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  1. 1. 3D Printing Technologies for Tissue Regeneration and Biomedical Science Jonathan T. Butcher, Ph.D. Department of Biomedical Engineering Cornell University July 10, 2013
  2. 2. Tissue Failure is a Tremendous Clinical Burden •  Approximately 5 million surgeries/yr in US to replace damaged tissues –  3M orthopaedic/reconstructive (bone, cartilage, soft tissue) –  1M cardiovascular (blood vessel, valve) –  300K internal organ –  200K neural •  Tissue transplant supply is insufficient •  Synthetic implants fail from wear, fatigue, biocompatibility “Rex”
  3. 3. Tissue  Engineering:  Living  Replacement   Tissues  Capable  of  Growth  and  Remodeling   Cell Isolation Expansion Scaffold Seeding In Vitro Conditioning Langer and Vacanti, Science 1993
  4. 4. Challenges of Tissue Engineering •  Cells, Scaffolds, Conditioning •  Rapid, scalable methods for fabrication of living tissues •  Minimize time, resources, cost, expertise needed for tissue production •  Cellular uniformity, QA/QC •  Fabrication of customized/ personalized tissues vs. “Off the shelf” replacements •  Effective business models –  FDA, Insurance reimbursement
  5. 5. Tissues Exhibit Complex Natural Engineering: The Aortic Valve S L O R L L S R = root, L = leaflet, S = sinus, O = Ostia Bicuspid Aortic Valve Valve Calcification How can we engineer this macro- and micro-scale complexity within living tissue replacements?
  6. 6. 3D Biofabrication Methods Injection Molding Tissue Injection Molding (Chang+, JBMR 2001) 3D Printing/FDM Tissue Printing (Cohen+ Tissue Eng 2006) Sintering/HIP Cell-Mediated Sintering (Mercier+ Ann Biomed Eng 2003) Spray Coating Tissue Painting (Roberts+ Biotech Bioeng 2005) Soft Lithography Living Lithography (Choi+ Nature Med 2007)
  7. 7. Tissue Injection Molding Tissue biopsy or stem cells Cells suspended in alginate solution + CaSO4 Intervertrebral Disc (Bowles et al, PNAS 2011) Ear (Reiffel et al, PLoS One2013) Trachea (Kojima et al, J Thoracic Cardio Surg 2002) Meniscus (Ballyns et al, Biomaterials, 2010) Mold from positive model Chang et al, J Biomed Mat Res 2001
  8. 8. Image-Guided Mold Design Mold DesignData ConversionµCT Image Molded Alginate Printed ABS Plastic Cultured Meniscus Implant Ballyns et al, Tissue Eng Part A 2008
  9. 9. 3D Tissue Printing Technology Micro CT/MRI Threshold Reconstruction Bioprinter Crosslinkable monomer Photoinitiator Cell Crosslinkable macromer UV LED Bioink Deposited and Crosslinked Bioink Cohen et al, Tissue Engineering 2006; Hockaday et al, Biofabrication 2012
  10. 10. 3D Printing “Inks” for Controllable Biological Response of Encapsulated Cells Me-HA MO0.05HA MO0.1HA Cell Cell adhesion site HA (MOHA) HA (MOHA)+Me-Gel Mw ↓ Me-HA (MOHA) Me-Gel PEGDA Stiffness ↑ Provide mechanical strength Provide cell adhesion cites Mimic ECM PEGDA+Me-Gel
  11. 11. UV LED Array Root Leaflets Nozzles Direct 3D Printing of Photocrosslinked Hydrogel Tissues Tri-Leaflet Heart Valves Gradient Tissues
  12. 12. Optimal Deposition Rate and Path Space Scale with Nozzle Diameter 0.000 0.002 0.004 0.006 400 600 800 1000 1200 DepositionRate Nozzle Diameter (µm) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 400 600 800 1000 1200Pathspace(mm) Nozzle Diameter (µm) Kang et al, Biofabrication 2013
