THE BONE TISSUE: MULTIDISCIPLINARY CHARACTERISATION IN EXPERIMENTAL ANIMALS AND SKELETAL DEVELOPMENT IN DOGS
Creators: name: ANDREIS, MARIA ELENA
Publisher: name: Universit� degli Studi di Milano
Year: 2019

description: Animals are considered sentient beings by law and the notion of animal welfare, both physical and psychological, is well-established (?Five Freedoms?). As a result, regulation on animal testing puts researchers through highly regulated and restrained protocols (to reduce the number of animals included in in vivo experiments and replace them with alternative procedures whenever possible. This project revolves around the study of the bone tissue in different species, grounding on the idea of cadaver ?recycle? applied to bone-related research. The bone, in fact, is particularly easy to study in cadavers since it suffers the early consequences of post-mortal decay to a lesser extent compared to other tissues. The bone is a highly specialized connective tissue characterized by a mineralized extracellular matrix, specifically designed for structural, mechanical and metabolic functions. Despite general shared features, interspecific differences exist in bone microscopic organisation, which have not yet been completely elucidated. Because of its peculiarities, a comprehensive evaluation of the bone includes several different techniques, such as histology, densitometry and biomechanics. Histologic techniques are particularly common, especially when it is necessary to evaluate tissue biocompatibility and integration in animal models of bone healing and regeneration. However, due to highly mineralized nature of its matrix, several problems arise during bone processing, especially during decalcification (the removal of mineral from the bone while preserving all the essential microscopic elements and tissue antigenicity), a process that is yet far from being standardized, but is necessary to prepare paraffin-embedded specimens. The first two aims of this project were the definition of species-specific and site-specific guidelines for bone specimens? decalcification and the characterization of swine and ovine bone tissue with a multidisciplinary approach. Basic protocols for trabecular bone decalcification in different species with different solutions were provided, showing that excellent morphologic results can be achieved with solution containing strong acids in rodents, sheep and pig. As a general rule, hydrochloric acid solution proved to be too aggressive, as previously reported. On the other hand, the combined use of hydrochloric acid and formic acid proved to be a very good compromise, shortening experimental time and preserving morphology. Finally, a combination of citric acid formic acid provided excellent morphology, but at the expenses of decalcification time. Collagen I was preserved in swine, canine and ovine samples that proved to have good/excellent morphology, while it wasn?t preserved in rats or mice, despite their excellent morphology. A multidisciplinary approach to the study of the bone in pigs and sheep was developed, evaluating several different parameters with different techniques on the same samples, also providing standard reference parameters about swine and ovine bone histology, densitometry and biomechanics, to be compared with future research results, to deepen the knowledge on the bone tissue and facilitate biomedical research on animal models. The histogenesis of bone can be divided in two modalities: endochondral and intramembranous, which share a common sequence of events. Bone growth starts during the fetal stage, and then continues after birth together with the growth of the entire individual. Special structures, called cartilage canals, are involved in the process of endochondral ossification, with nutrients supply to the growing cartilage and waste products elimination being their primary functions. Cartilage canals have been described in several species, but little information is known about them in dogs. The development of a newborn into an adult implies dramatic changes in both body size and shape. The dog, in particular, exhibits the highest intraspecific differences in body shape and size among mammals, and this might be reflected by different growth patterns. This is especially true when the skull is considered, as it is the most variable body part among different breeds. Little information is known about skull development in dogs in relation to their adult craniometric category (brachycephalic, mesaticephalic, dolychocephalic). Due to the extreme plasticity of young bones, moreover, skeletal development could potentially be influenced by the presence of orthopaedic diseases, which could modify the normal shape and size of the future adult bones. The last three aims of this project were the study of endochondral ossification in long bones, with particular emphasis on the role role of cartilage canals; the study of the effects of pathology (quadriceps contracture) on hindlimb development; and the study of skull development and its compared features in brachycephalic, mesaticephalic and dolychocephalic breeds. New insights on cartilage canals in small-sized dogs were provided, which confirmed their involvement in bone extracellular matrix production in dogs, as in other species, laying the basis for more extensive studies on the delicate mechanism of the formation of ossification centres and the regulation of ossification. The abnormalities induced by quadriceps contracture on hind limb skeletal development, mainly consisted of volume reduction and abnormal shape of several centres, were described in a litter of Dobermann. These alterations that should be carefully considered when evaluating puppies affected by the disease to plan a therapy. Finally, novel contribution in canine craniometry and skull development were provided, documenting morphometric differences among dolichocephalic, mesaticephalic and brachycephalic purebred puppies in the early neonatal period through a radiographic and anatomic study. Cadavers invariably proved to be an invaluable source of research material, providing a valid alternative to in vivo experiments and contributing to the reduction of the use of experimental animals.