A Proposed Analysis of Collagen Morphology Using

Electron & Soft X-ray Microscopy

Amy H. Brazin, NASA ApprenticeRussell Garman, Ph.D. Candidate

Stefan Judex, Ph.D.~~~~~~~

SUNY Stony Brook Dept. of Biomedical Engineering

August 1, 2005

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

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

Understanding Collagen Morphology

The following may elude to the elasticity and fracture toughness of the bone:

Diameter and density of fibersOrientation

Fibril diameters and distributions are associated with:

Tissue growth and maturationTissue 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.

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

Sample Preparation for AnalysisFixation

Decalcified BonesUndecalcified Bones

Post Fixation

Dehydration

Infiltration

Embedding

Sectioning, 100nm

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* Indicates that which is already completed

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Analyzing Collagen

Transmission Electron MicroscopyHigh resolutions allow the quantification of:

Collagen fibrils, subfibrils and microfibrilsDiameter distribution

Soft X-ray MicroscopyDifferentiates 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.

Acknowledgements

Special thanks to the following people, whom without, this research would not be possible:

Hoyan LamAdiba AliNASA SHARP