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review 3D bioprinting of tissues and organs Sean V Murphy & Anthony Atala Additive manufacturing, otherwise known as three-dimensional (3D) printing, is driving major innovations in many areas, such as engineering, manufacturing, art, education and medicine. Recent advances have enabled 3D printing of biocompatible materials, cells and supporting components into complex 3D functional living tissues. 3D bioprinting is being applied to regenerative medicine to address the need for tissues and organs suitable for transplantation. Compared with non-biological printing, 3D bioprinting involves additional complexities, such as the choice of materials, cell types, growth and differentiation factors, and technical challenges related to the sensitivities of living cells and the construction of tissues. Addressing these complexities requires the integration of technologies from the fields of engineering, biomaterials science, cell biology, physics and medicine. 3D bioprinting has already been used for the generation and transplantation of several tissues, including multilayered skin, bone, vascular grafts, tracheal splints, heart tissue and cartilaginous structures. Other applications include developing high-throughput 3D-bioprinted tissue models for research, drug discovery and toxicology. The invention of woodblock printing, and the subsequent devel- engineering, made possible by recent advances in 3D printing opment of the industrial-scale printing press in the 15th century, technology,cellbiologyandmaterialsscience.Arelateddevelopment facilitated rapid reproduction of text and images and the dissemina- was the application of 3D printing to produce medical devices such tion of information. Printing had a revolutionary effect on society, as stents and splints for use in the cli.ic affecting education, politics, religion and language across the globe. In 3D bioprinting, layer-by-layer precise positioning of bio- Over the past few decades, printing technology has advanced from logical materials, biochemicals and living cells, with spatial con- two-dimensional (2D) printing to an additive process in which suc- trol of the placement of functional components, is used to fabricate cessive layers of material are distributed to form 3D shapes,. The 3D structures. There are several approaches to 3D bioprinting, production of 3D structures with complex geometries by printing is including biomimicry, autonomous self-assembly and mini-tissue beingappliedbothtoenablerapidprototypingandmanufacturingin building blocks. Researchers are developing these approaches industryandtotheproductionofpersonalizedconsumerproductsin to fabricate 3D functional living human constructs with biologi- © 2014the home, such as bicycle parts, jewelry and electrical components. cal and mechanical properties suitable for clinical restoration of In addition to applications in the manufacturing and consumer sec- tissue and organ function. One important challenge is to adapt tors,3Dprintingistransformingscienceandeducation.Forexample, technologies designed to print molten plastics and metals to the npg archeologistsandanthropologistsproducereplicasofrareartifactsor printing of sensitive, living biological materials. However, the fossils that can be held, shared and distribut. Just as Watson and central challenge is to reproduce the complex micro-architecture Crick modeled the structure of DNA using a ball-and-stick model, of extracellular matrix (ECM) components and multiple cell types 3D printing is now being used to model complex molecules and in sufficient resolution to recapitulate biological function. protein interactions, and to fashion customized laboratory tools–7. Here we review the application of 3D bioprinting to tissue and 3D printing empowers students to design, visualize, hold and test organ engineering. We first consider the main strategies for printing their ideas in real spac. tissue constructs. Next, we describe the different types of bioprinters 3D printing was first described in 1986 by Charles W. Hull. In his andtheirinfluenceontheprintedtissueconstruct.Finally,wediscuss method, which he named ‘sterolithography’, thin layers of a material the stepwise process of printing a tissue, the limitations of current that can be cured with ultraviolet light were sequentially printed intechnologies and the challenges for future research. layers to form a solid 3D structure. This process was later applied to create sacrificial resin molds for the formation of 3D scaffolds 3D bioprinting approaches frombiologicalmaterials.Thedevelopmentofsolvent-free,aqueous- 3D bioprinting is based on three central approaches: biomimicry, based systems enabled the direct printing of biological materials intoutonomous self-assembly and mini-tissue building blocks. We dis- 3D scaffolds that could be used for transplantation with or without cuss these in more detail below. 10 seeded cells . The next step was 3D bioprinting as a form of tissue Biomimicry. Biologically inspired engineering has been applied to Wake Forest Institute for Regenerative Medicine, Wake Forest University Schoolologicalproblems,includingflight 12,materialsresearch , of Medicine, Winston-Salem, North Carolina, USA. Correspondence should be-culture methods 14and nanotechnology . Its application to 3D addressed to A.A. (firstname.lastname@example.org). bioprinting involves the manufacture of identical reproductions 15 Received 5 December 2013; accepted 12 June 2014; published online ofthecellularandextracellularcomponentsofatissueororgan .This 5 August 2014; doi:10.1038/nbt.2958 canbeachievedbyreproducingspecificcellularfunctionalcomponents nature biotechnology VOLUME 32 NUMBER 8 AUGUST 2014 773 review 20,21 of tissues, for example, mimicking the branching patterns of the smaller, functional building blocks or mini-tissues. These can vascular tree or manufacturing physiologically accurate biomaterial be defined as the smallest structural and functional component of a types and gradients. For this approach to succeed, the replication of tissue, such as a kidney nephron. Mini-tissues can be fabricated and biologicaltissuesonthemicroscaleisnecessary.Thus,anunderstand- assembled into the larger construct by rational design, self-assembly ing of the microenvironment, including the specific arrangement of or a combination of both. There are two major strategies: first, self- functionalandsupportingcelltypes,gradientsofsolubleorinsoluble assembling cell spheres (similar to mini-tissues) are assembled into a factors,compositionoftheECMaswellasthenatureofthebiological macro-tissueusingbiologicallyinspireddesignandorganization 