  13. 13. Comparison of 3D Biofabrication Technologies Injection Molding 3D Tissue Printing High spatial resolution Rapid fabrication Fewer “ink” material requirements Mold printed anywhere Resolution tied to nozzle diameter Significant “ink” material requirements “In-house” printing only (?) No ability to fabricate internal inclusions/voids Only homogeneous material formulations Must extract safely/sterily from mold Can fabricate virtually any geometry Can fabricate multiple materials and blends of materials No need to extract tissue
  14. 14. Image Based Quantification of Shape Fidelity Hockaday et al Biofabrication 2012 Surface Deviation Maps 80% ± 10% match Scaled Printed Valves Slice-by-Slice Overlay 74% Match 89% Match Inner Diameter 22mm 17mm 12mm
  15. 15. 70 80 90 100 0 5 %Accuracy Circular Diagonal Base Design Print Base Middle Top Design Print High Fidelity Micro-scale 3D Tissue Printing - Gradients Diagonal Gradient Spherical Gradient Middle Top
  16. 16. Dynamic Gradients of Cells in 3D Printed Hydrogel Tissues Cells Fluorescently Labeled Red or Green Printed in a 3D vertical gradient 50x 0 0.5 1 1.5 0 20 Intensity(au) Position (mm) High Throughput 3D Culture Screening
  17. 17. Density Thresholds for Material Regions Layer Specific Heterogeneous Material Domains Initial Layer Mid-print Final Heterogeneous printed valve shown in stages CT image slice Base Sinus Aorta Combined Macro- and Micro-Scale 3D Tissue Printing: Heart valves
  18. 18. Tissue Engineered Meniscus Ballyns et al, Tissue Eng Part A 2008 Cells remodel alginate and produce collagen in culture
  19. 19. Anatomically Appropriate Mechanical Stimulation CompressiveStrain Loading Platen Loading Tray Bioreactor Load Cell High Linear Poroelastic FE Model Low Ballyns et al, J Biomechanics 2010
  20. 20. Mechanical Conditioning Accelerates Biomechanical Remodeling Puetzer et al, Tissue Eng Pt A 2013
  21. 21. Tissue Engineered Intervertebral Disc via Hybrid Printing Bowles et. al., Tissue Eng Pt A 2010
  22. 22. In Vivo Evaluation in Rat Tail 6 Weeks 6 Months N = 24 N = 12 MRI Signal Disc Height Histology Mechanics N = 48 Discectomy N = 6 Native Disc Re-implant N = 6
  23. 23. TE-IVD Maintains Mechanical Integrity After 6 Months In Vivo Bowles et. al., PNAS 2011
  24. 24. TE-IVD Tissue Generation and in vivo Integration
  25. 25. Ear Reconstruction via Photogrammy Based 3D Printing •  Combined laser-scan and panoramic photograph –  Non-invasive, no ionizing radiation –  Scan time < 30 seconds, 250 micron resolution 3D Reconstruction Molded Tissue
  26. 26. 3 Months In Vivo Results in Cartilage-like Structure 26 Reiffel et al, PLoS ONE 2013 1 month 3 months
  27. 27. In Situ 3D Tissue Printing for Bone/Cartilage Defects Osteochondral DefectMounting and CT Scan In Line Scan and Print Cohen et al, Biofabrication 2010
  28. 28. Matrix Stiffness Directs Stem Cell Differentiation Cells differentiate on substrates mimicking native stiffness Reilly et al J Biomech. 2010, Kloxin et al Biomaterials 2010, Engler et al Cell 2006 Cells reside in matrix environments with specific stiffness ranges
