1. VARIATIONS IN MINERAL CRYSTALLITES OF BONE AT DISTINCT SKELETAL SITES:
POSSIBLE RELATIONSHIP WITH DISEASES LIKE OSTEOPOROSIS?
DR HASAN BABER
DOW UNIVERSIRY OF HEALTH SCIENCES
Web:
Supervisor
Maisoon Al Jawad

2. BACKROUND Weight bearing versus protective
Bones are made of hard tissues that form the human skeleton
Consist of both organic and inorganic mineral
Bones at different sites appear to respond differently to external stimuli.
Low levels mechanical load in limb – ‘disuse’ osteoporosis
Low levels mechanical load in skull – no osteoporosis
Weight bearing versus protective
Ulna
Calvaria

3. Osteoporosis is defined as a reduction in skeletal mass.
EVALUATING OSTEOPOROSIS AND ITS RELATIONSHIP TO BONE MINERAL ORIENTATION
Osteoporosis is defined as a reduction in skeletal mass.
Factors facilitating bone loss and osteoporosis
Hormonal imbalance
Calcium haemostasis imbalance
Loss of gonadal function
Ageing
Mechanical stress – ‘disuse osteoporosis’
Disuse osteoporosis corresponds to the loss of bone, as a result of mechanical unloading of the skeleton.
It could occur due to prolonged bed rest and space travel.
The skull not affected

4. What causes these differences in bone metabolism at these two functionally distinct sites?

5. Variations in bone at different skeletal sites
Major determinant of bone strength is due to
Amount of cancellous and cortical bone
Amount of organic matrix and collagen present
Amount and Composition of Mineral content present
Skull bone has a higher ‘safety factor’ compared with limb bone
ORIENTATION OF MINERAL CONTENT AND DIRECTION?

6. Research Question: Are there structural differences in the mineral content in bones from functionally different sites? Objectives: To identify bone mineral crystal orientation and morphology of Rat ulna, calvaria and whale rostrum, by X-ray diffraction and scanning electron microscope.

7. Materials & Methods Materials: Methods:
The animals were male (Sprague - Dawley) rats, Gms, which equates to approximately 5 weeks old.
Methods:
Samples for Scanning Electron Microscope were prepared by fixing and dehydration in graded alcohols and storing them in 100% ethanol .
X-ray diffraction carried at on X-Mas (BM28) Beam line, at the European synchrotron radiation facility using 100 micro thick sections of fixed tissue.
We first did calibrant of the samples, then did powder diffraction of each sample to produce diffraction patterns.
Data Analysis:
The diffraction patterns were then fitted in form of curves by software “ ORIGINS". Fitted curves were then plotted two theta, as a function of distance by software “SIGMAPLOT” .

8. Diagrammatic representation of the parts of bones that were scanned
Ulna
Calvaria
Rostrum

9. RESULTS
X-ray diffraction /SEM studies of rat ulna , calvaria and whale rostrum revealed some interesting findings.
BONE
CAVITY
BONE
ULNA
Rat ulna Composite grids, the composite grids demonstrate alternate patterns of bone/air samples. Lack of orientation is evident from the samples (absence of arcs in the inner circle).
BONE
CAVITY
CALVARIA
Section of rat calvaria samples, preferred orientation is evident from the samples (Presence of arcs in the inner circle) .
ROSTRUM
Composites of Whale Rostrum, there is presence of more orientation then rat calvaria

10. Complete circle in 002(arrow pointing) in bone, corresponding to absence of preferred orientation
Representation of 002 reflection arcs in bone samples, telling us about preferred orientation

11. Variation in intensity of 002 reflection of ulna
The variation in intensity around the Debye ring of the (002) Bragg reflection of a typical rat ulna diffraction pattern. No variation in intensity around the Debye ring of any Bragg reflections were observed in the rat ulna diffraction patterns

12. Variation in intensity of 002 reflection of calvaria
Figure 1.0: A typical 2D diffraction pattern from the rat calvaria. Diffraction arcs highlighting the variation in the (002) Bragg reflection
Graph 1.1: The variation in intensity around the Debye ring of the (002) Bragg reflection of a typical rat calvaria diffraction pattern. The arrows show the position from which the full-width half maximum values were extracted
002
Peak 2
Peak 1
Azimuthal Angle (0)

13. Variation in intensity of 002 reflection of rostrum
A typical 2D diffraction pattern from the whale rostrum. Diffraction arcs highlighting the variation in the (002) Bragg reflection.
The variation in intensity around the Debye ring of the (002) Bragg reflection of a typical whale rostrum diffraction pattern. The arrows show the position from which the full-width half maximum values were extracted
Azimuthal Angle (0)
Peak 2
Intensity (AU)
Peak 1
002

14. When integrating the specimen Azimuthally, with plotted graphs of FWHM(Y-axis) against Distance (X-axis), an increase in value of FWHM corresponds to reduction in preferred orientation .

15. Plotted graphs – Calvaria Specimen
Samples for track 1
Samples for track 2
Calvaria composite shown with added grid reflecting the diffraction pattern number
Distance in Microns μm
Variation in FWHM of the (002) reflection as a function of distance from the edge, with the Calvaria specimen. Track 1(51-55), Track 2(63-67).

16. Plotted graphs Rostrum specimen
Whale Rostrum composite shown with added grid reflecting the diffraction pattern number
Variation in FWHM of the 002 reflection as a function of distance from the edge, with the rostrum specimen. Track 1{64-73}

17. MICROSTRUCTURE OF BONE FROM DIFFERENT SITES
SEM IMAGE OF RAT ULNA
SEM sample of ULNA revealed non-uniform orientation of crystals.

18. Mag: 8000 x
Mag: x
SEM images of rat calvaria at different magnifications
The SEM images shown above of Rat Calvaria, demonstrate, bone crystallites following a direction

19. Mag 20000x
Mag x
Ultrastructural SEM image of crystallites. The crystallites seem to be stacked on top of each other.
SEM Image of whale Rostrum. Variation in crystallite orientation is evident.

20. DISCUSSION
This study demonstrates differences in skull bones when compared with ulna by both SEM and X-ray Diffraction.
Our studies revealed:
Presence of high crystallite orientation and texture in whale rostrum.
Rat calvaria crystal have less texture and orientation, then those in whale rostrum.
Little or no orientation in rat ulna samples.

21. Conclusion
SEM AND S-XRD revealed differences in crystallite orientation that varies from bone sites. The order of crystallite ordering decreases by the order of Whale Rostrum > Rat Calvaria > Rat Ulna The difference in crystal orientation in calvaria, whale rostrum and ulna is consistent with - but does not alone explain – differences in strength and susceptibility to osteoporosis in these anatomically/functional and embryologically diverse bones.

22. LIMITATIONS
Does these differences exist in a pathological bone samples like in osteoporosis that we have not explored in our current study We have compared normal bone via X-ray diffraction and SEM. We can further investigate their differential properties in a pathologic bone.