20,2; forces in the microenvironment is needed. The development of this second, accurate, high-resolution reproductions of a tissue unit are knowledge base will be important to the success of this approach and designed and then allowed to self-assemble into a functional macro- can be drawn from basic research in fields of engineering, imaging, tissue. Examples of these approaches include the self-assembly of biomaterials, cell biology, biophysics and medicine. vascular building blocks to form branched vascular networks 22,23 and the use of 3D bioprinting to accurately reproduce functional Autonomousself-assembly.Anotherapproachtoreplicatingbiological tissue units to create ‘organs-on-a-chip’, which are maintained and tissuesistouseembryonicorgandevelopmentasaguide.Theearlycel- connected by a microfluidic network for use in the screening of lularcomponentsofadevelopingtissueproducetheirownECMcom- drugs and vaccines or as in in vitro models of disease 24–26 . ponents, appropriate cell signaling and autonomous organization and Combinations of the above strategies are likely to be required to patterningtoyieldthedesiredbiologicalmicro-architectureandfunc- print a complex 3D biological structure with multiple functional, 16,17 tion .A‘scaffold-free’versionofthisapproachusesself-assembling structural and mechanical components and properties. The main cellular spheroids that undergo fusion and cellular organization to steps in the bioprinting process are imaging and design, choice of mimic developing tissues. Autonomous self-assembly relies on the cell materials and cells, and printing of the tissue construct (Fig. 1). astheprimarydriverofhistogenesis,directingthecomposition,locali- The printed construct is then transplanted, in some cases after a zation,functionalandstructuralpropertiesofthetissue 18,1.Itrequires period of in vitro maturation, or is reserved for in vitro analysis. anintimateknowledgeofthedevelopmentalmechanismsofembryonic tissuegenesisandorganogenesisaswellastheabilitytomanipulatethe Imaging and digital design environment to drive embryonic mechanisms in bioprinted tissues. Anessentialrequirementforreproducingthecomplex,heterogeneous architecture of functional tissues and organs is a comprehensive Mini-tissues. The concept of mini-tissues is relevant to both of the understanding of the composition and organization of their compo- above strategies for 3D bioprinting. Organs and tissues comprise nents. Medical imaging technology is an indispensable tool used Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 Imaging Design approach Material selection Cell selection Bioprinting Application © 2014 Nature America, Inc. All rights reserved. X-ray Biomimicry Synthetic polymers Differentiated cells Inkjet Maturation npg CT Self-assembly Natural polymers Plurpotent stem cells Microextrusion Implantation MRI Mini-tissues ECM Multipotent stem cells Laser-assisted In vitro testing Figure 1 A typical process for bioprinting 3D tissues. Imaging of the damaged tissue and its environment can be used to guide the design of bioprinted tissues. Biomimicry, tissue self-assembly and mini-tissue building blocks are design approaches used singly and in combination. The choice of materials and cell source is essential and specific to the tissue form and function. Common materials include synthetic or natural polymers and decellularized ECM. Cell sources may be allogeneic or autologous. These components have to integrate with bioprinting systems such as inkjet, microextrusion or laser-assisted printers. Some tissues may require a period of maturation in a bioreactor before transplantation. Alternatively the 3D tissue may be used for in vitro applications. Self-assembly image is reprinted from Mironov, V. et al. Organ printing: tissue spheroids as building blocks. Biomaterials 30, 2164–2174 (2014), with permission from Elsevier; mini-tissue image is reprinted from Norotte, C. et al. Scaffold-free vascular tissue engineering using bioprinting. Biomaterials 30, 5910–5917 (2009), with permission from Elsevier; the ECM image is adapted from ref. 132, with permission from Wiley; differentiated cells image is reprinted from Kajstura, J. et al. Evidence for human lung stem cells. N. Engl. J. Med. 364, 1795–1806 (2011), Massachusetts Medical Society, with permission from Massachusetts Medical Society; laser-assisted image is reprinted from Guillemot, F. et al. High-throughput laser printing of cells and biomaterials for tissue engineering, Acta Biomater. 6, 2494–2500 (2010), with permission from Elsevier. 774 VOLUME 32 NUMBER 8 AUGUST 2014 nature biotechnology review Figure 2 Components of inkjet, microextrusion and laser-assisted bioprinters. (a) Thermal inkjet printers electrically heat the printhead to produce air-pressure pulses that force droplets from the nozzle, whereas acoustic printers use pulses formed by piezoelectric or ultrasound pressure. (b) Microextrusion printers use pneumatic or mechanical (piston or screw) dispensing systems to extrude continuous beads of material and/or cells. (c) Laser-assisted printers use lasers focused on an absorbing substrate to generate pressures that propel cell-containing materials onto a collector substrate. Figure adapted from ref. 146. by tissue engineers to provide information on 3D structure and or might not be economically feasible for large-scale production. In function at the cellular, tissue, organ and organism levels. These these situations, computer-based models may entirely or partially technologies include most noninvasive imaging modalities, the most contributetoanatomicalstructuraldesign,analysisandsimulation . 37 commonbeingcomputedtomography(CT)andmagneticresonance Additionally,computermodelingcanassistinpredictingmechanical imaging (MRI). Computer-aided design and computer-aided manu- and biochemical properties of fabricated tissue constructs 37–39. To facturing (CAD-CAM) tools and mathematical modeling are also date, CT and MRI data have been used most often in regenerative used to collect and digitize the complex tomographic and architec- medicine to provide specific measurements of tissue dimensions to tural information for tissues. aid the design of a bioprinted construct. CT imaging, used for both diagnostics and interventional pro- Thecompletedtissueororganmodelisinterfacedwithnumerically cedures, is based on the variable absorption of X-rays by different controlled bioprinting systems for prototyping and manufacturing. tissues. The X-ray source rotates around the object, and as the Thisisachievedbyreversingthe2Dto3Dreconstruction,suchthatthe X-ray beam penetrates the body, sensors measure the transmitted 