Bio-3D Printing - University Of Florida
Bio-3D PrintingWei SunDrexel University, Philadelphia, USATsinghua University, Beijing, Chinasunwei@drexel.eduNSF Workshop on Frontiers of Additive ManufacturingResearch and EducationJuly 11, 2013
AcknowledgementsGraduate Assistants, Colleagues and CollaboratorsDrexel University and Tsinghua UniversityFunding Support:NSF, DARPA, NASANSFC and MOSTOrganizer:Prof. Yong Huang and Prof. Ming Leu
Bio – 3D PrintingUse biomaterials, cells, proteins orother biological compounds as buildingblock to fabricate 3D personalizedstructures or in vitro biological modelsthrough Additive Manufacturingprocesses3
Bio-3D PrintingAccording to the use of biomaterials, we cancategorize Bio-3DP into the following 4 level ofapplications:Level 1：medical models and medical devices non-biocompatible materialsLevel 2：medical implants biocompatible but not biodegradableLevel 3: tissue scaffolds biocompatible, biodegradable and bio-absorbableLevel 4: in vitro biological models cells and living biological compounds as materials
Bio-3D PrintingPersonalized Medical Modeling and ImplantsApplication：plastic surgery, surgical planning, prosthesis
Bio – 3D PrintingCustomized Implants
Challengesin 3DP of Medical Modeling,Devices and Implants
Personalization and CustomizationBiomodeling extractsmorphological and geometricalinformation from patient-specificmodalitiesdiffers fromconventional CAD designmodeling, in terms of bothmodeling approach andapplications.8
Process to arrive at aBio-CAD model fromCT/MRI data.CT/MRI images2D segmentation3D Region growingMedCAD interfaceIGES curvesReverse Engineering interfacePoint DataSurface Generation,rendering and Nurbs surfaceBio-modeling1) Accuracy2) ComplexitySTL interfaceGeoMagicsSurface renderingand Nurbs surface generationIGES formatCAD model9
Challenges for Bio-3DP of MedicalModeling, Devices and ImplantsPrecision engineering:- How to precisely manufacturingpatient specific geometrysurface engineering:- How to engineering the surfacetopography and surface chemistry toenhance cell-surface interaction10
Bio-3D PrintingAccording to the use of biomaterials, we cancategorize Bio-3DP into the following 4 level ofapplications:Level 1：medical models and medical devices non-biocompatible materialsLevel 2：medical implants biocompatible but not biodegradableLevel 3: tissue scaffolds biocompatible, biodegradable and bio-absorbableLevel 4: in vitro biological models cells and living biological compounds as materials
Scaffold Guided Tissue Engineering- an application example for craniofacial reconstructionBio-3DP ofTissue ScaffoldsCT/MRIReverse n12CMU – Bone Tissue engineering Website, Dr. L. Weiss
Tissue Scaffold Fabrication- Direct MethodsScaffoldAdditive Manufacturinng Imaging DataCT-ScanCADCT/MRI – CAD – SFF:––– FDMSLS3DP – Theriform ProcessAdvantages:– No restriction on shape– High control capability– Consistent – reproducibleMRI ScaffoldDisadvantages:– Limited resolution– Not a cell-friendly environment Harsh Heat Toxic Solvents Non-Sterile13
Scaffolds by Micro-SLASLA builds conceptverification models ofits tensegrity structures70 mm in diameter.Molecular Geodesics, Inc. Innovation through biologicalmimicry, www.molecgeodesics.com/contactUs.htmlSJ Lee & DW Cho (POSTECH, Korea), Miro-SLA for Tissue Scaffolds, MSEC 200714
Scaffolds by SLSAB(A) Original Condyle, (B) SLS FabricatedPCL Scaffold(Das & Hollister Group, UM)SLS PCL MechanicalTest Scaffold
Scaffolds by FDMD. Hutchmacher group16
TheriForm Process17
Precision Extruder Deposition (PED)(Drexel University)18
Scaffolds Fabricated by Precision ExtrusionDeposition TechniqueMaterial:Poly-e-Caprolactone (PCL)Average pore size: 200 mmSmallest strut: 100 mmDarling et al, JBMB, 2005Wang, et al, RPJ, 2005Starly et al, CAD 2006Shor et al, Biomaterials, 200719
Nude Mouse SC Osteogenesis(collaborate with Dr. H. An, MUSC)4 weeks6 weeksNude MouseSubcutaneous OsteogenesisPrinted poly e-Caprolactone scaffoldPCL scaffold bovine osteoblasts20
Low-temperature deposition for fabricationof multi-scale pores tissue scaffolds1. double nozzle multiple materials assemble2. particle-induced pore suitable living environment
Low-temperature deposition for fabricationof multi-scale pores tissue scaffoldsGradient PorosityMicrostructure of various levelsGradient StructureScaffold:PLA-TCPMulti - material : PLLA, PLGA, TCP, Gelatin, Collagen, etc.
