VARIATIONS IN MINERAL CRYSTALLITES OF BONE AT DISTINCT SKEL - PowerPoint Presentation

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VARIATIONS IN MINERAL CRYSTALLITES OF BONE AT DISTINCT SKELETAL SITES:

POSSIBLE RELATIONSHIP WITH DISEASES LIKE OSTEOPOROSIS?DR HASAN BABER DOW UNIVERSIRY OF HEALTH SCIENCESEmail:Relicpassion@yahoo.comWeb:www.scienctifica.comSupervisorMaisoon Al JawadSlide2

BACKROUND

Bones are made of hard tissues that form the human skeletonConsist of both organic and inorganic mineralBones 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 protectiveUlnaCalvariaSlide3

EVALUATING OSTEOPOROSIS AND ITS RELATIONSHIP TO BONE MINERAL ORIENTATION

Osteoporosis is defined as a reduction in skeletal mass.Factors facilitating bone loss and osteoporosisHormonal imbalanceCalcium haemostasis imbalanceLoss of gonadal functionAgeingMechanical 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 Slide4

What causes these differences in bone metabolism at these two functionally distinct sites?Slide5

Variations in bone at different skeletal sites

Major determinant of bone strength is due toAmount of cancellous and cortical boneAmount of organic matrix and collagen presentAmount and Composition of Mineral content present Skull bone has a higher ‘safety factor’ compared with limb bone ORIENTATION OF MINERAL CONTENT AND DIRECTION?Slide6

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.Slide7

Materials & Methods

Materials: The animals were male (Sprague - Dawley) rats, 110-120 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” .Slide8

Diagrammatic representation of the parts of bones that were scanned

CalvariaUlnaRostrumSlide9

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).

RESULTS X-ray diffraction /SEM studies of rat ulna , calvaria and whale rostrum revealed some interesting findings.Section of rat calvaria samples, preferred orientation is evident from the samples (Presence of arcs in the inner circle) .Composites of Whale Rostrum, there is presence of more orientation then rat calvaria CAVITYBONEBONEBONE

CAVITY

ULNA

CALVARIA

ROSTRUMSlide10

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 orientationSlide11

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 patternsVariation in intensity of 002 reflection of ulna Slide12

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 extracted002Azimuthal Angle (0)Peak 1Peak 2

Variation in intensity of 002 reflection of calvaria Slide13

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 extractedAzimuthal Angle (0)Peak 2

Intensity (AU)

002

Peak 1

Variation in intensity of 002 reflection of rostrum Slide14

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 .Slide15

Plotted graphs –

Calvaria SpecimenDistance in Microns μmVariation 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).Calvaria composite shown with added grid reflecting the diffraction pattern numberSamples for track 1Samples for track 2Slide16

Variation in FWHM of the 002 reflection as a function of distance from the edge, with the rostrum specimen. Track 1{64-73}

Plotted graphs Rostrum specimenWhale Rostrum composite shown with added grid reflecting the diffraction pattern numberSlide17

MICROSTRUCTURE OF BONE

FROM DIFFERENT SITESSEM IMAGE OF RAT ULNA SEM sample of ULNA revealed non-uniform orientation of crystals.Slide18

SEM images of rat calvaria at different magnifications

The SEM images shown above of Rat Calvaria, demonstrate, bone crystallites following a direction

Mag: 8000 x

Mag: 16000 xSlide19

SEM Image of whale Rostrum. Variation in crystallite orientation is evident.

Ultrastructural SEM image of crystallites. The crystallites seem to be stacked on top of each other.Mag 20000xMag 240000xSlide20

DISCUSSIONThis 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.Slide21

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 UlnaThe 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.Slide22

LIMITATIONSDoes 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.Slide23