Novel Polysaccharide-Based Hydrogel for Cell and Tissue Printing

Polysaccharide-Based Hydrogel for 3D Cell and Tissue PrintingGanesh SosaleHello Everyone, my name is Ganesh and my proposal is on polysaccharide based hydrogels for 3d cell and tissue printing1Why 3D Cell Printing?Over the last decade, about 76,650 people died due to a shortage of donated organsWith organ transplants, there is a risk of immune responseWith the use of hydrogels and 3D cell printing, supply could match demandIn the last decade, about 76,650 people died due to a shortage of donated organs. 2Hydrogels for 3D printing3D polymer networks that can be used as scaffolds for tissue engineeringThere are many different classifications of hydrogelsNatural or syntheticDurable or degradableCationic, anionic or neutral

Pros and Cons of Currently Used Hydrogels in 3D printingProsProvide good transport of nutrients to cellsBiocompatibleAre able to mimic ECM

ConsMechanical Properties Gelation Time (Desired range is from about 5 to 60 sec)Permeability

Here are the pros and cons of currently used hydrogels in 3D printing.

They provide good transport of nutrients, are biocompatible and are able to mimic ECM.

Cons are the mechanical propertiesThere is no gold standard so it is difficult to define what the desirable range is. Ideally, you would want mechanical properties that mimic the native tissue that you are trying to print. Currently researchers are having difficulty printing structures because once they get to a certain height, the structure is collapsing on itself. For one source, they reached up to half a cm.

Diagram for showing mechanical propertiesInclude range needed for 3D printing (desirable)Pectin doesnt gel unless its acidicChitosan tooPolysaccharide-based hydrogel due to GAGAgarose alginate HA(degrades within body)4Gellan GumFDA approvedGood mechanical propertiesGelation Time (about 5 to 45 sec)Permeability LevelLow costBiocompatibleCross-linkable with cell medium

In effort to collectively address those problems, I have chosen gellan gum because well, according to literature, polysaccharide based hydrogels are excellent candidates for biomedical applications because they are biocompatible and show minimum to no cytotoxicity. Gellan gum also has a gelation time required for 3D printing and

HistorySo many materials, why I chose gellan gumJustify why I think gellan gum will address all those problems.Define desirable range for each of the parameters5Specific Aim 1Formulate a polysaccharide-based hydrogel that collectively addresses all major problems associated with existing hydrogels for 3D printingMechanical PropertiesGelation TimePermeabilityMechanically test blood vessel and try to mimic native properties

Identify Variables to fit into ranges: Concentration of gellan gum, concentration of cell medium and identify optimum numbers of concentration of gellan gum and cell medium necessary to achieve ranges.

6Aim 1 ApproachExamine ratio of gellan and cell medium (cross-linker)Test cure rate (gelation time) and mechanical properties using rheometer for 1%, 2% and 3% Gellan Gum with and without cross-linkerRatios to be tested: 1:1, 1:2, 1:4 and 2:1, 4:1 Specific Aim 2Test the gellan gum formulation identified in aim 1 for 3D cell printing

Directly fabricate vasculature using endothelial cells

Cell phenotype stability do they change over time? Are they still endothelial cells? are they proliferating, are they gaining mechanical strength over time because they form tissue level structure based upon individually positioned.Mechanical property of construct time course, cell fusion, cell proliferation, growth of tissue. Cell hydrogel constructDo cells live happily in construct. In tissue level now. Do they gain mechanical strength over time when forming tissue level structure.

Need spheroids for spatial resolutionWhy we use cell spheroids as structure units for 3D printing. Spatial resolution. 8Aim 2 ApproachForm mixture of cell suspension with hydrogel Extrude cell spheroid (Endothelial Cells)Precisely pattern them on substrate using layer-by-layer addition to form a 3D structureEvaluate structural stability, cell phenotype stability, cell viability and functionality

Expected outcomes, pitfallsExpected OutcomeHydrogel is effective in producing the 3D tissue segments in vitro

PitfallsPrinted structure doesnt form tissue level structureCells die out over timeCell Seeding density may be too low

What are concerns that prevent you from achieving it: Dont see fusion of spheroids, cells die out over time, dont form tissue level structure. If they fail, can adjust cell seeding density, density may be too low and dont have contact with each other. Test different cell seeding density, which will lead to a different cell spheroid size. 10FutureSegments of structures printed may serve as building blocks for larger structures that can be used for applications involving tissue engineering, regenerative medicine, drug testing/screening and cell biology.Diagram how to join two segments togetherecm becomes common. Physically join two segments11ReferencesAlexander, Amit, et al. "Polyethylene Glycol (PEG)Poly(N-Isopropylacrylamide) (Pnipaam) Based Thermosensitive Injectable Hydrogels For Biomedical Applications."European Journal Of Pharmaceutics & Biopharmaceutics88.3 (2014): 575-585.Academic Search Complete. Web. 10 Feb. 2015.Boucard, Nadge, et al. "The Use Of Physical Hydrogels Of Chitosan For Skin Regeneration Following Third-Degree Burns."Biomaterials28.24 (2007): 3478-3488.Academic Search Complete. Web. 10 Feb. 2015.El-Sherbiny, Ibrahim M., and Magdi H. Yacoub. "Hydrogel Scaffolds For Tissue Engineering: Progress And Challenges."Global Cardiology Science & Practice2013.3 (2013): 1-27.Academic Search Complete. Web. 11 Feb. 2015.Favi, Pelagie M., et al. "Cell Proliferation, Viability, And In Vitro Differentiation Of Equine Mesenchymal Stem Cells Seeded On Bacterial Cellulose Hydrogel Scaffolds."Materials Science & Engineering: C33.4 (2013): 1935-1944.Academic Search Complete. Web. 10 Feb. 2015.Hoffman, Allan S. "Hydrogels For Biomedical Applications."Advanced Drug Delivery Reviews64.(2012): 18-23.Academic Search Complete. Web. 13 Feb. 2015.Ilkhanizadeh, Shirin, Ana I. Teixeira, and Ola Hermanson. "Inkjet Printing Of Macromolecules On Hydrogels To Steer Neural Stem Cell Differentiation."Biomaterials28.27 (2007): 3936-3943.Academic Search Complete. Web. 10 Feb. 2015.Imani, Rana, et al. "Evaluation Of Novel "Biopaper" For Cell And Organ Printing Application: An In Vitro Study."Iranian Journal Of Diabetes & Lipid Disorders10.(2011): 1-13.Academic Search Complete. Web. 10 Feb. 2015.Kobayashi, Jun, and Teruo Okano. "Thermoresponsive Thin Hydrogel-Grafted Surfaces For Biomedical Applications."Reactive & Functional Polymers73.7 (2013): 939-944.Academic Search Complete. Web. 10 Feb. 2015.Kwon, Jin Seon, et al. "Injectable Extracellular Matrix Hydrogel Developed Using Porcine Articular Cartilage."International Journal Of Pharmaceutics454.1 (2013): 183-191.Academic Search Complete. Web. 12 Feb. 2015.