Repair of bone defectCooperated with Fourth Military MedicalUniversity of PLA and the Institute ofChemistry of Chinese Academy ofScienceRadiographic images of the rabbitand canine implantation
Engineering of Blood Vessel(Dr. Zhang L et al)Animal Models(Mice and Dog-24 weeks)
Challenges for 3DP of TissueScaffoldsBiomaterialsStructureTopology
Biomaterials for Tissue Scaffolds- Topological design for structural cuesScaffold Materials: biocompatible, biodegradable, bioabsorable,Scaffold porosity and pore size: A large surface area favors cell attachment and growth; A large pore volume accommodates and delivers a sufficientnumber of cells; High porosity for easy diffusion of nutrients, transport andvascularization.5 mm for neovascularization,5-15 mm for fiberblast ingrowth,20 mm for the ingrowth of hepatocytes,20-125 mm for regeneration of adult skin,40-100 mm for osteoid ingrowth,100-350 mm for regeneration of bone,500 mm for fibrovascular tissueAgrawal, C.M.; Ray, R., . of BiomedicalMaterials Research, 55(2):141-50, 2001.26
Challenges for Biomaterials in Tissue Scaffold- a system design approach1) Biophysical requirements: scaffold structural integrity, strengthstability, and degradation; cell-specific pore, shape, size,porosity, and inter-architecture2) Biological requirements: cell loading and spatial distributions; cell attachment, growth and new tissueformation;3) Transport pore topology and inter-connectivity4) Anatomical requirements: anatomical compatibility; geometric fitting5) Manufacturability requirements: process ability (biomaterial availability,printing feasibility etc.) process effect (wrapping, distortion,structural integrity, etc.)It is a multi-constrainedSystem Design Problem27
Computer-Aided Tissue Engineeringfor Load Bearing Scaffold Design and Fabrication(NSF – 0427216; Sun, Shokoufandeh and Liebschner)Micro-CT/MRI ImageTissue PrimitivesBiomimetic designScaffold internal layout patternCAD-based Unit Cell ModelComputer modeling of bonetissue primitives and microstructureAlgorithm to determine optimalscaffold transportScaffold layered (TheriForm)freeform fabricationBiomimetic design andcomputational characterizationof designed tissue scaffoldsInterface of design and processtoolpath for freeform fabrication28of tissue scaffolds
Bio-3D PrintingAccording to the use of biomaterials, we cancategorize Bio-3DP into the following 4 level ofapplications:Level 1：medical models and medical devices non-biocompatible materialsLevel 2：medical implants biocompatible but not biodegradableLevel 3: tissue scaffolds biocompatible, biodegradable and bio-absorbableLevel 4: in vitro biological models cells and living biological compounds as materials
Multi-nozzle Direct Cell Deposition30
Directly Assembled 3D Biological Modelby Cell Printing/DepositionRGD Modified SurfaceUnmodified SurfaceFibroblasts (Encasulated)Endothelial (Applied to Direct Assembled Biological Modelfor AngiogenesisMorss-Clyne & Sun: 2008 Biomaterial Congress; Tissue Engineering, 2010, Biofabrication, 2010