3D-renderedmodelisdividedintothin2Dhorizontalslices(withcus- beam intensity and angle, and record the data as a compilation of tomizable size and orientation) that are imported into the bioprinter pixels that represent a small volume (voxel) of tissue. This imaging system. The anatomical and architectural information contained in modality produces closely spaced axial slices of tissue architecture the 2D horizontal slices provides the bioprinting device with layer- that, after surface rendering and stereolithographic editing, fully by-layer deposition instructions. Variations in the available bioprint- describe the volume of tissue. ingtechnologiesalsoaffecttissueandorgandesign.Somebioprinting Asecondapproach,MRI,alsocanprovidehighspatialresolutionin systems deposit a continuous bead of material to form a 3D struc- softtissue,withtheadvantageofincreasedcontrastresolution,which ture.Othersystemsdepositmultiplematerialsinshortinterruptedor is useful for imaging soft tissues in close proximity to each other, defined spaces. Tissue design must take into account the capabilities without exposure to ionizing radiation. MRI uses nuclear magnetic and properties of the bioprinting systems, which we discuss next. resonance: a strong magneticfieldcausesasmallfractionof nuclei in 28 thetissuebeingimagedtoalignthemselveswiththemagneticfield . Tissue bioprinting strategies Changes to energy states of nuclei produce radiofrequency signals, The main technologies used for deposition and patterning of © 2014which can be measured with receiver coils. The contrast of biological biological materials are inkjet40–43, microextrusion 44–46and laser- 47–49 structurescanbegreatlyincreasedwiththeuseofcontrastagentssuch assistedprinting (Fig.2).Differentfeaturesofthesetechnologies as barium 29 or iodine for CT scans and iron oxide , gadolinium 32 (Table1) should be considered in light of the most important factors npg or metalloproteins 33 for MRI scans. These agents attenuate X-rays in 3D bioprinting, which are surface resolution, cell viability and the or enhance magnetic resonance signals that are commonly used to biological materials used for printing. highlightstructures,suchasbloodvessels,whichotherwisewouldbe difficult to delineate from their surroundings. Inkjet bioprinting. Inkjet printers (also known as drop-on-demand Once raw imaging data have been acquired from these imaging printers) are the most commonly used type of printer for both non- modalities, the data must be processed using tomographic recon- biological and biological applications. Controlled volumes of liquid struction to produce 2D cross-sectional images. 3D anatomical aredeliveredtopredefinedlocations.Thefirstinkjetprintersusedfor representations can be produced for further analysis or modifica- bioprintingapplicationsweremodifiedversionsofcommerciallyavail- 50,51 tion. This process has been described as the transformation of ‘ana- able 2D ink-based printers . The ink in the cartridge was replaced lytical anatomy’ into ‘synthetic anatomy’34. One method to generate with a biological material, and the paper was replaced with an elec- computer-based 3D models of organ or tissue architectures is to tronicallycontrolledelevatorstagetoprovidecontrolofthezaxis 40,50 35 use CAD-CAM and mathematical modeling techniques . The 3D (the third dimension in addition to the x and y axes). Now, inkjet- anatomical representation produces views of organ anatomy while basedbioprintersarecustom-designedtohandleandprintbiological retaining the image-voxel information that can be used for volume materialsatincreasingresolution,precisionandspeed.Inkjetprinters rendering, volumetric representation and 3D image representation. use thermal 43or acoustic50,52,5forces to eject drops of liquid onto a Reconstructed images or models can be viewed in multiple ways, substrate, which can support or form part of the final construct. including as contour stacks, as wire-frame models, shaded models or Thermal inkjet printers function by electrically heating the print solid models with variable lighting, transparency and reflectivity36. head to produce pulses of pressure that force droplets from the If the aim is to produce an accurate reproduction of the imaged nozzle. Several studies have demonstrated that this localized heating, organ or tissue, 2D cross-sections or 3D representation can be used which can range from 200 °C to 300 °C, does not have a substan- directly for bioprinting applications. Alternatively, a direct copy of atial impact either on the stability of biological molecules, such as 52,53 patients’ own organ may not be desirable (due to disease or injury) DNA ,orontheviabilityorpost-printingfunctionofmammalian nature biotechnology VOLUME 32 NUMBER 8 AUGUST 2014 775 review 42,54 cells .Ithasbeendemonstratedthattheshortdurationoftheheat- Notwithstanding these drawbacks, inkjet-based bioprinters also ing (~2 µs) results in an overall temperature rise of only 4–10 °C in offer advantages, including low cost, high resolution, high speed and the printer head . The advantages of thermal inkjet printers include compatibility with many biological materials. Another advantage of high print speed, low cost and wide availability. However, the risk of inkjet printing is the potential to introduce concentration gradients exposing cells and materials to thermal and mechanical stress, low of cells, materials or growth factors throughout the 3D structure droplet directionality, nonuniform droplet size, frequent clogging by altering drop densities or sizes 68,6. Because of the availability of the nozzle and unreliable cell encapsulation pose considerable of standard 2D inkjet printers, researchers in many labs can read- disadvantages for the use of these printers in 3D bioprinting. ily access, modify and experiment with 3D inkjet–based bioprinting Many inkjet printers contain a piezoelectric crystal that creates an technology. Commercially available inkjet bioprinters are also rela- acoustic wave inside the print head to break the liquid into droplets tively cost-effective owing to their simple components and readily at regular intervals. Applying a voltage to a piezoelectric material available design and control software. The wide application of this induces a rapid change in shape, which in turn generates the pres- technology by many groups has accelerated advances in the capacity sure needed to eject droplets from the nozzle 56. Other inkjet printers of inkjet bioprinters to accurately deposit with high resolution and use an acoustic radiation force associated with an ultrasound field precision controllable droplet sizes with uniform cellular densities. 