1. A Proposed Analysis of Collagen Morphology Using Electron & Soft X-ray Microscopy Amy H. Brazin, NASA Apprentice Russell Garman, Ph.D. Candidate Stefan Judex, Ph.D. ~~~~~~~ SUNY Stony Brook Dept. of Biomedical Engineering August 1, 2005

2. Application to NASA Research Analyzing collagen in bone under the following conditions: Weightlessness-> astronauts experience this in space Mechanical Stimuli-> the countering force of bone loss due to weightlessness

3. Major structural protein that act as cables to strengthen body tissue 90% of body is Collagen Type I Triple helix structure Collagenous matrix correlates to bone fracture toughness Wang, X., Bank, R. et al. “The Role of Collagen in Determining Bone Mechanical Properties.” Journal of Orthopedic Research. 19(2001) 1021-1026 Role of Collagen in the Body

4. Understanding Collagen Morphology The following may elude to the elasticity and fracture toughness of the bone: Diameter and density of fibers Orientation Fibril diameters and distributions are associated with: Tissue growth and maturation Tissue response to physical stress Frank, C., Bray, D. “Electron Microscopic Quantification of Collagen Fibril Diameters in the Rabbit Medial Collateral Ligament: A Baseline for Comparison.” Connect Tissue Res. 19:11-25. 1989.

5. Current Task at Hand To develop a protocol to: Prepare calcified and decalcified bones Cut ultrathin sections using a microtome Establish standard resolution features for microscopy work

6. Sample Preparation for Analysis Fixation Decalcified BonesUndecalcified Bones Post Fixation Dehydration Infiltration Embedding Sectioning, 100nm * * Indicates that which is already completed * * * * * * *

7. Analyzing Collagen Transmission Electron Microscopy High resolutions allow the quantification of: Collagen fibrils, subfibrils and microfibrils Diameter distribution Soft X-ray Microscopy Differentiates the mineral quantities in bone as well as the crystalline properties of the bone matrices Frank, C., Bray, D. “Electron Microscopic Quantification of Collagen Fibril Diameters in the Rabbit Medial Collateral Ligament: A Baseline for Comparison.” Connect Tissue Res. 19:11-25. 1989.

8. Acknowledgements Special thanks to the following people, whom without, this research would not be possible: Hoyan Lam Adiba Ali NASA SHARP