The muscle spindle has been extensively studied ever since the detailed histological descriptions by Ruffini in 1898 which 50 years later were revived and fortified by Barker in 1948, Many physiologists as well as anatomists have devoted themselves enthusiastically to various aspects of these complex sense organs which are said to rank third to the vertebrate eye and ear in degree of complexity. It is perhaps because of the complexity in conjunction with technical difficulties that there are still many points of disagreement amongst physiologists and histologists and amongst histologists themselves.

In vertebrates, all trunk muscles, which includes the body and limb muscles, are derived from muscle progenitor cells originating in somites. Somites are segmentally repeated embryonic epithelial balls of cells that bud off from the pre-segmented paraxial mesoderm in a rostral -caudal progression along either side of the neural tube (Brand -Saberi et al., 1996, Dequeant and Pourquie, 2008, Pourquie, 2011) . During embryonic development, somites mature and differentiate into a variety of distinct tissues: a) the sclerotome, that gives rise to the vertebrae bone, connective tissue and cartilage surrounding neural structures; b) the dermomyotome, that gives rise to muscle progenitors, dermis and angiogenic cells; c) the syndetome, from which axial tendons are derived and d) the myotome, which is where muscle differentiation first occurs in the embryo. Limb muscles originate from somites only at limb -level and the formation of muscle involves several steps: migration, proliferation and differentiation (Duprez, 2002). At limb level, progenitor cells at the ventro-lateral lip of the dermomyotome delaminate and migrate into the limb bud. Delamination and migration occurs when a signal from the limb bud, HGF (hepatocyte growth factor)/SF (scatter factorL produced by mesodermal cells from the limb bud, interacts with C-met, a tyrosine kinase receptor expressed in the muscle progenitor cells localised at the ventro- Iateral lip (Dietrich et al., 1998). The progenitors migrate to two distinct regions in the limb: the dorsal muscle mass and the ventral muscle mass. Only at these two regions do the progenitor cells activate the myogenic programme and express muscle-specific genes such as the myogenic regulatory factors (MRFs). At transcriptional level, there are four key myogenic genes that have been implicated to be important for trunk muscle formation. These are the MRFs, a group of four basic helix-loop- helix transcription factors which includes MyfS, MyoD, myogenin and MRF4 (also known as Myf6). Although the first MRF was described back in 1987 (Davis et al., 1987L and the first in situ hybridisation experiments were performed using radioactive -labelled probes (Pownall and Emerson, 1992), the expression profile for the MRFs during embryogenesis was not comprehensive and remained fragmented in both chick and mouse.

The morphogenesis of muscle spindles in rat lower hindlimb muscles has been investigated using the electron microscope. The earliest detectable spindles are seen in the 19.5-day foetus and consist of a single myotube bearing simple nerve terminals of the large primary afferent axon from the nearby unmyelinated spindle nerve trunk. The capsule forms by an extension of the perineural epithelium of the supplying nerve fasciculus, and is confined initially to the innervated zone. Myonuclei accumulate in this region, so that the first intrafusal muscle fibre to develop is a nuclear-bag fibre. Myoblasts, that are present within the axial bundle throughout its development, fuse to form a smaller, less-differentiated myotube by the 20-day foetal stage. This matures in close association with the initial fibre, and by birth (21-22 days gestation) has formed the smaller intermediate bag fibre that has been identified histochemically and ultrastructurally in the adult. Nuclear-chain fibres develop in the same way; myoblasts fuse to form satellite myotubes that mature in apposition to one or more of the other fibres, lying within a common basement membrane. By the 4-day postnatal stage the full adult complement of 4 intrafusal muscle fibres is present, although the ultrastructural and histochemical variations, seen in the adult, are not present. The fusimotor innervation begins to arrive by birth, but is not fully established until the 3rd postnatal week, when the ultrastructural and histo chemical maturation of the axial bundle is complete. The maturation of the capsule and periaxial space occurs at the same time. It is suggested that the sequential development of the intrafusal fibres is a reflection of the decreasing morphogenetic effect of the afferent innervation, whereas the role of the fusimotor innervation is in ultra- structural and histochemical differentiation.

