agrawal, C. Bergmann, R. Schroder, M. N. Birkholz, G. Agrawal, C. Bergmann, R. Schroder, M. Castilho, C. Moseke, A. Ewald, U. Gbureck, J. Groll, I. Pires, C. Moseke, A. Ewald, M. Castilho, E. Vorndran, J. Tessmar, 1349. S. Patel, U. Klammert, L. M. Grover, M. Klarner, E. Vorndran, J. E. Barralet, U. Gbureck, J. F. Xing, M. L. Zheng, X. M.Duan, X. L.Zheng, M. Xing, J. F. J. W. Lee, J. W. 61 G. Brunello, S. Sivolella, R. Meneghello, L. Ferroni, C. Gardin, G. Brunello, S. Sivolella, R. Meneghello, L. Ferroni, A. Piattelli, B. Zavan, E. Bressan, b) X. Zhou, N. J. Castro, W. Zhu, H. Cui, M. Aliabouzar, K. Sarkar, K. Sarkar, H. Cui, M. Aliabouzar, Zhu, W. b) X. Zhou, N. J. Castro, L. G. Zhang, W. Zhu, M. Wang, Y. Fu, N. J. Castro, S. W. Fu, L. G. Zhang, G. L. Fu, S. W. J.Castro, N. Fu, Y. M. Wang, Zhu, W. B. Holmes, W. Zhu, J. Li, J. D. Lee, L. G. Zhang, Lee, L. G. Li, J. D. Zhu, J. B. Holmes, W. F. P. W. Melchels, M. A. N. Domingos, T. J. Klein, J. Malda, Klein, J. N.A. Domingos, T. M. Melchels, W. P. F. P. J. Bartolo, D. W. Hutmacher, Hutmacher, P. J. Bartolo, D. W. 106. M. K. Lee, A. Shkumatov, R. Raman, B. Bhaduri, M. Mir, G. Popescu, H. Kong, R. Bashir, 610. H. J. Donahue, S. H. Jariwala, G. S. Lewis, Z. J. Bushman, J. H. Adair, 3D Print. Add. Manufact. J. H. Park, J. M. Hong, Y. M. Ju, J. W. Jung, H. W. Kang, S. J. Lee, H. W. Jung, M. Ju, J. W. Y. J. H. Park, J. M. Hong, Cho, Kim, S. H. Kim, D. W. S. W. J. J. Yoo, J. P. Fisher, L. G. Zhang, J. P. Fisher, S. Miao, W. Zhu, N. J. Castro, M. Nowicki, X. Zhou, H. Cui, Zhu, N. J. Castro, S. Miao, W. Biomater. 5031. a) M. Guvendiren, J. Molde, R. M. D. Soares, J. Kohn, Eng. Sci. Biomater. X. Li, R. Cui, L. Sun, K. E. Aifantis, Y. Fan, Q. Feng, F. Cui, Q. Feng, Fan, X. Li, R. Cui, L. Sun, K. E. Aifantis, Y. F. Watari, 2014 [64] [67] [67] [51] [70] [71] [68] [69] [57] [53] [54] [56] [55] [59] [52] [58] [50] [49] [48] [72] [74] [76] [75] [73] [61] [60] [66] [65] [63] [62] [77] [78] Adv. Healthcare Mater. Adv. www.advancedsciencenews.com REVIEW 1601118 www.advhealthmat.de [108] [109] [114] [110] [113] [115] [112] [116] [117] [111] [118] [120] [121] [122] [123] [119] [134] [133] [127] [132] [125] [126] [128] [135] [130] [136] [137] [129] [131] [124] Chem. B M. A.Munusamy, K.Murugan,S.-T. Hsu, A.Umezawa, A. Higuchi,Q.-D.Ling,S.Kumar, Y. Chang,A.Alarfaj, V. Mironov, R. P.Visconti,V. Kasyanov, C.Gentile, J.Zhang,R.Markwald, I. S.Kinstlinger, J.S.Miller, C. S.Chen, P. A.Galie,D.H.Nguyen,C.K.Choi,M.Cohen, P.A.Janmey, 2016 M. R.Dokmeci,Oklu,A.Khademhosseini, S. M.Bakht,J.Yang, W. Jia,V. Dell’Erba,P.Assawes,S.R.Shin, Y. S.Zhang,K.Yue, J.Aleman,K.Mollazadeh-Moghaddam, 2011 A. Tan, R. Jeyaraj,N.G,G.Kirby, J.Rajadas,A.Mosahebi,M.Seifalian, S. J.Paulsen,Miller, E. C. Novosel, C. Kleinhans, P. J. Kluger, N. Thottappillil,P.D.Nair, a) A.H.Huang,L.E.Niklason, B. M. Nam, Y. G. Koh, J. Lee, I. J. G.Nemeno-Guanzon, S. Lee,J.R.Berg,Y. Yeo, H.Jo,J. E. 2557. 2103; b)M.J.Mulligan-Kehoe,Simons, Pflugers Arch P. Carmeliet, a) S.J. Paulsen, J. S. Miller, a) P. S.Briquez, Clegg, L. M. E. Martino, M. F. Gabhann, M. M. B.Elliott,S.Gerecht, 956345. L. E.Niklason, P. K.Wu, B.R.Ringeisen, J. A.Hubbell, Cardiol. D. G.Seifu,A.Purnama,K.Mequanint,Mantovani, J. A.Lewis, D. B.Kolesky, R.L.Truby, A.S.Gladman,T. A. Busbee, K.A.Homan, Q. Gao,Y. He,J.-z.Fu, A.Liu,L.Ma, Biomater. Sci. Y. Zhang, Y. Yu, Akkouch, A. A. Dababneh, F. Dolati, I. T. Ozbolat, Y. Luo,A.Lode,M.Gelinsky, 020902. G. Dai, V. K. Lee, D. Y. Kim, H. Ngo, Y. Lee,L. Seo, S. Yoo,S. P. A. Vincent, Y. Zhang,Y. Yu, I.T. Ozbolat, S. N.Bhatia,C.Chen, D. M.Cohen, E.Toro, A.Chen,P.Galie,X.Yu, R.Chaturvedi, J. S.Miller, K.R.Stevens,M.T. Yang, B.M.Baker, D.H.Nguyen, Natl. Acad.Sci.USA D. B.Kolesky, K.A.Homan,M.Skylar-Scott, J. A.Lewis, R. S.Tuan, H. Lin,D.Zhang,P.G.Alexander, G.Yang, J.Tan, A.W. Cheng, C. Xu,W. Chai,Y. Huang,R.Markwald, 2016 J. P.Fisher, C. A.Kraynak,L.K.Kimerer, A.Krieger, P.Kim,C. K.Breuer, A. J.Melchiorri,N.Hibino,C.Best,T. Yi,Y. U.Lee, Mater. A. Barbetta,M.R.Dokmeci,Dentini, A.Khademhosseini, C. Colosi, S.R.Shin,V. Manoharan, S.Massa,M.Costantini, 109 Biomaterials Q. Pi,B.Byambaa,M.R.Dokmeci, S.R.Shin,A.Khademhosseini, W. Jia, P. S.Gungor-Ozkerim, Y. Zhang,K. S. Yue, K. Zhu, W. Liu, B. Holmes,K.Bulusu,M.Plesniak,L.G.Zhang, (28 of29) , 3152. , doi:10.1007/s10439-016-1612-8. , , 63 27