57,58 to eject liquid droplets from an air-liquid interface . Ultrasound Droplet size and deposition rate can be controlled electronically, and parameters, such as pulse, duration and amplitude, can be adjusted can range from <1 pl to >300 pl in volume 70,71with rates of 1–10,000 to control the size of droplets and the rate of ejection. Advantages of dropletspersecond .Patternsofsingledrops,eachcontainingoneor 61 acoustic inkjet printers include the capability to generate and control twocells,inlines~50µmwide,havebeenprinted .Futureadvances a uniform droplet size and ejection directionality as well as to avoid will continue to adapt this technology to handle and deposit other exposure of cells to heat and pressure stressors59–61.Additionally, the biologically relevant materials, in a manner that both facilitates their sheer stress imposed on cells at the nozzle tip wall can be avoided by printing and provides the essential biological, structural and func- using an open-pool nozzle-less ejection system . This reduces the tional components of the tissue. Additional complexities, such as the potential loss of cell viability and function, and avoids the problem requirement for multiple cell types and materials, will also have to of nozzle clogging. Acoustic ejectors can be combined as multiple be addressed. ejectorsinanadjustablearrayformat,facilitatingsimultaneousprint- Notable examples of the inkjet bioprinting approach include the 62 72 73 ing of multiple cell and material types . Even so, there remain some regeneration of functional skin and cartilage in situ. The high concerns regarding the 15–25 kHz frequencies used by piezoelectric printing speed of the approach enables direct deposition of cells and inkjet bioprinters and their potential to induce damage of the cell materials directly into skin or cartilage lesions. These applications 43 membrane and lysis . Inkjet bioprinters also have limitations on achievedrapidcrosslinkingofthecell-containingmaterialviaeithera materialviscosity(ideallybelow10centipoise)owingtotheexcessive biocompatiblechemicalreactionoraphotoinitiatorandcrosslinking force required to eject drops using solutions at higher viscosities .3 through exposure of the material to ultraviolet light. The inkjet One common drawback of inkjet bioprinting is that the biologi- approachfacilitatedthedepositionofeitherprimarycellsorstemcell cal material has to be in a liquid form to enable droplet formation; types with uniform density throughout the volume of the lesion, and as a result, the printed liquid must then form a solid 3D structure maintainedhighcellviabilityandfunctionafterprinting.Thesestud- with structural organization and functionality. Our group and oth- iesdemonstratethepotentialofinkjet-basedbioprintingtoregenerate ers65 have shown that this limitation could be addressed by using functional structures. © 2014 Nature America, Inc. All rights reserved. materials that can be crosslinked after deposition by printing using Layeredcartilageconstructshavealsobeenfabricatedinvitrousing chemical, pH or ultraviolet mechanisms. However, the requirement acombinationofelectrospinningandinkjetbioprinting 74.Thehybrid for crosslinking often slows the bioprinting process and involves electrospinning–inkjetbioprintingtechniqueenabledthefabrication npg chemical modification of naturally occurring ECM materials, which of a layered construct that supported cell function and maintained changes both their chemical and material properties. Additionally, suitablemechanicalandstructuralproperties.Inkjetbioprintershave somecrosslinkingmechanismsrequireproductsorconditionsthatare also been used to fabricate bone constructs , matured in vitro before 66 toxic to cells, which results in decreased viability and functionality . implantationintomice.Theseconstructscontinuedtomatureinvivo Another limitation encountered by users of inkjet-based bioprinting andformedhighlymineralizedtissueswithsimilardensityasendog- technologyisthedifficultyinachievingbiologicallyrelevantcellden- enous bone tissue. sities.Often,lowcellconcentrations(fewerthan10millioncells/ml) 42 are used to facilitate droplet formation, avoid nozzle clogging and Microextrusionbioprinting.Themostcommonandaffordablenon- 60 reduce shear stress . Higher cell concentrations may also inhibit biological 3D printers use microextrusion. Microextrusion bioprint- some of the hydrogel crosslinking mechanisms . 67 ersusuallyconsistofatemperature-controlledmaterial-handlingand Table 1 Comparison of bioprinter types Bioprinter type Inkjet Microextrusion Laser assisted Refs. Material viscosities 3.5–12 mPa/s 30 mPa/s to >6 × 10 mPa/s 1–300 mPa/s 48,63,78,107 Gelation methods Chemical, photo-crosslinking Chemical, photo-crosslinking, sheerChemical, photo-crosslinking 64,85,106,110 thinning, temperature Preparation time Low Low to medium Medium to high 38,64,94,107 Print speed Fast (1–10,000 droplets per second) Slow (10–50 µm/s) Medium-fast (200–1,600 mm/s) 49,58,76,90 Resolution or droplet size<1 pl to >300 pl droplets, 50 µm wide 5 µm to millimeters wide Microscale resolution 49,68,69,76 Cell viability >85% 40–80% >95% 42,54,80,104 Cell densities Low, <106 cells/ml High, cell spheroids Medium, 10 cells/ml 42,49,88,89 Printer cost Low Medium High 77 776 VOLUME 32 NUMBER 8 AUGUST 2014 nature biotechnology review dispensing system and stage, with one or both capable of movement bioprinting field. Some groups have used solutions comprised only along the x, y and z axes, a fiberoptic light source to illuminate the ofcellstocreate3Dtissueconstructswithmicroextrusionprinting 87. deposition area and/or for photoinitiator activation, a video camera Multicellularcellspheroidsaredepositedandallowedtoself-assemble forx-y-zcommandandcontrol,andapiezoelectrichumidifier.Afew into the desired 3D structure 20,88,. Tissue spheroids are thought systems use multiple print heads to facilitate the serial dispensing of to possess material properties that can replicate the mechanical and severalmaterialswithoutretooling 20,7.Nearly30,0003Dprintersare functionalpropertiesofthetissueECM.Dependingontheviscoelastic soldworldwideeveryyear,andacademicinstitutionsareincreasingly propertiesofthebuildingblocks,theapposedcellaggregatesfusewith purchasing and applying microextrusion technology in tissue and eachother,formingacohesivemacroscopicconstruct.Oneadvantage organ engineering research . Industrial printers are considerably oftheself-assemblingspheroidstrategyispotentiallyacceleratedtissue more expensive but have better resolution, speed, spatial control- organization and the ability to direct the formation of complex lability and more flexibility in the material they can print. structures.Thisapproachshowspromiseinenablingthegenerationof