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Silk_ Scaffolds_ and Stem Cells

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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 [8]
   It yields a high surface area to volume ratio preferable for cellular
    infiltration




                                                               [1]
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 [9]
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
[1] Sell S, Bowlin G, et al, Polymer International
   56:1350,1358 (2007).
[2] Bashur C, Dahlgren LA and Goldstein AS, Biomaterials
   27:5681 (2006).
[3] Lee CH, et al, Biomaterials 26:1261 (2005).
[4] Altman, G H, Kaplan, DL, et al, Biomaterials 24:402-406
   (2002).
[5] Zhidao X, Triffitt JT, Biomed. Matter 1:R1-R9 (2006).
[6] Zigang G, Lee EH, et al, Journal of Biomedical Materials
   Research Part A 77A: 639-652 (2006).
[7] Functional Soft-Tissue Examination and Treatment by
   Manual Methods Author: Warren I. Hammer pp 18-20
[8] 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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posted:10/13/2012
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