2016

Mater. Sci.Eng.CMater. Biol.Appl. Biomaterials 2013

, 300. , 064001. 2015 Biomaterials Adv. Mater. Proc. Natl.Acad.Sci.USA , Adv. HealthcareMater. Expert Opin.Biol.Ther.

2016 , 28

Cell

, 2015 10 3 Curr. Opin.Chem.Eng. 2009 Nat. Rev. Mater. , 677. , 8032. , 410.

, 2011 wileyonlinelibrary.com 106 ,

3 , 2014 2016 , 134. , 58. , 457

2014

146 2013 J. Mater. Chem.B Nat. Mater. Dev. Dyn. , , , 963;c)M.Potente,H.Gerhardt, Biofabrication 35 113 , 873. Vasc. HealthRiskManag. Lab Chip , , , 8092. Adv. HealthcareMater. 26 Dev. Dyn. 34 , 3179. J. Nanotechnol.Eng.Med. , 3124.

, 331. J. Biomed. Biotechnol. 2016

2016 2010 2015 Cell. Mol.LifeSci.

2012 2016 Biomaterials

2014 , 2014

, , , 1 2015 2010 5 10 , 15006;b)S.Egginton, 244 , , , 319. Biotechnol. Bioeng.

, 2016 2015 11 16 , Adv. Drug Delivery Rev. , 409. © 111 3 , 629. Circulation , , , 768. , 2025. 2016WILEY-VCHVerlag GmbH&Co. KGaA,Weinheim , 68. 2 244 , 014111. , 7968. , , Ann. Biomed.Eng. 4

54 2015 , 629; b) L. Gui, , 3443. Nanotechnology

, 225. 2013

2015

, 2012

61 2014 , 2014 Nat. Rev.

