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Fabrication of novel Bioink for 3D printing
- 1. Under guidance of- Dr. Joyita Sarkar By- Jidnyasa Chintamani Roll no- J19IMT626 Fabrication of Novel Bioink for 3D Printing IPT 2 Presentation
- 2. Rational of study: Fabrication of novel bioink for 3D printing Citrus peels are rich source of pectin and cellulose. Blend them with Gelatin Methacrylate (GelMA) Use this technique to print hepatic tissues and liver-like structures.
- 3. 1.Biocompatible material 2.Cells Advantages:- •Potential for accurate cell distribution and high resolution cell deposition. •Scalability •Cost-effectiveness What is 3D Bioprinting?
- 4. Process flow of 3D Bioprinting Pre-Processing • Designing • 3D modeling Processing • Bioink preparation • Bioprinting Post processing • Cell Culture and/or Differentiation • Maturation of tissue Applications • Tissue engineering • Regenerative Medicine
- 5. Pre-Processing Designing • A liver has two types of cells Hepatic cells Endothelial cells • Two types of cell–cell interaction Homogeneous Heterogeneous • After designing the structure we can print it in two types:- Array of liver lobules Stack 3D Modeling Liver lobule Hepatocytes Hepatic artery Bile duct Portal vein Central vein Endothelial cells Hepatocytes Portal triad: • Bile duct • Portal vein • Hepatic artery Sinusoids Central vein Hepatocyte Kupffer cell Array Stack Hepatic lobule
- 6. • Bioink development objectives i. Biocompatibility ii. Minimize the adverse effect on cell viability • Factors involved in the fabrication of bioink i. Ink rheology, ii. Cytotoxicity iii. Crosslinking parameters, iv. Bioprintablity Bioink Processing
- 7. Polymers Why do we use Polymers in bioink? Types of Polymers • Natural - ex-: Alginate, Gelatin, Fibrin , Collagen, etc. • Synthetic - ex:-Polycaprolactone (PCL), Polyethyleneglycol (PEG) etc. Polymers and Bioactive components in citrus peels • Pectin • Cellulose • Limonoids • Phenolic acids • Flavonoids • Polyphenoliccompounds
- 8. Pectin Extraction Cellulose Extraction Washing Drying Willing Pressing Boiling add dilute acid Filtration Add alcohol to solution Filtration II Drying and milling Standardize Peel soaking in organic solvent Filtration Drying NaOH treatment Filtration Drying NaOH treatment Filtration and drying Acid hydrolysis
- 9. 1. Preparative High-Performance Liquid Chromatography (Prep-HPLC) 2. Preparative Thin Layer Chromatography (Prep-TLC) 3. Silica Gel Column Chromatography 4. High-Speed Countercurrent Chromatography (HSCC) 5. Flash Chromatography 6. Supercritical Fluid Chromatography 7. Reverse-Phase High-Performance Liquid Chromatography 8. Size Exclusion Chromatography Isolation techniques
- 10. Nanocellulose • Advantages High specific surface area Right mechanical properties Biocompatibility Non-toxicity High tensile strength • Applications Inks for 3D printing in tissue engineering Drug delivery Wound healing. Packaging Electronic sensors
- 11. GelMA Why do we use GelMa? • Pectin and cellulose are non cytoadherent • Liver cells are adherent • Gelatin has RGD (arginine glycine aspartic acid) motif that helps to attach to cytoskeleton. Why methacrylate? • Methacrylate helps in photocrosslinking with assistance of UV-A light of range 400–315 nm which is not harmful to the cell viability.
- 12. 3D Bioprinters There are 4 main types of Bioprinters 1. Inkjet Bioprinter 2. Extrusion Bioprinter 3. Laser-assisted Bioprinter 4. Stereolithography based Bioprinter
- 13. Type Mechanism Advantage Disadvantages Application Effects Inkjet printer • Thermal • Piezoelectric • Availability • Low cost • High speeds • Bioink can solidify • nozzle clogging Creating quick skin grafts • Cellular distortion • Harm cell viability Extrusion printer • Mechanical • Pneumatic • High viscosity Bioink • High cell densities • Low resolution • Slow print speed Creating large 3D structure. Loss of cellular viability & structure Laser assisted printer • Noncontact nozzle- free. • Laser pulses through a ribbon containing bioink • Micro patterned peptides DNA . • High resolution 108 cells/m • No clogging and viscosity limitations • high cell viability • Time-consuming ribbon fabrication process. • Cannot print multiple layers easily. • High cost of laser system. Placing cells precisely in two solid structures Laser assisted printing is a more force neutral bioprinting method that preserves multipotency Stereolit- hography Printer Photo-polymerization • High degree of fabrication accuracy • Low printing time • Lengthy post-processing • Lack of compatible materials. Regenerative medicinal and biomedical applications Use of high intensity UV light might harm cells
- 14. Extrusion Based Bioprinting It can print high viscosity bioink Print complex geometries with great precision. We will be printing 2 types of liver cells. Hepatic cells and Endothelial cells in ratio 10: 1 at the same time using a two nozzle Extrusion printer.