Enabling Cell PrintingTechniques
Enabling Engineering Processesfor Cell d outerInterdigitated rings PiezoelectricSubstrateAcoustic DropletEjectionInkjet(thermal, piezo)Dimatix, ClemsonU. ting(extrusion through syringe)Envisiontech, OrganovoTsinghua, DrexelUIUC(J. Lewis)Invivo BAT- LDW-Clemson
Inkjet Cell PrintingCells NozzleImage courtesy by Dr. T. BolandEndothelial cells printed onmatrigelNSF EFRI (2008-2011): Emerging Frontiersin 3-D Breast Cancer Tissue Test SystemsAwarded to: Burg, Boland, Leadbetter andDreauProf. Thomas Boland (Clemson/UTEP)
Bioplotter - robotic bioprinterUniversity of FreiburgEnvisiontec ( 100K per system)Envisiontech: http://www.envisiontec.de35
Acoustic Nozzle-Free Droplet Ejection(This slide is by courtesy of Dr. Utkan Demirci. Harvard-MIT)ddropletAirdhFocusEjectionLiquidHd outerInterdigitated rings PiezoelectricSubstratelithium niobate, lithiumtantalate, and quartzSAWlow “Pf”high “Pf”100mmCellencapsulation363D Cell Encapsulating Microgel Printing for Tissue Model
Laser assisted cell printing37(Courtesy of Dr. Guillemot, INSERM)
cell as materialsWhat we can do
3D Printing of Heart PatchCell/Protein3DPRadisic, 2004, NationalAcademy of Sciences美国科学院， 2004The Body shape:Part human, partmachine,replacementmaybe one dayextend your life
3D Printing of 3D Cell Aggregates asPhysiological Model for Disease StudyGriffith L., Naughton, G., Science. 295 (5557),2002, pp 1009-1014Image courtesy by Dr. T. Boland, ClemsonFunded in part by: NSF EFRI (2008-2011):Emerging Frontiers in 3-D Breast CancerTissue Test SystemsBurg, Boland, Leadbetter and Dreau40Griffith & Swartz, Nature Review, 200640
3DP Cells for in vitro Micro-Liver Model浓度变化od n/od ��组0d2d4d6d8d培养时间 dEffect of Alcoholic on Liver Function
Bioprinting Adipose-derived stromal (ADS) Cell and Cellassembled Drug Model for Metabolic syndrome (Diabetes)(NSFC-30800248; Tsinghua)pancreatic isletsADSc derived endothelialcells along the edgesA simulated physiological modelAdipocytes cells fill insideA simulated physiologicalmodelXu et al: Biomaterials, 2010Adipocytes cells Irregularlydistributedfalse physiological model 4242
Bioprinting Micro Liver Organ for NASAPharmacokinetic Study(NSAS-USRA-09940-008)NASA’s Interest - Safe plenary exploration & Mars LandingBioprinted Micro-organsMicrofluidic channelsA’AA’’LiverDrug ATargetOrgan7-Ethoxy-4(trifluoromethyl) coumarin(EFC)7-Hydroxy-4(trifluoromethyl) coumarin(HFC)Micro-Organ DeviceMicro-Organ Device for drug conversion study (A, A’, A’’ with multiple micro-organs)Schematic drug metabolic conversion from EFC HFC43
Sinusoid Flow Pattern Design to BiomimicLiver c PhysiologyMediumin 500um20- 40um100 250mmEndothelial CellHepatocytesNon-biodegradable,highly porousbiomaterialMultiple Cell Architecture hepatic vascular system (capillaries) is configured in sinusoidal pattern design the sinusoidal micro-fluidic channel patterns to biomimic invivo liver microstructure. Channel dimensions and strut widths vary from 50mm to 250mm,flow varies from 1ml/min to 5ml/min.