Microextrusionprintersfunctionbyroboticallycontrolledextrusion anintraorganbranchedvasculartreein3Dthicktissueororgancon- of a material, which is deposited onto a substrate by a microextrusion structsbypatterningself-assemblingvasculartissuespheroids,in3D head. Microextrusion yields continuous beads of material rather than bioprinted organs. The most common technology used for scaffold- liquiddroplets.Smallbeadsofmaterialaredepositedintwodimensions, less tissue spheroid bioprinting is mechanical microextrusion. asdirectedbytheCAD-CAMsoftware,thestageormicroextrusionhead Cell viability after microextrusion bioprinting is lower than that ismovedalongthezaxis,andthedepositedlayerservesasafoundation with inkjet-based bioprinting; cell survival rates are in the range of for the next layer. A myriad of materials are compatible with microex- 40–86%, with the rate decreasing with increasing extrusion pres- trusion printers, including materials such as hydrogels, biocompatible sure and increasing nozzle gauge 76,8. The decreased viability of cells copolymers and cell spheroids 3. The most common methods to deposited by microextrusion is likely to result from the shear stresses extrude biological materials for 3D bioprinting applications are pneu- inflicted on cells in viscous fluids. Dispensing pressure may have a matic 65,78–8ormechanical(pistonorscrew) 44,81,dispensingsystems. more substantial effect on cell viability than the nozzle diameter 90. Mechanicaldispensingsystemsmightprovidemoredirectcontrolover Although cell viability can be maintained using low pressures and the material flow because of the delay of the compressed gas volume large nozzle sizes, the drawback may be a major loss of resolution in pneumatic systems. Screw-based systems might give more spatial and print speed. Maintaining high viability is essential for achieving control and are thought to be beneficial for the dispensing of hydro- tissuefunctionality.Althoughmanystudiesreportmaintenanceofcell gels with higher viscosities, although pneumatic systems could also be viability after printing, it is important for researchers to demonstrate 78 suited to dispense high-viscosity materials . Pneumatically driven thatthesecellsnotonlysurvive,butalsoperformtheiressentialfunc- printershavetheadvantageofhavingsimplerdrive-mechanismcompo- tions in the tissue construct. nents,withtheforcelimitedonlybytheair-pressurecapabilitiesofthe Increasingprintresolutionandspeedisachallengeformanyusers system. Mechanically driven mechanisms have smaller and more of microextrusion bioprinting technology. Nonbiological microex- complex components, which provide greater spatial control but often trusion printers are capable of 5 µm and 200 µm resolution at linear at reduced maximum force capabilities. speeds of 10–50 µm/s (ref. 75). Whether these parameters can be Microextrusion methods have a very wide range of fluid prop- matchedusingbiologicallyrelevantmaterialswhilemaintaininghigh erties that are compatible with the process, with a broad array of cell viability and function is yet to be seen. Use of improved biocom- 91,92 © 2014 Nature America, Inc. All rights reserved.iterature. Materials with patiblematerials,suchasdynamicallycrosslinkedhydrogels ,that viscosities ranging from 30 mPa/s to >6 × 10 7 mPa/s (ref. 77) have are mechanically robust during printing and that develop second- been shown to be compatible with microextrusion bioprinters, with ary mechanical properties after printing might help to maintain cell higher-viscosity materials often providing structural support for viablity and function after printing. Single-phase, dual-phase and npg the printed construct and lower-viscosity materials providing a continuous-gradation scaffolds are also being designed using similar suitable environment for maintaining cell viability and function. principles. Additionally, improvements in nozzle, syringe or motor- For microextrusion bioprinting, researchers often exploit materi- control systems might reduce print times as well as allow deposition 82 als that can be thermally crosslinked and/or possess sheer-thinning of multiple diverse materials simultaneously . properties. Several biocompatible materials can flow at room tem- Microextrusion bioprinters have been used to fabricate multiple perature, which allows their extrusion together with other biological tissue types, including aortic valves , branched vascular trees and 95 96 components, but crosslink into a stable material at body tempera- in vitro pharmokinetic as well as tumor models . Although the ture83,84. Alternatively, materials that flow at physiologically suitablefabrication time can be slow for high-resolution complex structures, temperatures (35–40 °C), but crosslink at room temperature may constructs have been fabricated that range from clinically relevant also be useful for bioprinting applications 76,8. Materials with tissue sizes down to micro-tissues in microfluidic chambers. shear-thinning properties are commonly used for microextrusion applications. This non-newtonian material behavior causes a Laser-assisted bioprinting. Laser-assisted bioprinting (LAB) is decreaseinviscosityinresponsetoincreasesinshearrate .Thehigh based on the principles of laser-induced forward transfer 97,9. shear rates that are present at the nozzle during biofabrication allow Initially developed to transfer metals, laser-induced forward transfer these materials to flow through the nozzle, and upon deposition, technology has been successfully applied to biological material, the shear rate decreases, causing a sharp increase in viscosity. The such as peptides, DNA and cells 99–102. Although less common than high resolution of microextrusion systems permits the bioprinter to inkjet or microextrusion bioprinting, LAB is increasingly being used accuratelyfabricatecomplexstructuresdesignedusingCADsoftware for tissue- and organ-engineering applications. A typical LAB device and facilitate the patterning of multiple cell types. consistsofapulsedlaserbeam,afocusingsystem,a‘ribbon’thathasa The main advantage of microextrusion bioprinting technology is donortransportsupportusuallymadefromglassthatiscoveredwith the ability to deposit very high cell densities. Achieving physiologi- a laser-energy-absorbing layer (e.g., gold or titanium) and a layer of cal cell densities in tissue-engineered organs is a major goal for the biological material (e.g., cells and/or hydrogel) prepared in a liquid nature biotechnology VOLUME 32 NUMBER 8 AUGUST 2014 777 review Box 1 Ideal material properties for bioprinting The selection of appropriate materials for use in bioprinting and their ▯performance