, J. Mater. 2 , 203. 2013 11 , 777. ,

, 2012 2012 , 79. , Proc. 129 Adv. , 71 4 , , , , ,

[169] [168] [167] [165] [166] [164] [162] [163] [159] [160] [158] [157] [153] [154] [156] [155] [161] [152] [150] [149] [151] [138] [139] [140] [142] [141] [143] [147] [144] [145] [146] [148] X. Jia,M.C.McAlpine, Y. L.Kong,E.A.Engel,K.D. Krick,A.Ju,F.Meng,L.W. Enquist, B. N.Johnson,K.Z.Lancaster, G.Zhen,J.He,M.K.Gupta, S. Yoo, Y. B.Lee,S.Polio,W. Lee,G.Dai,L.Menon,R. S. Carroll, R. N.Shah, A. E.Jakus,B.Secor, A.L.Rutz,S.W. Jordan,M.C.Hersam, 2013 C. M. Owens, F. Marga, G. Forgacs, C. Heesch, M. S. Huang,J.He,Chen,Y. Du,M.Gou, Y. Hu,Y. Wu, Z.Gou, J. Tao, J.Zhang,Q.Liu, T. Kang,S.Jiang, Microelectron. Eng. C. Luo, L. Liu, X. Ni, L. Wang, S. M. Nomura, Q. Ouyang, Y. Chen, J. W. Haycock, P. P. Robinson, S. Rimmer, F. Boissonade, F. M. Claeyssens, C. J.Pateman,A.Harding,Glen,S.Taylor, C.R.Christmas, Biomater. Sci. S. Knowlton,Y. Cho,X.J.Li,A.Khademhosseini, S. Tasoglu, Biomed. Microdevices S. Suri,L.H.Han,W. Zhang,A.Singh,S.Chen,C.E.Schmidt, J. L.Curley, S.R.Jennings,M.J. Moore, S. Yoo, W. Lee,J.Pinckney, V. Lee, J.H.Lee,K.Fischer, S. Polio,J.K.Park, e2636. D. B.Chrisey, A.Szabo,Nogradi, B. Hopp,T. Smausz,N.Kresz,Barna,Z.Bor, L.Kolozsvari, M. inhetPanhuis, C. J.Ferris, K.J.Gilmore,S.Beirne,D.McCallum, G.Wallace, (Lond) materials to3DFabrication Techniques L. G.Zhang,D.Kaplan, Soc. Rev. G. A.Saracino,D.Cigognini,Silva,Caprini, F.Gelain, T. Boland, T. Xu, C. Gregory,A. P. Molnar, X. Cui, S. Jalota, S. Bhaduri, B. F. Y. Hsieh,S.H.Hsu, C. Egles, K. Belanger, T. M.Dinis,S.Taourirt, G.Vidal,D.L.Kaplan, W. Zhu, C. O’Brien,J. R. O’Brien, L. Zhang, G. Delivery Rev. A. Faroni, S.A.Mobasseri,P.J.Kingham,Reid, X. Gu,F.Ding,D.Williams, W. G.Sawyer, T. E.Angelini, T. Bhattacharjee,S.M.Zehnder, K.G.Rowe,S.Jain,R.M.Nixon, Adv. H. J.Shue,M.Ramadan,A.R.Hudson,W. Feinberg, T. J.Hinton,Q.Jallerat,R.N.Palchesko,H.Park,M.S.Grodzicki, H. Kruger, W. Meyer, S.Engelhardt,E.Novosel,B.Elling,M.Wegener, Biofabrication M. S.Kindy, C.J.Drake,W. S.Argraves,R.Markwald,Y. Mei, a) Y. Tan, D.J.Richards,T. C.Trusk, R.P. Visconti,M.J.Yost, B. David,S.Jürgen,L.Robert, B. Stefan,N.Franziska, S.C.Ligon,N.Anneliese,B.Helga, R. Markwald,G.Forgacs, C. Norotte,F.S.Marga,L.E.Niklason,G.Forgacs, 2864. X. Liu,B.Pi,H.Wang, X.-M.Wang, 2009 B. Hubbard,S.Colbert, F.Gabor, F. Marga,K.Jakab,C.Khatiwala,B.Shepherd,S.Dorfman, X. Gu, X. Jin,T. Yamashita, C. Stichel,H.W. Muller,

2015 , , 5 30

Front. Med. 2014 , 045007.

