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Imaging of Collagen Gels for Tissue Engineering By: Allison Throm Advisor: Rebecca Kuntz Willits, PhD BE@SLU, Summer 2009
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Background Injured neurons do not always regenerate properly in vivo, so alternative methods are needed for their regeneration. One proposed method is use of scaffolds; in this study, collagen gels are of particular interest. Collagen I provides favorable conditions for cell adhesion and growth. However, collagen I is not alone in the extracellular matrix (ECM). Other types of collagens and noncollagenous proteins are present. Of particular interest are chondroitin sulfate, collagen IV and laminin. Chondroitin sulfate is a glycosaminoglycan (GAG), consisting of a protein core with polysaccharide chains. Collagen IV is found only in basement membranes. Laminin is found in conjunction with collagen IV and functions in cell attachment. |
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Methods Collagen gels of concentrations varying from .4 to 2mg/ml were prepared by mixing NaHCO3, H2O, collagen I, NaOH, F12K, HEPES, and an additive were mixed over ice. They were allowed to set in a dry incubator and then fixed with 2% glutaraldehyde. The gels were then washed three times with PBS. Samples were then dehydrated with ethanol by removing the liquid on the gels and replacing it with the next higher concentration of ethanol before placing it on a mixer plate for 10 minutes. This was done with concentrations of 10% ethanol up to 100% ethanol. Specimens were then critical point dried and mounted on a stub for scanning electron microscope (SEM). Specimens were then sputter coated and viewed with an SEM. Images were analyzed for fiber diameter with ImageJ. Results were statistically analyzed using a student’s t-test. |
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SEM images depicting plain collagen gels |
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There was no significant difference between the fiber diameter of the plain collagen gels |
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Results |
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SEM images depicting .4mg/ml collagen gels with various additives. |
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Depicted above are images for .4mg/ml collagen gels. 1mg/ml and 2mg/ml gels appeared similar and had the same results. Laminin had no significant effect on fiber diameter. Both collagen IV and chondroitin sulfate significantly decreased fiber diameter. In addition, the gels with chondroitin sulfate and collagen IV had a matted appearance. |
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Discussion The results for the gel with chondroitin sulfate are consistent with previous studies. This may be due to alteration of nucleation sites, which causes end to end aggregation of collagen molecules. Laminin is responsible for cell attachment in the basement membrane. These images were blurrier than the others and were harder to focus, possibly due to the laminin; this may have altered the results of the measurements. Collagen IV decreased fiber diameter. # |