in a particular application depend on several features. These are listed below. • Printability Properties that facilitate handling and deposition by the bioprinter ma▯y include viscosity, gelation methods and rheological properties. • Biocompatibility Materials should not induce undesirable local or systemic responses from▯ the host and should contribute actively and controllably to the biological and functional components of the construct. • Degradation kinetics and byproducts Degradation rates should be matched to the ability of the cells to produ▯ce their own ECM; degradation byproducts should be nontoxic; materials should demonstrate suitable swelling or contractile characteri▯stics. • Structural and mechanical properties Materials should be chosen based on the required mechanical properties o▯f the construct, ranging from rigid thermoplastic polymer fibers for strength to soft hydrogels for cell compatibility. • Material biomimicry Engineering of desired structural, functional and dynamic material prope▯rties should be based on knowledge of tissue-specific endogenous material compositions. solution, and a receiving substrate facing the ribbon. LAB functions depositnano-hydroxyapatiteinamousecalvaria3Ddefectmodel 111. using focused laser pulses on the absorbing layer of the ribbon to In these studies, a 3 mm diameter, 600 µm–deep calvarial hole was generateahigh-pressurebubblethatpropelscell-containingmaterials filled as a proof of concept. Laser 3D printing has been used to fabri- toward the collector substrate. catemedicaldevices,suchasacustomized,noncellular,bioresorbable The resolution of LAB is influenced by many factors, including tracheal splint that was implanted into a young patient with localized the laser fluence (energy delivered per unit area), the surface tracheobronchomalacia 1.Futurestudiesmightusematerialsthatcan tension, the wettability of the substrate, the air gap between the directly integrate into a patient’s tissue. Additionally, incorporating ribbon and the substrate, and the thickness and viscosity of the the patients’ own cells may facilitate the applicability of these types biological layer103. Because LAB is nozzle-free, the problem of of constructs to contribute to both the structural and functional clogging with cells or materials that plague other bioprinting components of the tissue. technologies is avoided. LAB is compatible with a range of viscosities (1–300 mPa/s) and can print mammalian cells with negligible effect Materials and scaffolds oncellviabilityandfunction 104–10.LABcandepositcellsatadensity Initially, 3D printing technologies were designed for nonbiological of up to 10 cells/ml with microscale resolution of a single cell per applications, such as the deposition of metals, ceramics and thermo- © 2014 Nature America, Inc. All rights reserved.f 5 kHz, with speeds up to plastic polymers, and generally involved the use of organic solvents, 1,600 mm/s (ref 49). hightemperaturesorcrosslinkingagentsthatarenotcompatiblewith Despite these advantages, the high resolution of LAB requires rapid living cells and biological materials. Therefore, one of the main chal- gelationkineticstoachievehighshapefidelity,whichresultsinarelatively lenges in the 3D bioprinting field has been to find materials that are npg lowoverallflowrate 10.Preparationofeachindividualribbon,whichis notonlycompatiblewithbiologicalmaterialsandtheprintingprocess oftenrequiredforeachprintedcellorhydrogeltype,istime-consuming butcanalsoprovidethedesiredmechanicalandfunctionalproperties and may become onerous if multiple cell types and/or materials for tissue constructs. havetobeco-deposited.Becauseofthenatureoftheribboncellcoating, Materials currently used in the field of regenerative medicine for it can be difficult to accurately target and position cells. Some of thesepair and regeneration are predominantly based on either naturally challengesmightbeovercomebyusingcell-recognitionscanningtech- derived polymers (including alginate, gelatin, collagen, chitosan, nologytoenablethelaserbeamtoselectasinglecellperpulse.Thisso- fibrin and hyaluronic acid, often isolated from animal or human tis- called‘aim-and-shoot’procedurecouldensurethateachprinteddroplet sues) or synthetic molecules (polyethylene glycol; PEG 112–115). The containsapredefinednumberofcells.However,statisticalcellprinting advantages of natural polymers for 3D bioprinting and other tissue- canbeachievedusingaribbonwithveryhighcellconcentrations,avoid- engineering applications is their similarity to human ECM, and their 49 ing the need for such specific cell targetin. Finally, metallic residuesinherentbioactivity.Theadvantageofsyntheticpolymersisthatthey arepresentinthefinalbioprintedconstruct,owingtothevaporizationof can be tailored with specific physical properties to suit particular themetalliclaser-absorbinglayerduringprinting.Approachestoavoid applications.Challengesintheuseofsyntheticpolymersincludepoor thiscontaminationincludetheuseofnonmetallicabsorbinglayersand 108,109 biocompatibility, toxic degradation products and loss of mechanical modifyingtheprintingprocesstonotrequireanabsorbablelayer . properties during degradation. Even so, synthetic hydrogels, which The high cost of these systems is also a concern for basic tissue- are both hydrophilic and absorbent, are attractive for 3D bioprinting engineering research, although as is the case with most 3D printing regenerative-medicine applications owing to the ease of controlling technologies, these costs are rapidly decreasing. their physical properties during synthesis. The application of LAB to fabricate a cellularized skin construct As the variety of biological materials for medical applications demonstrated the potential to print clinically relevant cell densities increases, the list of desirable traits for printable materials has in a layered tissue construct, but it is unclear whether this system canbecome more specific and complex (Box 1). Materials must have 110 be scaled up for larger tissue sizes . In vivo LAB has been used to suitable crosslinking mechanisms to facilitate bioprinter deposition, 778 VOLUME 32 NUMBER 8 AUGUST 2014 nature biotechnology review must be biocompatible for transplantation over the long-term, and The degradation products should be nontoxic, readily metabolized must have suitable swelling characteristics and short-term stability. and rapidly cleared from the body. Toxic products can include small Short-term stability is required to maintain initial mechanical prop- proteinsandmoleculesbutalsononphysiologicalpH,temperatureor erties, ensuring that tissue structures such as pores, channels and otherfactorsthatcanbedetrimentaltocellviabilityandfunction.For