1. Collagen Fibrillogenesis Mary Beth Bird Mentor: Ramana Pidaparti, PhD. Mechanical Engineering Department July 23, 2007 Using Voltage to Optimize Protein Growth

2. Goals of Project To trigger self-assembly mechanisms in collagen To trigger self-assembly mechanisms in collagen To establish an experimental setup that will support fibrillogenesis To establish an experimental setup that will support fibrillogenesis To monitor and alter variables in experiment to determine the best environmental conditions for fibrillogenesis To monitor and alter variables in experiment to determine the best environmental conditions for fibrillogenesis To replicate experimental data in computational modeling derived from research data To replicate experimental data in computational modeling derived from research data

3. Background on Collagen Most common form of protein in the human body Most common form of protein in the human body Triple helical molecule consisting of 3 alpha chains of amino acids Triple helical molecule consisting of 3 alpha chains of amino acids There are 13 different kinds of collagen, Type I – XIII There are 13 different kinds of collagen, Type I – XIII Cross-linking during fibrillogenesis causes unique properties amongst the different types Cross-linking during fibrillogenesis causes unique properties amongst the different types Collagen can be found in all connective tissues; ie. bones, tissues, cartilage, …. Collagen can be found in all connective tissues; ie. bones, tissues, cartilage, ….

4. The Original Idea Use metal substrate as a voltage conductor coated with SDS (Sodium Dodecylsulfate) Use metal substrate as a voltage conductor coated with SDS (Sodium Dodecylsulfate) Layer with a 1-2cm sq of collagen in an acidic solution Layer with a 1-2cm sq of collagen in an acidic solution Apply voltage across metal conductor Apply voltage across metal conductor Monitor growth formation using electron microscope Monitor growth formation using electron microscope Variables: pH, time, voltage, concentration of collagen solution Variables: pH, time, voltage, concentration of collagen solution

5. Problems Encountered Type of substrate Type of substrate Determining collagen Type and form to be used Determining collagen Type and form to be used Use of SDS (Sodium Dodecylsulfate, ionic surfactant) Use of SDS (Sodium Dodecylsulfate, ionic surfactant) Use of Hydroxylapatite (mineral) Use of Hydroxylapatite (mineral) Application of voltage using Corrosion Data Program Application of voltage using Corrosion Data Program Physical experimental setup Physical experimental setup Ability to monitor pH and temperature Ability to monitor pH and temperature Method of examining collagen formation Method of examining collagen formation Method of quantifying and understanding data Method of quantifying and understanding data Ability to confirm presence of fibrils Ability to confirm presence of fibrils Prevention of corrosion Prevention of corrosion

6. Images from the Lab

11. Concerns Corrosion Corrosion How can we prevent corrosion? How can we prevent corrosion? Alter voltage? Alter pH using NaOH? Alter substrate? Alter voltage? Alter pH using NaOH? Alter substrate? Substrate Surface Substrate Surface How can we improve the surface of the substrate to prevent false images? How can we improve the surface of the substrate to prevent false images? Analyzing Images Analyzing Images How do we confirm presence of fibrils? How do we confirm presence of fibrils? What is the best way to take images? What is the best way to take images? SEM, AFM, Optical Microscope SEM, AFM, Optical Microscope

12. Future of the Project Continue to refine methods to decrease the number of experimental errors Continue to refine methods to decrease the number of experimental errors Control and alter experimental variables ie. pH, temperature, voltage Control and alter experimental variables ie. pH, temperature, voltage Form a better method to quantify and examine data Form a better method to quantify and examine data Expand project to include DNA Expand project to include DNA

13. Acknowledgements Project Team Members: - Sumeet Lall, HSURP - Dr. Ramana Pidaparti Mechanical Engineering Dept., VCU - Dr. Gary Bowlin Biomedical Engineering Dept., VCU Biomedical Engineering Dept., VCU - Dr. Peter C. Moon School of Dentistry, VCU School of Dentistry, VCU - Eddie McCumiskey, Grad Student NanoMan Lab Mechanical Engineering Dept., VCU - Dr. Stephen Fong’s Lab Chemical and Life Sciences Engineering Dept., VCU

14. References 1. Encyclopedia of Human Biology. 2nd ed. San Diego, CA: Academic Press; 1997. 2. Du C, Cui F, Zhang W, Feng Q, Zhu X, de Groot K. Formation of calcium phosphate/collagen composites through mineralization of collagen matrix. Journal of Biomedical Materials Research [serial online]. 2000;50:May 31, 2007. 3. Sini P, Denti A, Tira M, Balduini C. Role of decorin on in vitro fibrillogenesis of type I collagen. Glycoconjugate Journal. 1997;14:871-874.