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Viability of endothelial cells on different surface coatings of silk sponges Matthew BouwerAuthentic Science Research Program Manchester-Essex Regional High School, Manchester-by-the-Sea, MA 01944 • The materials used for the silk processing • -sodium Chloride powder, • -Silk cocoons, • lithium bromide. • For the cleaning of the silk solution I used • -dialysis cassettes. • For the sponges • 500-600 micron salt crystals were used with the 6% silk solution. • Laminin, Collegen, and RGD were the added to the sponges. • For the assay • a Pico Green assay kit was used along with a fluorescence plate reader which determined which sets of scaffolds had the most living cells on it. • Cells • I used passage 9 human dermal micro vascular endothelial cell line • Methods • 500-600 micron sized salt crystals to create the sponges (Meinel et al., 2004). • Pre-weighed amounts of silk poured into 16 well plates • After 12 hrs, sponges were placed into spinning distilled water, which allowed all the salt to diffuse out, leaving silk sponges (personal communication). • Sponges were dehydrated by replacing the water with ethanol • Sponges were divided into 4 groups of matrix proteins: • control • laminin • Collagen • RGD (arginine, glycine,and aspartic acid) • All proteins were covalently bound to the silk surface. • Sponges were added to a 16 well plate and secured with a rubber O ring • Each row contained 1 of the 4 groups of scaffolds • Cells were seeded onto the sponges, and fed regularly for 3 days. • After three days, sponges were cut-up using sterile scissors • Cells were eluted and lysed • Pico green assay was performed on cells from each type of sponge (Biotechniques) Results/Conclusions The pico green assay’s results were non-conclusive. These results could have been due to several factors: - It is possible that I did not use enough cells - The cells were older cells (passage 9). - Errors while completing the pico green assay. - The plate reader had an error If this experiment were to be repeated successfully, it would provide very useful information regarding the type of matrix protein required to grow culture cells for engineered tissue. Researches looking to create vascularization using silk could utilize these results in order to determine which coating is most effective, or if the least expensive one, or easiest one still provided enough cell seeding for their experiment. Methods Abstract Viability of endothelial cells on different surface coatings of silk sponges Matthew BouwerManchester-Essex High School, Manchester-by-the-Sea, MA Teacher, Dr. Maria Burgess, Manchester-Essex High School Mentor, Dr. Bruce Pantelatis, Tissue Engineering Research Center, Tufts Univ, Medford, MAIf silk is used as a biomaterial for vascularization then it would be important to understand which surface coating if any would offer the most when seeding cells. Silk sponges were coated in laminin, collagen, or RGD(arginine,glycine, and aspartic acid), and one was used without a coating for a control. These were seeded with endothelial cells and then analyzed using a pico green assay. The Picogreen assay gave non-conclusive results. If the results had been conclusive, researches looking to create vascularization using silk could utilize these results in order to determine which coating is most effective, least expensive one, or the easiest one to use for their experiment. Materials Figure 2. Silk worms were used to harvest the silk used in the scaffolds of this experiment. Literature cited Nakazawa Y, Sato M, Takahashi R, Aytemiz D, Takabayashi C, Tamura T, Enomoto S, Sata M, Asakura T. Development of Small-Diameter Vascular Grafts Based on Silk Fibroin Fibers from Bombyx mori for Vascular Regeneration. J Biomater Sci Polym Ed. 2010 Jun. Charu Vepari, David L. Kaplan. Silk as a biomaterial. Progress in Polymer Science 2007. Rouwkema J. Rifron NC, Van Blitterswijk CA. Vascularization in tissue engineering. Trends Biotechnol. 2008. Biotechniques 20, 676 (1996) Figure 3. Dialysis cassettes were used to clean the isolated silk. Introduction Tissue engineering has been an important topic of research for years, and as such, many tissues can now be cultured in the lab. Silk is a common substrate used to engineer tissues in the lab. Thus far, only very small tissue samples can be engineered due to the need for closely adjacent nutrients. A common problem with engineering viable tissue is the absence of vessels to transport necessary nutrients. This limits the tissue sample size to small pieces often insufficient to replace diseased or missing tissues. Without the ability for the tissue to feed its cells via vascularization, it dies (Rouwekema, 2008). If vascularization were to be achieved using silk, it would be important to understand which surface coating, if any would allow for best cell attachment and viability. The purpose of this study was to determine which protein would best yield viable engineered tissues on silk substrate. Figure 5. Silk sponge scaffolds at 37oC, 95% humidity at ~95% oxygen in sterile cell culture incubator. Acknowledgments I thank Lindsay Wray for being my summer mentor, and helping me in the lab. Tufts university, and the TERC center for letting me work there, and also Dr.Burgess. For further information Please contact Matthew.Bouwer@gmail,com. More information on this and related projects can be obtained at http://ase.tufts.edu/terc/. Supported by a grant from the Spaulding Education Fund. Figure 4. Immunohistochemical staining of cells grown on silk scaffolds. Yellow denotes actin cytoskeletal fibers blue shows nuclei and green shows myosin cytoskeletal fibers. Figure 1. Silk can be constructed into a variety of templates on which to culture cells for tissue engineering. Figure 6. Dishes containing cells and red culture media, which provided nutrients for the growing cultures.