networks do not collapse. As bioprinted tissues develop in vivo, they example,somelarge-molecular-weightpolymersthatareinitiallyinert should be amenable to remodeling, facilitating the formation of can be broken down into oligomers or monomers that can be recog- structures driven by cellular and physiological requirements. Most nized by cells and cause inflammation and other detrimental effects. importantly,materialsmustsupportcellularattachment,proliferation Swelling and contractile characteristics of materials are especially and function . We now discuss in more detail the key attributes of of concern in the fabrication of tissue-engineering products. Overly printability, biocompatibility, degradation kinetics and byproducts, swelling materials can potentially result in absorption of fluid from structural and mechanical properties, and material biomimicry. the surrounding tissues, and contraction may result in the closing of poresorvesselsthatareessentialforcellmigrationandnutrientdeliv- Printability. An important property of a suitable material is that it ery. Moreover, it is important to understand these responses when canbeaccuratelyandpreciselydepositedwiththedesiredspatialand applying multiple materials with dissimilar swelling or contractile temporal control. Some types of bioprinting technology, such as behavior because this could potentially result in loss of layer integrity inkjet, have limitations on material viscosity, whereas others, such as or deformation of the final construct. microextrusion, may require the material to have specific crosslink- ingmechanismsorshear-thinningproperties.Processingparameters, Structural and mechanical properties. If a material is essential such as nozzle gauge, determine the shear stress to which cells are for the maintenance of a 3D structure, in resisting or producing spe- exposed 90as well as the time required for the material to be deposited cific forces or as an anchoring point for mechanical leverage, then 64 to form a 3D structure . For example, inkjet printing requires maintenance of these properties is essential for continued function materials with a rapid crosslinking time to facilitate the layering of of the construct. Materials must be carefully selected based on the a complex 3D structure. Microextrusion, however, can incorporate required mechanical properties of the construct, and different struc- highlyviscousmaterialstomaintaina3Dshapeafterdeposition,with tural requirements will be needed for diverse tissue types ranging 64,102 120 121 final crosslinking occurring after fabrication. from skin and liver to bone . One approach to overcome The choice of material may also be influenced by the ability this limitation is the use of sacrificial materials that can provide the of the material to protect cell viability during the printing process. required structural and mechanical properties over a given period of Thermal inkjet printing and LAB both involve the localized heating time. This sacrificial material either may be used at the time of print- of the material to deposit cells. Materials with either low thermal ing to allow sufficient crosslinking to occur in the construct22,12or conductivity 116ortheabilitytocushionthecellsduringdeliverymay alternatively could be incorporated into the construct, functioning increase cell viability and function after printing17. Although post- until the endogenously produced materials can sufficiently carry out printing cell viability can range markedly based on printer specifica- this function. With this approach, care must be taken to design a tions,materialproperties,resolutionandcelltypes,inkjetbioprinting material with specific structural and degradation properties while studies usually quote cell viabilities in excess of 85%, microextrusion avoiding potential foreign body responses or toxic degradation printing studies report viability ranges of 40–80% and LAB studies byproducts in the construct. 42,54,80,104 © 2014 Nature America, Inc. All rights reserved.. Material biomimicry. The importance of biomimicry for biocom- Biocompatibility. With the advent of tissue engineering, the goal for patibility has only recently been studied. The ability to incorporate biocompatibility has changed from needing an implanted material biomimetic components into a bioprinted construct can have an npg to coexist with the endogenous tissue without eliciting any undesir- active effect on the attachment, migration, proliferation and func- able local or systemic effects in the host, to implanted materials being tion of both endogenous and exogenous cells. It is well established expected to passively allow or actively produce desirable effects in the that materials have a large influence on cell attachment 104,124,125 118 126 host . Biocompatibility in bioprinting includes the expectation of an as well as cell size and shape , and these principles may be use- activeandcontrollablecontributiontothebiologicalandfunctionalcom- ful in controlling the proliferation and differentiation of cells in a ponentsoftheconstruct.Thiscouldincludeinteractionwithendogenous scaffold. The addition of surface ligands to a material has the tissues and/or the immune system, supporting appropriate cellular potentialtoincreasecellattachmentandproliferationonthematerial activityandfacilitationofmolecularormechanicalsignalingsystems,all substrate125. The presence of nanoscale features such as ridges, steps of which are essential for successful transplantation and function. andgroovesalsoaffectscellattachment,proliferationandcytoskeletal assembly 127,12.The3Denvironmentinatissue-engineeredconstruct 129,130 Degradation kinetics and byproducts. As a material scaffold can influence cell shape and affect the differentiation process . degrades,theembeddedcellssecreteproteasesandsubsequentlypro- Nanoscale characteristics of materials can affect cell adhesion, cell duce ECM proteins that define the new tissue 119. The degradation orientation, cell motility, surface antigen display, cytoskeletal con- kinetics of the materials must be understood and controlled. There densation and modulation of intracellular signaling p131ways that are several aspects of degradation that must be considered. The first regulate transcriptional activity and gene expression . is the ability to control degradation rates, ideally matching the rate of A biomimicry approach to engineer materials with specific physio- degradation with the ability of cells to replace the materials with theirlogicalfunctionsrequiresanunderstandingofthenaturallyoccurring ownECMproteins.Thisischallengingbecausematerialswithsuitable tissue-specific composition