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Biomineralisation is the synthesis of inorganic materials in biological systems. Many biominerals - such as bone! teeth, and shells - are high-performance composites synthesised with extreme precision under physiological conditions. Understanding biomineralisation is expected to inspire 'green' methods for the manufacture of novel materials. Diatoms are eukaryotic algae that mineralise an external cell wall, or frustule, composed of hydrated silica. Silicification depends upon the uptake of soluble silicon (silicic acid) from the local environment by specific silicic acid transport proteins (SITs). This unusual family of integral membrane proteins are relatively uncharacterised. This project aimed to express and purify 5113 from the diatom Thalassiosira pseudonana (TpSIT3) for further characterisation in vitro, and to explore whether synthetic SIT3 proteoliposomes could be used as a model mineralisation system with potential applications in nanotechnology. TpSIT3 was successfully overexpressed in yeast and purified in the solubilising detergents Fos-choline 12 and octyl glucoside. The purified protein was successfully reconstituted into synthetic liposomes and silicic acid uptake was assessed using two fluorescent assays including a novel method which utilised zinc silicate fluorescence. This method was used to determine that silicic acid transport by TpSIT3 displayed Michaelis-Menten kinetics with a Km of 6.1 ± 2.7 μM, similar to silicic acid uptake studies in diatom cultures. The structure and function of a silicifying cationic peptide were also characterised for the first time. Peptide-mediated silicification only proceeded at ≥2 mM silicic acid when pH was >6.4 and peptide concentration was ≥2.5 mM. These results underpinned efforts to synthesise silica within the int erior lumen of peptide-loaded SIT proteoliposomes. Preliminary electron microscopy and elemental analysis suggested that such an approach was feasible. This thesis thus establishes a series of novel methods that can be used to study silicic acid transport and silica mineralisation in vitro

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Resorption of calcium phosphate (CaP) biomaterials is traditionally assessed using an osteoclast (QC) resorption assay where resorption pits formed on the CaP surface are analysed by microscopy techniques and quantified on the basis of pit number, pit area or pit volume. Pit area measurements (20) have become common practice when assessing CaP biomaterial resorption in vitro. Apart from the time consuming nature of pit area analysis techniques it is not a precise indicator of resorption; variations in pit depth are not taken into consideration and it is unsuitable for use on porous materials where visualisation of internal structures is difficult or for use on materials with rough surfaces where determination of individual pits would be difficult. A 3D quantification of bioresorption is available but requires specialised, expensive equipment. An appropriate measure of resorption was required to be more efficient and more cost-effective than the current available in vitro methods, but most importantly, to directly correlate with pit area measurements and have the ability to be used in a broad range of in vitro experiments. An QC resorption assay was developed and optimised through a series of experiments. The established assay was used to assess several outcome measures as potential indicators of resorption in vitro, namely; the correlation of percentage area resorbed in vitro with Ca and P concentration in cell culture medium, QC number and QC activity. A dense substrate free from surface anomalies was used to accurately correlate pit area with the alternative outcome measures. This body of work has established two main outcome measures of bioresorption in vitro that correlate with pit area measurements; Ca and P ion concentration in culture medium and QC specific enzyme activity. These outcome measures will prove invaluable for improving the fundamental understanding of QC resorption of CaP biomaterials.

Rupture of the cranial cruciate ligament (CCL) in dogs and the anterior cruciate ligament (ACL) in human beings, with ensuing development of osteoarthritis (OA) is a common orthopaedic problem in these species. Many risk factors have been identified in the development of cruciate rupture and OA; these include obesity, trauma, gender, age and genetics. Differences in physiological hormone levels have been associated with some of these risk factors. Obesity is a major risk factor for OA and leptin levels are positively correlated to fat mass. More recently, it has been shown that the tissues of the synovial joint express leptin and the leptin receptor, indicating that leptin may have an autocrine or paracrine role in these tissues.