Final Report Summary - MODELLING_JOINT_DEV (Modelling joint development: Integrating biological and mechanical influences)
The overall aim of this project was to explore the influence of mechanical forces on joint morphogenesis, i.e. shape development, in the embryonic limb. Whilst the original proposal had a mainly computational methodology it was decided to address the same research topic with a mainly experimental approach, due to financial and operational constraints. The revised objectives of the project were defined as follows:
1. to characterise the morphology of the embryonic hip joint in three dimensions over a range of developmental stages;
2. to alter the mechanical environment of the developing limb in order to assess the effects on hip shape development;
3. to identify mechanoregulatory pathways underlying the mechanobiology of embryonic hip joint development.
The key methodological approaches that were used were three-dimensional imaging of embryonic limbs stained for cartilage, embryonic chick immobilisation, histological and immuno-histological staining.
For the application of three-dimensional imaging of embryonic limbs, embryonic limbs were stained for cartilage using alcian blue and were scanned in three dimensions using Optical projection tomography (OPT). For each limb, a Visualisation toolkit (VTK) representation of the limb was obtained, and imported into the Paraview application. Relevant information may be found in www.vtk.org and www.paraview.org respectively. Two-dimensional slices were obtained for each limb in a standardised orientation and position. For the database of chick and mouse hip morphologies the specimens best representing the developmental stage were chosen as 'reference' limbs for the stage.
The embryonic chick immobilisation was applied via a pre-specified procedure. Each day, from day five of incubation onwards, fertilised eggs were 'windowed', and 100 µl of saline, i.e. controls, or 0.5 % neuromuscular blocking agent decamethonium bromide (experimental) were dropped onto the growing embryo. Control and experimental embryos were harvested at days seven, eight and nine. From an initial sample of 120 eggs that were incubated, 27 control and 30 experimental embryos were harvested. Mortality rates were largely due to rupturing of the membrane rather than due to the treatment itself.
The histological and immuno-histological staining process involved the following steps. For each embryo analysed, the right limb was retained for histological or immuno-histological staining. The limbs were embedded in a standard orientation in paraffin and sectioned into 7 µm thick microtome sections. Serial sections were taken so that every second section was placed on a different slide. Every second slide was stained for cartilage using alcian blue and for other tissues using Haematoxylin and eosin (H&E). Every other slide was reserved for immuno-histochemical staining for proliferation using an antibody specific to phospho-histone-h3 (phh3), a mitosis marker. The protocol that was used was published by Roddy et al. in 2011.
The project results were as follows:
1. Three-dimensional imaging of embryonic limbs: Large numbers of hindlimbs were scanned, reconstructed and processed for both chick and mouse embryos. Once the VTK images were obtained 'virtual sections' of each limb were obtained using Paraview in order to easily compare between stages. The chick dataset was for between Hamburger Hamilton (HH) stages 26 and 34, while the mouse dataset was for between Theiler stages (TS) 22 to 25. While the data was still under preparation for publication by the time of the project completion, the change in complexity of joint shape in the developing chick limb was evident even over small time scales.
2. Embryonic chick immobilisation: features of developmental dysplasia or Dislocation of the hip (DDH) were detectable in the immobilised hip joints. At embryonic day seven, when hip joint morphogenesis was at a very early stage, a slight difference was already detectable in the immobilised limbs, with immobilised embryos displaying a less protuberant femoral head. At embryonic day eight the immobilised hip joints had a decreased indentation at the femoral neck and a reduced protrusion of the femoral head, when compared to stage matched controls. Similarly, while control femora at embryonic day nine had a distinctly defined femoral neck and protuberant femoral head, the proximal femora of immobilised limbs had a very shallow indentation of the femoral neck and much less protuberant femoral heads. In some cases, the femoral head of immobilised embryos was undefined.
3. Histological and immuno-histological staining: sections of non experimental control mouse and chick embryos and of immobilised and control chick embryos were successfully stained using alcian blue and H&E. The researcher planned to photograph and analyse these slides in the coming months so as to prepare them for publication.
1. to characterise the morphology of the embryonic hip joint in three dimensions over a range of developmental stages;
2. to alter the mechanical environment of the developing limb in order to assess the effects on hip shape development;
3. to identify mechanoregulatory pathways underlying the mechanobiology of embryonic hip joint development.
The key methodological approaches that were used were three-dimensional imaging of embryonic limbs stained for cartilage, embryonic chick immobilisation, histological and immuno-histological staining.
For the application of three-dimensional imaging of embryonic limbs, embryonic limbs were stained for cartilage using alcian blue and were scanned in three dimensions using Optical projection tomography (OPT). For each limb, a Visualisation toolkit (VTK) representation of the limb was obtained, and imported into the Paraview application. Relevant information may be found in www.vtk.org and www.paraview.org respectively. Two-dimensional slices were obtained for each limb in a standardised orientation and position. For the database of chick and mouse hip morphologies the specimens best representing the developmental stage were chosen as 'reference' limbs for the stage.
The embryonic chick immobilisation was applied via a pre-specified procedure. Each day, from day five of incubation onwards, fertilised eggs were 'windowed', and 100 µl of saline, i.e. controls, or 0.5 % neuromuscular blocking agent decamethonium bromide (experimental) were dropped onto the growing embryo. Control and experimental embryos were harvested at days seven, eight and nine. From an initial sample of 120 eggs that were incubated, 27 control and 30 experimental embryos were harvested. Mortality rates were largely due to rupturing of the membrane rather than due to the treatment itself.
The histological and immuno-histological staining process involved the following steps. For each embryo analysed, the right limb was retained for histological or immuno-histological staining. The limbs were embedded in a standard orientation in paraffin and sectioned into 7 µm thick microtome sections. Serial sections were taken so that every second section was placed on a different slide. Every second slide was stained for cartilage using alcian blue and for other tissues using Haematoxylin and eosin (H&E). Every other slide was reserved for immuno-histochemical staining for proliferation using an antibody specific to phospho-histone-h3 (phh3), a mitosis marker. The protocol that was used was published by Roddy et al. in 2011.
The project results were as follows:
1. Three-dimensional imaging of embryonic limbs: Large numbers of hindlimbs were scanned, reconstructed and processed for both chick and mouse embryos. Once the VTK images were obtained 'virtual sections' of each limb were obtained using Paraview in order to easily compare between stages. The chick dataset was for between Hamburger Hamilton (HH) stages 26 and 34, while the mouse dataset was for between Theiler stages (TS) 22 to 25. While the data was still under preparation for publication by the time of the project completion, the change in complexity of joint shape in the developing chick limb was evident even over small time scales.
2. Embryonic chick immobilisation: features of developmental dysplasia or Dislocation of the hip (DDH) were detectable in the immobilised hip joints. At embryonic day seven, when hip joint morphogenesis was at a very early stage, a slight difference was already detectable in the immobilised limbs, with immobilised embryos displaying a less protuberant femoral head. At embryonic day eight the immobilised hip joints had a decreased indentation at the femoral neck and a reduced protrusion of the femoral head, when compared to stage matched controls. Similarly, while control femora at embryonic day nine had a distinctly defined femoral neck and protuberant femoral head, the proximal femora of immobilised limbs had a very shallow indentation of the femoral neck and much less protuberant femoral heads. In some cases, the femoral head of immobilised embryos was undefined.
3. Histological and immuno-histological staining: sections of non experimental control mouse and chick embryos and of immobilised and control chick embryos were successfully stained using alcian blue and H&E. The researcher planned to photograph and analyse these slides in the coming months so as to prepare them for publication.