1. Analysis of bone structure in brittle bone disease
John Jameson
ALS User Meeting – Tomography Workshop
10/09/2012

2. Osteogenesis imperfecta (OI)
Genetic fragility disorder
25-50,000 in US
Mutations cause impaired collagen synthesis and assembly
Limb deformity, reduced stature, respiratory problems, connective tissue laxity…
8 known types*
Mild, Type I: 50% of all cases
Moderate-Severe, Types II-VIII
High degree of variability
1:5-10K worldwide, 25-50K US
Limb deformity, short stature, respiratory problems, hearing loss…
Type 1 most common (~50%), but least severe
Byers 1994, Chiasson 2004

3. Bone hierarchical structure
Collagen
Mineralized triple helices
(<1 µm)
Fibrils
Staggered bundles
of molecules
(~1 µm)
Fiber arrays
“Plies” at different
orientations
(~5-20 µm)
Osteons
Lamellae + vasculature
( µm)
Solid material
Cortical bone
(>3000 µm)
Trabeculae
“Plates and rods”
( µm)
Trabecular bone
Whole bone
Femur

4. BONE FRAGILITY -How does bone structure affect mechanical performance?
-How do changes to OI bone cause fragility?
MOUSE
Micro
HUMAN
200 µm
Trabecular (spongy):
-decreased quantity
-more isotropic
-poor organization
50 µm
25 µm
Cortical (compact):
-higher porosity
-thinner canals
Cortical (compact):
-higher porosity
-larger pores w/higher connectivity
10 µm
5 µm
<1 µm
Nano

5. Additional considerations
Tomography
Preventing tissue damage
SAXS/WAXD
Collagen spacing, mineral thickness
In situ mechanical testing?
Similar to hot cell, but much simpler experiment
Notch and apply different loads
Can do this in ESEM now, but only get 2D info => 3D with tomo after

6. Data collection pipeline
Preparing for beamtime
Beamtime
Average and peak data rates
10-15 scans/beamtime => 11 GB/scan => GB/session
Only person doing data collection, analysis
Real-time feedback would be helpful in assuring quality of images
Working on noise study to provide feedback to users on proper settings to use
After beamtime
Reconstruction (1 hr/dataset)
Filtering and segmentation (1-2 hrs first dataset)
Bilateral filtering => Usually have to downsample images, convert to 8-bit so it doesn’t take forever
Binary morphological operations: Opening/closing
Data processing (0.5 day/dataset)
Sphere-fitting algorithms, skeletonization, surface meshing
Visualizations
Fiji and/or Avizo => Requires downsampling so it doesn’t crash all the time

7. Quantifying “resolution”
Resolution is difficult to measure
Noise
Instrument alignment
Imaging parameters/setup:
Lens, #angles, counts (exp. time/scintillator), dithering…
Fourier ring correlation (FRC)
Cardone et al (2005), J Struct Biol 151:p
Shows contribution of a single image
Can give you an idea about the quality of your data sets and how they change over time
Example: #Angles
513 => 6.7 µm
1025 => 3.5 µm
2049 => 2.1 µm
Tradeoff b/w resolution and data set size

8. The RERC Grant Objective
0-2 years:
-µCT
-Mechanical testing
-Gait analysis
2-5 years:
-SIMM
-FE mesh refinement
5+ years:
-Patient-specific fracture risk
-Guided surgery
-Assessment of existing/new therapies

9. Mechanical & Imaging Data: Closing the Gap
Microhardness indentation
Notched mechanical testing
Haversion Canals
Crack
Koester et al 2008
α tip geometric factor
E modulus
H hardness
P applied tip load
c total crack length
Tomography
-3D crack visualization
-Crack volume

10. NERSC areas of interest
Reconstruction:
Feedback on image quality/FRC
Filtering:
Reduce amount of downsampling
Software:
Fiji –macro capability
Avizo – advanced visualization of crack propagation
Matlab – FRC for noise/resolution calculations
Igor Pro – SAXS/WAXD