- 15. • Photo-crosslinking provides scaffold for growth • Achieved by methacryloylation of gelatin • Crosslinking of GelMA can be achieved by adding a water-soluble photo- initiator followed by exposure to UV-A of range 400–315 nm. Photo-crosslinking Scaffold GelMA+ cells UV light PhotoCrosslinked Scaffold with cells Extrusion nozzle
- 16. Cell culture Individual cells isolated from tissue Primary cell culture Liver tissue Liver tissue Contact Inhibition Some cells transferred to new medium Secondary cell culture Transformed or individual cells isolated from a tumor Continous cell line Post-Processing Primary cell culture • Adherent • Suspension Cell lines • Finite cell lines • Continuous cell lines Stem cell culture • Embryonic • Amniotic
- 17. Analysis of Hepatic functionalities MTT- Measure cell proliferation (Mosamann, 1983) 3 most important functional properties of a liver to be analyzed are:- • Synthetic Property • Drug Metabolism • Detoxification Techniques used :- • Biochemical Assay using ELISA and Spectrofluorometery • Gene expression using RT-PCR
- 19. Final outcomes Successful implementation of the project will lead to fabrication of novel ink for 3D bioprinting procured from citrus fruit waste - therefore a step towards sustainability. An efficient in vitro tissue engineering hepatic platform for drug screening. Reducing animal usage for the same.
- 20. IPT in MIT • Rotational moulding • Injection moulding • CNC machine • VMC machine • 3D printing • Solid Edge • Solid Works.
- 21. Key learnings 3D bioprinting Research Methodologies Printing techniques 3D model designing Time management skills Molding Methods
- 22. Extracurricular Did 8+ certified courses • SOLIDWORKS: Mold Design • SOLIDWORKS 2021 Essential Training • Package Design Project: Paperboard Food Packaging • Learning Bitcoin and Other Cryptocurrencies • Learning 3D Printing • Design Your First Logo • Creating a 3D Logo in Photoshop • Defining and Achieving Professional Goals. Individual Participation in ICT Mumbai’s Fest VORTEX 8.0 Participation in National level poetry and arts competition. Conducted competitions in college being the second year secretary of ‘DARSH’, ICT MARJ. Attended several online webinars on food safety, sustainable development ,financial investing etc Learning to play guitar and improvising on my creative skills like Graphic designing, painting, doodle art, poetry writing etc. Freelancing for an organic food company as a content writer ,SMGD.
- 23. Notes on Important Papers Sr. Research Paper References Material Used as Reference 1. Polymeric Bioinks for 3D Hepatic Printing https://doi.org/10.3390/chemistry301 0014 Advantages of synthetic polymers. ,Natural polymers 2. Bioink properties before, during and after 3D bioprinting DOI: 10.1088/1758-5090/8/3/032002 Information about 3D printers 3. Natural Polymers for Organ 3D Bioprinting DOI: 10.3390/polym10111278 Natural polymers and their properties. 4. Synthetic Polymers for Organ 3D Printing DOI: 10.3390/polym12081765. Synthetic polymers and their properties. 5. 3D Bioprinted Nanocellulose-Based Hydrogels for Tissue Engineering Applications: A Brief Review DOI: 10.3390/polym11050898 Nano cellulose ,its types and Synthesis 6. Nanocellulosic materials as bioinks for 3D bioprinting DOI: 10.1039/c7bm00510e 3D printing process Using nanocellulose 7. Novel bioprinting method using a pectin 2 based bioink DOI: 10.3233/THC-160764. Making of bioink using pectin 8. Bioinks for 3D Bioprinting: An Overview DOI: 10.1039/c7bm00765e. About gelatin and GelMA in bioink
Editor's Notes
- Citrus peels cannot be directly fed to cattle's as they have various antinutritional properties. we extract these polymers from them and since these polymers are non-cytoadherent and GelMA helps cells and other bioactive components to stick together and fabricate a novel bioink from it.