Bioprinting Micro Liver Organ forNASA Pharmacokinetic Study(NSAS-USRA-09940-008)PositivePressureComputer Aided Tissue nges with cells and solubleextracellular matrix &scaffold1.0 cmcomputer3D MotionArmBioprinted TissueBioprinted TissueChang and Sun, Tissue Engineering, 2007 & 2008, Biofabrication, 201045
Integration of Bioprinted Liver Chamberwith Microfluidic Chang and Sun, Tissue Engineering, 2007 & 2008, Biofabrication, 2010
Bio – 3D Printing Cells- What we can do ? Assemble cells and biomaterials in theright spatial position: accelerated cellmigration Fabrication in vitro biological model withdesigned cell micro-environment47
Fabrication in vitro Biological Model with designed Cell Microenvironment by Directly Assembling Cells and BiomaterialsRGD Modified SurfaceUnmodified SurfaceFibroblasts (Encasulated)Endothelial (Applied to Direct Assembled Biological Modelfor AngiogenesisMorss-Clyne & Sun: 2008 Biomaterial Congress; Tissue Engineering, 2010, Biofabrication, 2010
Single Cell Printing/AssemblyImmune responses( 20mm)Nature Reviews Drug Discovery 2005; 4, 399-409assemble cells together (within 20mm) tostudy the immune response49
Assembly Cancer Cells for InVitro Tumor Model(NSFC 2012 E05 Key Research Project; Tsinghua)B1, normal cellsB2, abnormal cells;B3, cancer cells;Clinic Cancer Res 2006; 12(4): 1121.
cell as materialsThe limitations
Printing Cells - 3DP Process Prepare cells and cell delivery medium- Bioink Printing cells- 3D assemble at temperature controlledenvironment Post printing– bioreactor for 3D culture52
Printing Cells - 3DP Process Prepare cells and cell delivery medium- Limited selection: Hydrogel, Alginate,Collagens, MetraGel etc Printing cells- cell injury Post printing– structure integrity, strength and stability- 3D co-culture to simulate human physiology53
Cell delivery medium viscosity changeswith temperatureWe have very limited cell delivery medium andthey are temperature 876543210Gelation temperaturerange0.40.35PP25 SCTR Ag/G D0 10.3ViscosityPP25 SCTR Ag/G D0 1PP25 SCTR Ag/G D0 2Viscosity0.25ViscosityViscosityPP25 SCTR Ag/G D0 2PP25 SCTR Ag/G D0 3Viscosity0.2ViscosityPP25 SCTR Ag/G D0 3Viscosity0.150.10.050200510 15 20 25Temperature T303540 C253035Temperature T4045 C 5050Viscosity changes with temperature for Gelatin/Alginate hybrid materials. Concentration forGelatin is 10% (w/v) and for Alginate is 1% (w/v). The uprush region means the gelationtemperature range, which is sensitive to temperature.
Cell will injury when printingMechanical Disturbances byCell Deposition ProcessBio-deposition ofcell-hydrogel threadDeposition PressureDeposition SpeedNozzle DiameterNozzle LengthHydrogel PropertiesHealthy cellwith desiredphenotypeInjuredNormal nucleusRecoveryAltered phenotypeDeadKaryolysisFragmentation andissolutionof nucleusApoptosisPyknosisContraction ofnuclear chromatinfollowed bynuclearcondensation55
Optical Microscopy Image for Single Cell InjuryConstructs counterstained with DNA stain Hoechst 33342 andcell-impermeant Alexa Fluor 594 wheat germ agglutinin (WGA)Printing at 150 m nozzle and p 5psiBefore printingFluorescentMicroscopyshowing normalcell50 µ50 µFluorescentMicroscopy showingcondensed nucleussuggesting possiblereversible injuryOptical MicroscopyOptical MicroscopyBlue: nucleusRed: cell membranePrinting at 150 mnozzle and p 40psi50 µOptical MicroscopyFluorescent Microscopyshowing disintegratednucleus suggestingirreversible injury andcell death5656
Bio-deposition Induced Effect on Living Cells(NSF-0700405)Cell apoptosisafter printing(40psi)Nair, et al:BiotechnologyJournal, 2009Cell viability and recovery as function ofdeposition pressureCell images for Hepatocytes in 3% Alginate (1million cells/ml) deposited at 40 psi pressure with100 μ diameter nozzlesChang, et al: Tissue Engineering 2007,Tissue Engineering 2008Stem Cell expedited differentiation after57printing
Most importantlywe do not have theneeded BIOLOGYyet !!!