  29. 29. Mechanical Tunability PEGDA/Me- HA/Me-Gel Hydrogels PEGD700/Me- HA/Me-Gel PEGD3350/Me- HA/Me-Gel PEGD8000/Me- HA/Me-Gel Irgacure 0.1% Irgacure 0.05% Irgacure 0.025% 0 20 40 60 80 100 120 Young'sModulus(kPa)
  30. 30. A.Lc (human) A.Sc (human) P.Sc (porcine) P.Lr (porcine) PEGDA3350/ Me-Gel/Alg PEGDA8000/ Me-Gel/Alg P.Sc (pediatric) P.Lc (pediatric) A. Aortic P. Pulmonary L: Leaflet S: Sinus c: circumferential r: radial Material Formulations that Mimic Physiological Valve Tissue Mechanics 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 80 100 Stress(MPa) Strain (%) k
  31. 31. 0 25 50 75 100 0.5 0.75 1 Viability[%Live] VA086 Concentration [w/v%] 0 25 50 75 100 0.05 0.075 0.1 Irgacure 2959 Photoinitator Concentration [w/v%] Sensitivity to Encapsulation Conditions Dependent on Cell Type and Photoinitiator DAY 7 A P<0.05 A B A B B A A A A AB AA A AA B B HAdMSC HAVIC HAsSMC
  32. 32. Stiffness and Adhesion Control Myofibroblast Phenotype of VIC 0 2 4 6 Relative Expression αSMA 0 2 4 6 Relative Expression Vimentin 0 5 10 15 20 25 30 Relative Expression Periostin 0 5 10 15 20 25Relative Expression Hyaluronidase I MO0.1HA MO0.05HA Me-HA MO0.1HA/Me-Gel MO0.05HA/Me-Gel Me-HA/Me-Gel
  33. 33. Stiffness Directs Stem Cell Differentiation Towards Heart Valve Phenotypes
  34. 34. Fabricated chamber C 3D Printed Fluid Bioreactor Enables Direct Stimulation of TEHV in Minimal Volumes Bioprosthetic “Stiff” Valve Physio-Valve
  35. 35. 3D Printed Vascularized Tissue Grafts for Reconstructive Surgery Wound MRI CAD Print Design Print Implant
  36. 36. Colloidal Gels Hydrogels ‘Fugitive’ Inks Barry, Shepherd et. al (2009) Therriault, Shepherd et. al (2005) Printing ~1 µm hydrogel filaments under UV light. Next Generation Designer “Inks” Hanson-Shepherd et. al (2010) pHEMA Primary rat neuron cells
  37. 37. µ-Fluidic Particle Synthesis for Novel 3D Printing Nozzles Shepherd et. al, Adv. Mat. (2008) Shepherd et. al, Langmuir (2006) *unpublished Single Emulsion: Sheath Flow Double Emulsion: Co-flow Microcapillary Single Phase: Stop Flow Lithography
  38. 38. Where We Are Now Skin: Michael+ PLoS One 2013 Ear: Reiffel+ PLoS One 2013 Heart Valve: Hockaday+ Biofabrication 2012 IVD: Bowles+ PNAS 2012 Meniscus: Ballyns+ Tissue Eng 2010 Bone: Ciocca+ Comp Med Imag 2009
  39. 39. •  Total body scan (data storage) •  Marrow stem cell biopsy Cell storage Cell-seeded polymer “ink” Tissue printer Living implant Data Gathering Injury/Disease/Defect Treatment Where We Hope to Be
  40. 40. How Do We Get There? •  New 3D Printing Technology – Multiple printing modes – Controllable curing systems – Direct clinical printing options – Cost and revenue models •  Improved “inks” for printing – Significant but KNOWN material requirements – Shear thinning for more rapid deposition •  Improved Image based geometry/material retrieval and deposition algorithms
  41. 41. Acknowledgments Cornell Prof. Hod Lipson Prof. Larry Bonassar Prof. Rob Shepherd Duan Bin, PhD Robby Bowles, PhD Jeff Ballyns, PhD Bobby Mozia Heeyong Kang Laura Hockaday CWMC Roger Härtl, MD Harry Gephard, MD Jason Spector, MD Alyssa Reiffel HSS Suzanne Maher, PhD Tim Wright, PhD Russ Warren, MD Hollis Potter, MD