9/15/2017. Prof. Steven S. Saliterman Department of Biomedical Engineering, University of Minnesota

Transcription

1 Department of Biomedical Engineering, University of Minnesota Prof. Angela Panoskaltsis-Mortari s BMEn 5920, Special Topics in 3D Bioprinting Tissue engineering Pathway for bioprinting 3D tissue Workflow Bioprinting Modalities & Bioink Design Scaffolds Modeling Role of Imaging Segmentation Toolpaths Bioprinting example - bronchus Brief Introduction Design & Imaging Tissue Engineering Components: The type or types of living cells being implanted (e.g. somatic, embryonic stem cells, adult stem cells, or induced-pluripotent stem cells). Type of scaffolds supporting the cells (i.e. the mechanical cues provided to the cells). Type of drugs, extra-cellular matrix (ECM), and growth factors conditioning the cells, (the additives that provide chemical cues to the cells). 3D Printing: Computer assisted process for depositing biomaterials and living cells in a determinate configuration in order to produce a defined 3D biological structure. Bioinks consist of various polymer materials, cells and additives. Mosadegh, B., G. L. Xiong, S. Dunham, and J. K. Min. "Current Progress in 3d Printing for Cardiovascular Tissue Engineering." Biomedical Materials 10, no. 3 (Jun 2015). 1

2 Murphy, S. V., and A. Atala. "3d Bioprinting of Tissues and Organs." Nature Biotechnology 32, no. 8 (Aug 2014): Our focus today Teodori, L., A. et al. "Three-Dimensional Imaging Technologies: A Priority for the Advancement of Tissue Engineering and a Challenge for the Imaging Community." Journal of Biophotonics 10, no. 1 (Jan 2017): Anatomically correct constructs from medical imaging data. Porous structures. Co-culturing of multiple cell types. Precise patterning of cells and ECM. Controlled deliver of growth factor and genes. Potential for high-throughput fabrication. Suitable vascularization - eventually! Remember the need for a vascular network in relationship to the diffusion length of oxygen. 2

3 1:17 Today 1) Design What are your trying to achieve? What bioinks and printing method? 2) Images - Useful from the subcellular to organ level. Apply CAD tools for segmentation, freeform and other space-filling methods. 3) Slicer - G-code generation for controlling toolpath, speed, valves, droplet patterns, laser pulse, photoinitiator lights (e.g. Ingracure and other gels), temperature etc. 4) Special setups - Extruder, tips, light source (specific nm), pressure & calibration, cooling (Pluronic Gel) etc. 5) Bioprinting 6) Bioreactor - Incubation, nutrients, growth factors, oxygen supply, environment, etc. (Your 4 th dimension time!) 7) Observation - Fluorescent and transmitted light (confocal microscopy, bright-field, dark-field, confocal laser microscopy etc.). Automatic imaging with control of ambient air, humidity and temperature. 8) Characterization - Histology, growth, mechanical properties etc. Bioprinted hydrogel examples Hospodiuk, M. et al. "The Bioink: A Comprehensive Review on Bioprintable Materials." Biotechnology Advances 35, no. 2 (Mar-Apr 2017):

4 Hospodiuk, M. et al. "The Bioink: A Comprehensive Review on Bioprintable Materials." Biotechnology Advances 35, no. 2 (Mar-Apr 2017): Hydrogels Decellularized matrix Microcarriers Tissue spheroids Cell pellets Tissue strands The goal of all of these materials is to fabricate viable constructs with high physiological relevance. Dorothy, Wizard of OZ, Toto. I ve a feeling Ozbolat, I.T. 3D Bioprinting Fundamentals, Principles and Application. Elsevier, Amsterdam Biobots Ozbolat, I.T. 3D Bioprinting Fundamentals, Principles and Application. Elsevier, Amsterdam

5 Giannitelli, S. M., et. al.. "Current Trends in the Design of Scaffolds for Computer-Aided Tissue Engineering." [In English]. Acta Biomaterialia 10, no. 2 (Feb 2014): Underlying methods in CAD systems: Constructive solid geometry (solid primitives and boolean operators) Boundary representation (vertices, edges and faces) Spacial enumeration (cubic elements) Image-based design Implicit surfaces Space-filling curves Irregular porous structures Giannitelli, S. M., et. al.. "Current Trends in the Design of Scaffolds for Computer-Aided Tissue Engineering." [In English]. Acta Biomaterialia 10, no. 2 (Feb 2014): Ozbolat, I.T. 3D Bioprinting Fundamentals, Principles and Application. Elsevier, Amsterdam

6 Nam Jet.al. Computer aided tissue engineering for modeling and design of novel tissue scaffolds. Computer-Aided Design & Applications 2004;1: Honeycomb pores Hilbert recursive curves Giannitelli, S. M., et. al.. "Current Trends in the Design of Scaffolds for Computer-Aided Tissue Engineering." [In English]. Acta Biomaterialia 10, no. 2 (Feb 2014): Giannitelli, S. M., et. al.. "Current Trends in the Design of Scaffolds for Computer-Aided Tissue Engineering." [In English]. Acta Biomaterialia 10, no. 2 (Feb 2014):