and localization of ECM components functional and mechanical characteristics for a given tissue may not in the tissue of interest. Recent advances in tissue decellularization match the ability of the cellular components to replace the material methods 132 could provide intact ECM scaffolds for detailed analy- upondegradation.Degradationbyproductsarealsoimportantbecause sis of ECM compositions, localization and biological functions. they often define the biocompatibility of any degradable material. Thisprocessinvolvesthelysisandremovalofthecellularcomponents nature biotechnology VOLUME 32 NUMBER 8 AUGUST 2014 779 review of a tissue, usually via perfusion with deionized water or mild deter- comprise multiple cell types with specific and essential biologi- gents, while leaving behind the tissue-specific ECM. The ability to cal functions that must be recapitulated in the transplanted tissue. reproduce identical ECM scaffolds using a bioprinting approach In addition to the primary functional cell types, most tissues would be useful in tissue engineering and regenerative medicine. also contain cell types that provide supportive, structural or barrier Challenges in tissue decellularization include striking a balance functions, are involved in vascularization or provide a niche for stem between complete removal of cellular components and maintenance cell maintenance and differentiation. Current options for printing of the fine vascular and other tissue structures. Additionally, some cells involve either the deposition of multiple primary cell types into toxicity has been observed when cells are grown on decellularized patterns that faithfully represent the native tissue or printing stem tissue scaffolds, potentially due to the retention of the decellulari- cells that can proliferate and differentiate into required cell types. zation detergent within the ECM 13. In mammals, there are more Cellschosenforprintingshouldcloselymimicthephysiologicalstate than300ECMproteinsaswellasmultipleECM-modifyingenzymes, of cells in vivo and are expected to maintain their in vivo functions ECM-binding growth factors and other ECM-associated proteins 13. under optimized conditions 135. The most abundant and understood proteins are the collagens, pro- Any cell type chosen for printing should be able to expand teoglycans and glycoproteins. These proteins provide strength and into sufficient numbers for printing. Precise control of cell prolif- space-filling functions, bind growth factors, regulate protein com- eration in vitro and in vivo is important for bioprinting. Too little plexes, promote cell adhesion, participate in cellular signaling and proliferationmayresultinthelossofviabilityofthetransplantedcon- may have additional functions. A ‘scaffold-free’ approach to bioprin- struct, whereas too much proliferation may result in hyperplasia or ting may be another interesting take on the concept of material apoptosis. Efforts to control cellular proliferation in the transplanted biomimicry.AscellsproduceanddepositthetissueECM,bioprinted construct are essential to achieve physiological ratios of functional self-assembling cellular spheroids may produce an ECM environ- and supporting cells. In addition, the timing of cellular proliferation ment best suited for their own function. Engineering these dynamic is important. Initially, a high cellular proliferation rate may be desir- ECM mechanisms into materials offers further control over cell able to populate the construct, but over the long term, proliferation behavior. One challenge is to develop methods to incorporate these must be maintained at a rate suitable to achieve tissue homeostasis, materialsintoconstructsusingbioprintingtechnology,ensuringthat albeit without hyperplasia. Some approaches to solve this problem the materials have suitable degradation times and byproducts, and involveviraltransfection 136 oruseofsmallmolecules 137,13toinduce thatthesematerialshavewell-understoodandcontrollablestructural cell proliferation and prevent senescence. In vivo, endogenous stem and functional biological effects in the construct. cells function to replace terminally differentiated cells following nor- mal cell turnover or injury. For a bioprinted construct to maintain Cell sources function long-term, after transplantation, bioprinted tissues must The choice of cells for tissue or organ printing is crucial for cor- be able to maintain cellular homeostasis, self-renew and respond to rect functioning of the fabricated construct. Tissues and organs tissue damage or injury. Improved understanding of the nature and Box 2 Next steps for 3D bioprinting For 3D bioprinting to realize its potential, advances are needed in several aspects of the technology and in our understanding of the biology © 2014 Nand biophysics underlying regenerative processes in vivo. Table 2 details some of the specific areas where further research is needed. Table 2 Issues to be addressed npg Area Focus for future research Bioprinter technology Compatible with physiologically relevant materials and cells Increased resolution and speed Scale up for commercial applications Combining bioprinter technologies to overcome technical challenges Biomaterials Complex combinations or gradients to achieve desired functional, mechani▯cal and supportive properties Modified or designed to facilitate bioprinter deposition, while also e▯xhibiting desired postprinting properties Use of decellularized tissue-specific ECM scaffolds to study ECM compo▯sitions, and/or as printable material Cell sources Well-characterized and reproducible source of cells required Combinations of cell phenotypes with specific functions Greater understanding required of the heterogeneous cell types present i▯n the tissues Direct control over cell proliferation and differentiation with small mo▯lecules or other factors Vascularization Well-developed vascular tree required for large tissues May have to be engineered in the bioprinted construct Capillaries and microvessels required for tissue perfusion Suitable mechanical properties for physiological pressures and for surgi▯cal connection Innervation Innervation is required for normal tissue function May be inducible after transplantation using pharmacologic or growth fac▯tor signaling Simulation before transplantation could be achieved using bioreactors Maturation Time required for assembly and maturation Bioreactors may be used to maintain tissues in vitro Provide maturation factors as well as physiological stressors Potential for preimplantation testing of constructs 780 VOLUME 32 NUMBER 8 AUGUST 2014 nature biotechnology review Figure 3
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