, 5910. 2013 Macromol. Biosci. , Exp. Neurol. Neuroreport Biomaterials 1 J. Funct. Biomater. , , e1500758. ACS Nano

2015 9

, 2016 2014 , 859. Biomaterials 42 , , 225.

2015 , 82–83 , Biomater. Sci. 2011 4 6 J. Biochem.

2011 , 768. , 024111;b)K.Jakab,A.Neagu,V. Mironov,

2009 2010

2015 Organogenesis 2006 Adv. Funct. Mater. , , , 160. 9 88 , 401. ,

Adv. Healthcare Mater.

2016 , Prog. Neurobiol. , 13 2015 Neural Engineering:fromAdvancedBio , 1707. , , 20 Sci. Adv. 223

9 Proc. Natl.Acad.Sci.USA 27 2012 , 983. , 4636. Biomed. Mater. , 798. Biomaterials

, 3580. , 2016 , 645. ,

2013 16 49 Biofabrication www.advancedsciencenews.com , 3 , 472. Front. Mater. Sci. , 77. Tissue Eng.

, 257. ,

2015 , Springer, Heidelberg, 2015 , 159 1 , 224.

Sci. Rep. 2015 , 491. , ,

1 1998 2014 11 , e1500655. J. Vis.Exp.

2011 , 153. ,

2005 25 2012 , ,

56 2017

35 , 6205. 2016 , , 119. , 6143. 6 ,

, Nanomedicine 2014 Biofabrication , 055003. 11 4 , Biomaterials , 022001. ,

6

, 1817. 2004 6 Adv. Drug , 1601118 2011 , 32184. , 9 Chem. , 1. , 2016 , 101 Sci. 48 . - , ,

REVIEW

, , Proc. 1601118 2016 2016 Tissue

, 045004. J. Bioact. 2 , 2010

(29 of 29) , 1, doi: 10.1007/ , 21685. , 376. 6 , LB424. , www.advhealthmat.de Nanotechnology , 83. 2016 29 499

Trends Biotechnol. Trends 12 , , , 2016

ACS Appl. Mater. Interfaces Mater. ACS Appl. 2015 2016 Biofabrication , 952.

2006

22 , Sci. Rep. , 2206. 2016 FASEB J.

113 , , 249. Tissue Eng. Ann. Biomed. Eng. Int. J. Pharm. wileyonlinelibrary.com 24 , 014101. , 2016

8 , , 872.12. 2009

27 , 2016

2013

, 30017. 8 , , 315103; c) X. Zhou, W. Zhu, M. Nowicki, S. Miao, H. Cui, , 315103; c) X. Zhou, W. , 722; b) W. Zhu, N. J. Castro, H. Cui, X. Zhou, B. Boualam, Zhu, N. J. Castro, , 722; b) W. X. Wang, Y. Yan, Y. Pan, Z. Xiong, H. Liu, J. Cheng, F. Liu, F. Lin, H. Liu, J. Cheng, F. Pan, Z. Xiong, Y. Yan, Y. X. Wang, R. Zhang, Q. Lu, R. Wu, R. Chang, K. Emami, H. Wu, W. Sun, W. Emami, H. Wu, R. Chang, K. S. Li, Z. Xiong, X. Wang, Y. Yan, H. Liu, R. Zhang, H. Liu, Yan, Y. S. Li, Z. Xiong, X. Wang, Polym. Compat. S. B. Nupura, M. Vijayan, M. Solange, T. Ali, G. Masoumeh, T. M. Vijayan, M. Solange, S. B. Nupura, A. Nasim, S. Su Ryon, C. Giovanni, Shrike, Qi, Z. Yu M. Mario, L. K. Ali, R. D. Mehmet, A. Anthony, E. B. Colin, D. S. Thomas, Biofabrication J. W. Jung, J. S. Lee, D. W. Cho, Jung, J. S. Lee, D. W. J. W. D. Nguyen, J. Robbins, C. Crogan-Grundy, V. Gorgen, V. C. Crogan-Grundy, D. Nguyen, J. Robbins, King, S. Lin, C. Khatiwala, S. O. Creasey, P. Bangalore, D. Perusse, C. Halberstadt, S. Presnell, J. B. Robbins, V. Gorgen, P. Min, B. R. Shepherd, S. C. Presnell, Gorgen, P. Min, J. B. Robbins, V. FASEB J. X. Ma, X. Qu, W. Zhu, Y. S. Li, S. Yuan, H. Zhang, J. Liu, P. Wang, H. Zhang, J. Liu, P. Wang, S. Li, S. Yuan, Zhu, Y. X. Ma, X. Qu, W. F. Sheikh, S. Chien, S. Chen, S. Feng, C. S. Lai, F. Zanella, G. Natl. Acad. Sci. USA a) W. Peng, D. Unutmaz, I. T. Ozbolat, Peng, D. Unutmaz, I. T. a) W. A.-V. Do, A. Akkouch, B. Green, I. Ozbolat, A. Debabneh, A.-V. A. K. Salem, S. Geary, s10439-016-1648-9. G. Jonathan, A. Karim, 27 L. G. Zhang, B. Holmes, R. I. Glazer, Engineering Part C: Methods S. Miao, W. Zhu, N. J. Castro, J. Leng, L. G. Zhang, Zhu, N. J. Castro, S. Miao, W. 2016 34 L. G. Zhang, R. McGrane, R. I. Glazer, [185] [191] [184] [187] [186] [189] [188] [190] [195] [192] [193] [194]

