| dc.description.abstract | Interfaces of hard and soft tissues in the body play a crucial role in processes such as skeletal growth, as well as distributing stresses during load bearing ac- tivities. The mechanically dissimilar tissues are able to be studied individually, but how they integrate at the interface, both by collagen, and mineralisation, is an under explored research area. This is of importance due to these interfaces being particularly prone to damage. In the case of the endplate, hyperminerali- sation of the cartilaginous endplate has been correlated with degeneration of the intervertebral (IVD) discs and chronic lower back pain. For the skull of infants, abnormalities in mineralisation of the cranial sutures leads to deformities of the skull, resulting in increased inter-cranial pressure, and developmental complica- tions for the child. Specific questions addressed in this thesis include, how does the osteocyte lacunae canaliculi network (OLCN) in irregular bones compare to the previously studied long bone?, how are collagen fibres arranged at the soft- hard tissue interfaces?, and how does the mineral density change with distance from the soft-hard interfaces?
This PhD project has investigated these research questions via experimental methods, with the spine experiments using the central section of the 1 year old Lumbar 4-5 ovine samples in the coronal plane, to assess the vertebral body - endplate - IVD interfaces cranial to the IVD. The skulls used intact 6 week old murine samples to assess the suture-cranial plate interface for the interfrontal, sagittal, squamous, and cranial sutures. These were dissected, dehydrated, stained, embedded, and polished in polymethylmethacrylate, followed by multi- modal imaging. The imaging techniques used have been confocal laser scanning microscopy, to assess the OLCN, scanning electron microscopy to map the spa- tial distribution of minerals, and second harmonic generation for investigating the collagen across these mechanically complex tissues.
Analysis for the OLCN in the spine has used Python scripts to quantify the net- work density, the lacunae density, and the direction of the network with respect to the nearest blood vessel. Quantification of minerals in the skull used Quantitative Backscattered Electron Imaging to get the calcium weight % from the pixel in- tensity. Polarised second harmonic generation was used to quantify the principle direction of the collagen bundles, as well as the dispersion of the collagen fibres making up the bundles.
Results have been both qualitative and quantitative in this project. Minerali- sation patterns in the vertebral endplate (VEP) show heterogeneity, with higher degrees of mineralisation in the mineralised cartilage. The values for canaliculi density within the VEP range from 0.05-0.14 μm/μm3, similar values reported in long bone, and the collagen across the cartilage and bone interface has the same principle direction, but the cartilage has a greater degree of dispersion. For the suture-cranial plate interface, the mineral density values ranged between 15-22%, with higher values located at the sites of growth, and edges close to non-mineralised tissues. The collagens have continuity across the mineralisation face, with changes in collagen structure to become more ordered once within the bone tissue, or as Sharpey’s fibres which span the soft-hard interface. The soft-hard interface, which defines the boundary of mineralised tissue, is spatially distinct from the interface between the major collagen types: type I and type II. This observation is seen in both the spine and the cranial sutures.
This thesis outlines reliable methods to image and quantify the OLCN, miner- alisation, and collagen in mechanically dissimilar tissues, and establishes a base- line for future experiments to expand on how these features may change with age or disease. The results are in agreement with similar findings in literature, and are novel in that these specific tissues have not been quantified by their OLCN, min- eralisation, and collagen arrangement at this scale before. Findings in this thesis show that there are multiple spatially distinct interfaces of the different constituent components as tissues transition from mineralised to non-mineralised. | en_GB |