尺度单位：m10-1Cell/Tissue Printing for 3Din vitro Biological Model10-310-4Cell Printing10-610-9In vitro Biological Model
Difference in Engineering f it looks like an engine,it probably is an engine.Form-based Engineering DesignIt looks like a heart,but it does not have the functionTime-based Cellular Squences(Functionality is designed throughForm and Material)(Functional regeneration throughcellular tissue engineering process)60
3D Printing of Blood VesselsNSF-FIBR: Frontiers of IntegrativeBiological Research; 2007-2012“Understanding Cell Assembly”Gabor Forgacs, U. of Missouri6161
Knowledge Gap“Classical Biology is based on the understanding of developmental physiology,i.e., growth from cells in Petri dishes. What if biologists are given cell printing3D cellular biological models”Image courtesy by Dr. V.Mironov, MUSCWe do not have enough BIOLOGY!!!
Opportunities:From Tissue Engineering toTissue Science andEngineering
Evolving of Tissue Engineering NSF definition of Tissue Engineering (1989):“Tissue engineering is the application of principles and methods ofengineering and life sciences toward the fundamental understandingof structure-function relationships in normal and pathologicalmammalian tissues and the development of biological substitutes torestore, maintain, or improve tissue function”New for Tissue Science & Engineering*(2007)“The use of physical, chemical, biological, and engineering processes tocontrol and direct the aggregate behavior of cells”* Advancing Tissue Science &Engineering: A multi-angencystrategic plan, June 200764
From Tissue Engineering to Tissue Scienceand Engineering Regenerative Medicine more on cells, particularly on Stem Cells 3D Physiological or Disease Models For better study disease pathogenesis and fordeveloping molecular therapeutics Pharmacokinetic Models to replace animal testing For drug screening and testing Cell/Tissue on Chip For detection of bio/chemical threat agents* Advancing Tissue Science & Engineering: A multi-angency strategic plan, June 200765
Fabrication of In Vitro 3D TissueConstructs as Drug Testing ModelsIntroduce testmoleculesTissue ChamberMetabolism of primarychemical compoundsMedia PumpRPVTissue-on-ChipVRecycleloopWVDRVAnimal on-ChipWRVPElectrophoreticChamberRRWVDVPDWVW 66
Manufacturing CellularMachinesBy creating the building blocks — component cell types of biological machines toproduce integrated cellular systems, such as biosensors, cell-on-chips, bio-robots, etcNSF Research Center - EmergentBehaviors of Integrated CellularSystems(MIT, GT, UIUC)Mechanical Engineering Magazine, Nov. 2010
DARPA/NIH Joint Program– 150M/year for 5 yearsA 5 year plan to develop an in vitroplatform of engineering 3D humantissue constructs that accuratelypredicts the safety, efficacy, andpharmacokinetics of drug/vaccinecandidates prior to their first use inmanBroad Agency AnnouncementMicrophysiological SystemsDSODARPA-BAA-11-73, Sept. 15, 201168
“Human-on-a-chip”Wyss Institute to Receive up to 37 Million from DARPA to IntegrateMultiple Organ-on-Chip Systems to Mimic the Whole Human c-the-whole-human-bodyCell Printing Novel Biomaterials Micro-fluidictechnology will provide a vital tool to this development.
Printing Body PartsA machine that prints organs is coming to marketFeb 18th 2010, The Economist print edition70
Frontier Research- What We Need A new generation of biomaterials - Bio-Ink: gowith cells (structure as cell delivery medium),grow with cells (support as cell ECM) andfunction with cells (as biomolecules)； Developmental Engineering (vs DevelopmentalBiology) to fill the biological knowledge gap; Bio-3DP manufacturing tools: viable, reliableand reproducible, and capable of makingheterogeneous structures; 4-D 3DP model: embedded time into Bio-3DPmodel: printing Stem Cells with control releasedmolecules for complex tissues, Organs, CellularMachines and Human-on-a-Chip devices
Thank you!72
Bio-3D Printing According to the use of biomaterials, we can categorize Bio-3DP into the following 4 level of applications: Level 1：medical models and medical devices non-biocompatible materials Level 2：medical implants biocompatible but not biodegradable Level 3: tissue scaffolds biocompatible, biodegradable and bio-absorbable
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