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Silk, Scaffolds, and Stem Cells Rachel M. Beard 2008 Summer Experience Virginia Commonwealth University BBSI Mentor: Dr. Gary Bowlin Need for Ligament Tissue Engineering Knee ligaments cannot self repair High injury rate, especially the anterior cruciate ligament (ACL) > 200,000 ACL (ACL) surgeries/year > 5 billion dollars Surgery options Disease transfer Tissue rejection Poor mechanical strength (current synthetic grafts) Research on the Rise Design a scaffold that will mimic the native ACL tissue Mechanical support Collagen growth Collagen Electrospun Adipose Derived production from Stem Cells Scaffold stem cells Ligament composed of collagen bundles Silk Properties Yield a Promising Scaffold Choice Biocompatibility Fibroin protein polymer already spun into fibers by the Bombyx Mori silkworm Distinguishing mechanical properties Slow degradation in vivo http://www.dkimages.com/discover/previews/975/85004290.JPG Silk Preparation from Cocoons Silkworm Cocoon 2L boiling water 4.24g Na 2 CO 3 5g cocoon pieces Heater 30 minutes Drying in hume hood 3rd 2nd 1st 1L Milli -Q water 20 minutes 20 minutes 20 minutes Tufts University Department of Biomedical Engineering Removing Sericin from Silk Dissolving in LiBr solution 7g of dried cocoon 12mL of fibroin LiBr sol. 20 mL syringe 60 °C, 4 h 9.3 M LiBr solution 28mL Dialysis cassette Department of Biomedical Engineering and Technology Center Science Dialysis, 48 h Centrifuge 9,000 rpm 20 minutes Stirrer bar 2 times Stirrer Tufts University Department of Biomedical Engineering PCL (Polycaprolactone) Properties Yield a Promising Scaffold Choice Biocompatible Slow degradation in vivo Stretchy/mechanically strong polymer Able to formulate polymer blends http://web.mit.edu/rutledgegroup/projects/electrospinning.html Electrospinning Polymers Electrospinning is just one novel of creating nanofiber polymer scaffolds that mimic the native tissues of the extracellular matrix  It yields a high surface area to volume ratio preferable for cellular infiltration  Electrospun Silk Scaffolds Magnified 500X Magnified 1500X SEM Images Punching the Silk and Silk/PCL Scaffolds 3 pure silk sheets were electrospun 2 50/50 (by volume) silk/PCL sheets were electrospun Do Not Let the Cells Die! ADSCs (Adipose Derived Stem Cells) Cells were fed culture media every 2 days and split into 3 separate flasks as each flask became confluent with cells Culturing Stem Cells Scaffolds Collagen Assay Testing on Disk Scaffolds at Day 7 If cells like their environment, they will produce collagen Before producing collagen, cells proliferate and migrate on the scaffolds Cells are set on the scaffold Cells are not yet situated Hypothesis Collagen production No collagen production Collagen Assay Standards are in Acceptable Range Lots of collagen detected on scaffold Minimal collagen detected on scaffold Standard Curve Color Absorbance Concentration of Dye Attached to Collagen (µg/ml) Silk Collagen Assay Results After 1 Week 400 350 Collagen Production 300 250 200 150 100 50 0 Control Stem Cells Macrophages Silk/PCL Collagen Assay Results After 1 Week 300 Collagen Production 250 200 150 100 50 0 Control Stem Cells Macrophages Stretching the Scaffolds to Failure Mechanical Properties of Ligaments Elastic Regions Slope Toe Region: initial loading, = Modulus collagen crimp is initially straightened in ligaments Linear Region: collagen crimp is completely straightened out Plastic Region Ligament failure occurs when the collagen fibers break Binkley (1989) Physc asdf 23.424  Silk Graphs Similar to Actual Ligament Adipose Stem Cells Mean Modulus for Silk/PCL Scaffolds 1 Week After Seeding Cells 6 Stem Cells No Cells 5 Modulus (MPa) 4 3 2 1 0 1 2 3 4 Type of Cells on Scaffold Silk/PCL Graphs Resemble Ligament Adipose Stem Cells Mean Modulus for Silk/PCL Scaffolds 1 Week After Seeding Cells 60 50 Stem Cells Modulus (MPa) 40 No Cells 30 20 10 0 1 2 3 4 Type of Cells on Scaffold The Project is Still Underway… Histology pictures that show the ADSCs on the scaffolds should be in the mail very soon Day 14 results will be assembled today Day 21 results will be assembled next Tuesday References  Sell S, Bowlin G, et al, Polymer International 56:1350,1358 (2007).  Bashur C, Dahlgren LA and Goldstein AS, Biomaterials 27:5681 (2006).  Lee CH, et al, Biomaterials 26:1261 (2005).  Altman, G H, Kaplan, DL, et al, Biomaterials 24:402-406 (2002).  Zhidao X, Triffitt JT, Biomed. Matter 1:R1-R9 (2006).  Zigang G, Lee EH, et al, Journal of Biomedical Materials Research Part A 77A: 639-652 (2006).  Functional Soft-Tissue Examination and Treatment by Manual Methods Author: Warren I. Hammer pp 18-20  Bowlin G, Materials Today 7:64 (2004). Thanks for a BBSI Summer! Dr. Gary Bowlin for his lab, time, resources, and lunch! Graduate students for their time, patience, and helpful responses to my questions Scott Sell, Koyal Garg, Tricia Wolfe, Michael McClure, Yas Maghdouri Moghaddam, Anna Bulysheva, Parth Madurantakam, and Michael Frances for the stem cells Dr. Jeff Elhai, Dr. Sherry Baldwin, and Billy Budd for directing VCU BBSI Dr. Wan-Ling Chiu for the Bombyx Mori silkworm cocoons National Science Foundation for the grant money!
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