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Mechanical integrity and architecture of bone relative to osteoporosis, ageing and drug treatment

Deliverables

A CSM Nano-Hardness tester was used to assess the tissue stiffness of the different samples by nano-indentation of the prepared sample surface. We have assessed the stiffness for the three groups and the different time-points. Thus, we got insight in the changes in material during ageing, and caused by the ovariectomy. The Tibolone treated group provided information on how this medicine interacts with ovariectomy and ageing on tissue level. Further, we have distinguished the effects on surface tissue and inner tissue. At all time points, for the whole material, there is a significant difference between healthy and ovariectomised (p<0.05) and ovariectomised, drug-treated and ovariectomised (p<0.05). It is thought that the rapid stiffening of the edge material from the ovariectomised rats could be due to increased mineralisation of fewer individual trabeculae under unchanging physiological loads. The hormone replacement treatment seems to negate the affects of the ovariectomy and even those of natural ageing.
We were able to follow the remodelling process in the bones of the rats at the level of single trabeculae. We could see resorption of bone at certain locations, while at the same time bone was formed at other locations, sometimes within the distance of just one or two trabeculae. We could see bone remodelling leading to the formation of complete new trabecular structures - this has been impossible to show until now.
In order to assess the mechanical properties and fatigue behaviour of single trabeculae, a micro-tensile/fatigue testing apparatus has been developed that minimizes errors due to misalignment and stress concentrations at the grips. A method for calculating strain in the specimen has been developed involving solid modelling and finite element analyses at micro-resolutions. We use the method to test the hypothesis that the strength of single trabeculae will differ for normal, ovariectomised, and drug treated rat bones over the course of ageing. By measuring the tensile strength of individual trabeculae, we have found that the bone tissue of trabeculae is significantly stronger in osteoporotic bone compared to normal, and that Tibolone treatment normalizes the strength to normal levels. These differences may be an effect of inhibiting bone resorption during drug treatment and or due to a change in the material properties of osteoporotic bone tissue in order to compensate for bone loss.
A precise segmentation of the scan images into binary bone/non-bone images is a crucial step to successfully detect changes between the in-vivo scans of different time points. Standard algorithms were not sufficiently accurate for the in-vivo data and we developed an automatic algorithm to perform this task. The new algorithm yields very accurate results of the true surface of the bone. The algorithm has been accepted as a journal publication. The software itself is available free of charge on the group's website. The software will be made available to others in order to further improve this research area in general. A code for improved segmentation was developed and made public (publication and placed on website). The code is already in use by plm. 10 other researches groups and significantly improve the segmentation of the micro-CT scans.
In the first 4 weeks BV/TV rapidly decreased by 31% in the OVX animals, which gradually proceeded to 83% after 54 weeks. Bone loss started in the more centrally located trabecula. Besides bone loss, also new bone formation was observed at the endosteal cortex. Non-resorbed trabecula increased in thickness (from 139 mm to 153 mm) for all animals and even complete new trabecular structures were formed. In the sham operated rats, BV/TV slowly decreased at all time points reaching a loss of 37% at week 54, while the thickness of the remaining trabecula increased, again for all animals. The changes in bone structure for both groups followed a similar pattern. Decrease in bone volume was followed by an increase in trabecular thickness. The similarity between age related bone loss and bone loss caused by estrogen depletion might indicate that similar mechanisms are at work, and that estrogen depletion resulted in an increase in the speed at which these mechanisms inflict changes in the ovx group. The fact that some trabecula increase in thickness while at the same time their neighbours are being resorbed raises questions on how bone cells sense spatial differences. The well-known mechanical responsiveness of bone might make it plausible that the mechanism at work is an ongoing mechanical adaptation of the bone.
In connection with RUCA software package was bought (MIRIT, KULeuven, Leuven, Belgium). It was used as a basis for matching different data sets. A methodology was designed to use this software package on growing rat bones that experience drastic changes in shape over time. It provided surprisingly good and valuable results. We consider this as a major step forward in bone research and these results are of crucial importance for the success of MIAB. A crucial step to successfully detect changes between the in-vivo scans of different time points is precise segmentation of the scan images into binary bone/non-bone images. Standard algorithms were not sufficiently accurate for the in-vivo data and we developed an automatic algorithm to perform this task. The new algorithm yields very accurate results of the true surface of the bone (2). The presented method adds new possibilities to skeletal research, since it offers the possibility to follow biological processes in detail as they occur. We expect that this new tool will contribute greatly to experimental rat/mouse studies on pharmacological intervention and transgenic models.
The Skyscan 1076 in-vivo micro-CT scanner is a very innovative new imaging tool. Thanks to it, it is possible to obtain new and interesting scientific results. The machine became available for the market within the project period. It has become a big commercial success for Skyscan and more than ten machines have already been sold to various research labs world-wide.

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