- 8. 12. 2021
- 8. 12. 2021
- Pre-bioprinting Pre-bioprinting is the process of creating a model that the printer will later create and choosing the materials that will be used. One of the first steps is to obtain a biopsy of the organ. Common technologies used for bioprinting are computed tomography (CT) and magnetic resonance imaging (MRI). To print with a layer-by-layer approach, tomographic reconstruction is done on the images. The now-2D images are then sent to the printer to be made. Once the image is created, certain cells are isolated and multiplied.[3] These cells are then mixed with a special liquefied material that provides oxygen and other nutrients to keep them alive. In some processes, the cells are encapsulated in cellular spheroids 500μm in diameter. This aggregation of cells does not require a scaffold, and are required for placing in the tubular-like tissue fusion for processes such as extrusion
- It is made up of cellular materials additives and a supportive scaffold Shear thinning, Composition of the gel, Ink rheology, Structure stability test, Mechanical strength, Crosslinking parameters, Optimization of the cell viability, etc. Bioink development objectives: Biocompatibility. Minimize the effect of printing on cell viability without compromising the shape and stability of tissue construct. Methacrylated gelatin has been widely used as a tissue engineering scaffold material in cell laden bioinks. Reasons why we use GelMA • It is potential as a viable bioink material due to its suitable biocompatibility and readily tunable physicochemical properties. • Its tunable rheology and rapid thermocrosslinking of bioink improved shape fidelity after bioprinting. • It is self supporting after printing due to its higher complex modulus and yield stress induced by GelMA in the system.
- 8. 12. 2021
- 8. 12. 2021
- Nanocellulosic materials display many interesting properties for application in tissue engineering. These include cost-effectiveness, sustainability, environmental friendliness, biocompatibility, biodegradability and non-toxicity. Cellulose nanofibers and nanocrystals have been employed as inks for 3D printing in tissue engineering, drug delivery and wound healing. Material degradability is another advantageous property of cellulose nanofibers.
- Pectin and cellulose are non cytoadherent, Liver cells are adherent so we need to provide it a surface to which it can adhere Gelatin has RGD (arginine glycine aspartic acid) motif which are recognized by the integrins of the cells . Integrins are trans membrane protein. Inside cell- ATTACHEDT to cytoskeleton. Outside part to rgd motif. Why methacrylate- so that we can photocrosslink using UV light of range 400–315 nm which is not harmful to the cell viability. The polymer is also highly biocompatible, cytoadherent, and non-immunogenic. It also contains the tripeptide motif, Arg-Gly-Asp, which is recognized by integrins on the cell membrane for attachment. But crosslinking of gelatin can be too toxic to cells so we prefer photocrosslinking.
- 8. 12. 2021
- It’s geometrical and fabrication parameters can be easily changed to match the requirements of the scaffold, like modulus strength, structural integrity. Can also change the printing parameteDisadvantage of extrusion bioprinting is that cell viability is lower than that with inkjet-based bioprinting (40–86%).
- For stabilization of extruded filaments, a photoinduced crosslinking mechanism is used by adding photoinitiators to the formulation of bioink. These molecules when exposed to light produce reactive species that initiate the polymerization process. UV-A and visible light irradiation at wavelengths that don’t cause significant DNA damage is most commonly used. Photocrosslinking has also been achieved by methacryloylation of gelatin engendering gelatin methacrylate (GelMA). UV irradiation.The most commonly used photoinitiator for this is 2-hydroxy-1-[4-(2-hydroxyethoxy) phenyl]-2-methyl-1-propanone (Irgacure 2959), which has an aqueous solubility of 5 mg/mL . The stiffness and cell viability of the bioprinted GelMA structure highly depends on the concentration of polymer, photoinitiator, and UV light.
- Removal of cells from animals or plants. Cells are further placed in a favorable artificial environment for their growth. Obtained from tissues directly Disaggregated by enzymatic or mechanical means before cultivation. Widely used in diagnosing infections, testing new drugs, studying diseases like cancer, etc.
- Post-bioprinting[edit] The post-bioprinting process is necessary to create a stable structure from the biological material. If this process is not well-maintained, the mechanical integrity and function of the 3D printed object is at risk.[3] To maintain the object, both mechanical and chemical stimulations are needed. These stimulations send signals to the cells to control the remodeling and growth of tissues. In addition, in recent development, bioreactor technologies[8] have allowed the rapid maturation of tissues, vascularization of tissues and the ability to survive transplants.[4] Bioreactors work in either providing convective nutrient transport, creating microgravity environments, changing the pressure causing solution to flow through the cells, or add compression for dynamic or static loading.