7 A. Femur from CT. B. Printed in alginate. C. Uniaxial tensile testing. D. Coronary from MRI. E. Arterial tree in alginate. F. Arterial tree in fluorescent alginate. G. Zoom of vessel wall H. Darkfield positioning. I. Time-lapse dye flow Hinton, TJ et al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Science Advance 23 Oct (2) Our focus today Nam, S. Y., et. al. "Imaging Strategies for Tissue Engineering Applications." Tissue Engineering Part B-Reviews 21, no. 1 (Feb 2015): Magnetic Resonance Imaging (MRI) Human max. is 3T (Tesla) resolution of 250µm x 250µm 0.5mm. High spatial resolution µmri, 7-10T, 5-200µm. Magnetic nanoparticles. Computed tomography (CT) Computer Axial Tomography Typical resolution of mm. µct, resolution of 1-200µm. Ultrasound Resolution of 1mm x 1.mm x 0.2mm. PET Positron emission tomography SPECT Single photon emission computed tomography Optical Coherence Tomography (OCT) Traditional optical techniques. 7

8 Mayo Foundation for Medical Education and Research CT scan/pet Scan/ Combined Mayo Foundation for Medical Education and Research 8

9 Purpose To delineate and isolate anatomical features within an imaging database- e.g. bone, cartilage, soft tissue, edema; muscle, lung, brain & other organs, and tumors. Method Extract images from DICOM files (ITK-Snap, Onis) and possible deindentifying them for HIPPA regulations (DICOMCleaner). Segmentation Software (ITK-Snap, Materialise Mimics, Materialise 3- matic). Pre-segmentation Phase - identify parts of image as foreground and background. Active Contour Phase - manual and semiautomatic methods. Editing and fixing mesh files (.STL) - Autodesk Meshmixer. Slicer software Simplify3D and Repetier. G-coding for the specific bioprinter - e.g. Slic3R (printer customized interface to control what happens in a sequence of control steps.) Sagittal or Median Parasagittal (Yellow) Frontal or Coronal Transverse or Axial Image, Wikipedia Manual Segmentation 9

10 10

11 Import the STL Mesh file generated by ITK-Snap. Edit feature here slicing in a plane, bottom view. Bioprinting rasters may be in Cartesian vs parametric* form. Consider extrusion-based (EBB) rather than droplet-base (DBB) or laser-based bioprinting (LBB) which are less common and based on manufacturer specific tool paths. Why use one method vs the other? Issues arise with resulting printed gradients as excess accumulation of bioink can occur at directional changes. Parametric* modeling/toolpath may be helpful for lumen and other hollow shape object printing. Control of porosity (e.g. bones) *Parametric implies a variable is dependent on other variables commonly used to express the coordinates of the points that make up a geometric object such as a curve or surface. A. Design of a continuous toolpath and bilayer bioprinted vertebra. B. Toolpath for graded wound device and bilayer printed device. C. Comparison of toolpath using cartesian vs parametric coordinates. Ozbolat, I.T. 3D Bioprinting Fundamentals, Principles and Application. Elsevier, Amsterdam

12 A. CAD model of aorta with controlled material composition along the parametric distance u. B. Femur model with toolpath for controlled porosity along the distance u; sample double layer structure bioprinted using sodium alginate hydrogels. Ozbolat, I.T. 3D Bioprinting Fundamentals, Principles and Application. Elsevier, Amsterdam Biobots, Repetier Host and Slic3R setup for BioBot1 Pluronic F127 is a sacrificial bioink that can be used for temporary support or to create channels, vessel or vasculature for bioprinting purposes. Liquid when cold, gel when warmed. Streamlined printable biomaterial that supports 3D printed cellular constructs. Biobots, Pluronic F

13 Open Repetier Host and load your CAD File. Biobots, Repetier Host and Slic3R setup for BioBot1 Biobots, Repetier Host and Slic3R setup for BioBot1 Print pressure and temperature are set in BioBots software after the sliced G code file is saved and uploaded. Biobots, Repetier Host and Slic3R setup for BioBot1 13

14 1. 3D Bioprinting is a useful tool for tissue engineering. 2. Anatomically correct constructs from imaging data can be made. 3. Desired tissue properties determine design and technologies employed. 4. Workflow includes design, optionally image import and segmentation, slicer software, special setups, bioprinting, bioreactor, observation and characterization. 5. Design methodologies include CAD-based, imagebased, implicit surfaces, space-filling curves and irregular porous structure. 6. Types of imaging: CT, MRI, PET and ultrasound. 7. Segmentation is used to delineate and isolate anatomical features within an imaging database- e.g. bone, cartilage, soft tissue, edema; muscle, lung, brain & other organs, and tumors. 8. Toolpaths may be either Cartesian or parametric, with parametric favoring gradient control, hollow shapes and porosity. 14