, , , . 10 Acta , 2016 2016 2013

2013

J. Biomed. , 48. Biofabrication 71 , , 1782. 33 2015

, , 035005. 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim GmbH & Co. 2016 WILEY-VCH Verlag 4 © Med. Hypotheses , 2012 , 99, 1, doi: 10.1109/ , 99, 1, doi:

Adv. Healthcare Mater. Adv. PP 2012

, , 9218. Biomaterials , doi: 10.1007/s10439-016-1607-5. , Rice University, Houston, , Rice University, 32 , 2016

Biomaterials 2016

2011

, 1601118 Nat. Rev. Gastroenterol. Hepatol. Nat. Rev. Biofabrication , 1255. 6 , , 264. 101 67 , , 1836. , 2017

10 , 2013 Biomaterials

2015

Anatomy & Physiology 2014 Ann. Biomed. Eng.

, 015001. 6 , , 30. , 1429. L. A. Hockaday, K. H. Kang, N. W. Colangelo, P. Y. Cheung, P. Y. Colangelo, K. H. Kang, N. W. L. A. Hockaday, H. Lipson, L. N. Girardi, L. J. Bonassar, B. Duan, E. Malone, J. Wu, Butcher, C. C. Chu, J. T. 697. T. Ikegami, Y. Maehara, Ikegami, Y. T. B. Duan, L. A. Hockaday, K. H. Kang, J. T. Butcher, Butcher, K. H. Kang, J. T. B. Duan, L. A. Hockaday, Mater. Res. A Res. Mater. B. Duan, E. Kapetanovic, L. A. Hockaday, J. T. Butcher, Butcher, J. T. B. Duan, E. Kapetanovic, L. A. Hockaday, Biomater R. Gaetani, P. A. Doevendans, C. H. Metz, J. Alblas, E. Messina, R. Gaetani, P. A. Doevendans, C. H. Metz, J. A. Giacomello, J. P. Sluijter, G. Steinhoff, Q. Gu, E. Tomaskovic-Crook, R. Lozano, Y. Chen, R. M. Kapsa, Chen, Lozano, Y. R. Q. Gu, E. Tomaskovic-Crook, Crook, J. M. Q. Zhou, G. G. Wallace, 5 A. Seifalian, D. Radenkovic, A. Solouk, F. Y. Hsieh, H. H. Lin, S. H. Hsu, Hsieh, H. F. Y. Biomaterials 87 J. G. Betts, R. Gaebel, N. Ma, J. Liu, J. Guan, L. Koch, C. Klopsch, M. Gruene, R. Gaebel, N. Ma, J. Liu, Li, W. Chichkov, B. Wang, F. P. Mark, Wang, W. A. Toelk, B. Duan, E. M. Stewart, M. in het Panhuis, M. Romero-Ortega,E. M. Stewart, M. in het G. G. Wallace, R. Lozano, L. Stevens, B. C. Thompson, K. J. Gilmore, R. Gorkin 3rd., 3rd., Gorkin K. J. Gilmore, R. Stevens, B. C. Thompson, R. Lozano, L. 2014 S.-J. Lee, W. Zhu, L. Heyburn, M. Nowicki, B. Harris, L. Zhang, M. Nowicki, B. Zhu, L. Heyburn, S.-J. Lee, W. Biomed. Eng. IEEE Trans. TBME.2016.2558493. Martin, K. R. Hutchings, I. M. K. Hsiao, W. B. Lorber, Adv. Healthcare Mater. Adv. www.advancedsciencenews.com [181] [180] [183] [182] [179] [174] [175] [173] [176] [178] [177] [172] [171] [170]