A cysteine-containing elastin-like polypeptide (ELP2cys) was successfully synthesized and purified, and was shown to behave in a similar fashion to other well-characterized ELPs. Incorporating the ELP2cys as a crosslinking agent into a solution of sulphated hyaluronan (CMHA-S) not only decreased the gelation time of the solution but also increased the crosslinking density of the resultant hydrogel, in turn increasing both the resiliency and stiffness of the construct. Preliminary in vitro work involved culture of human disc cells, followed by their encapsulation within the hydrogel. Unfortunately the results were inconclusive, although it appeared as though the addition of ELP2cys to the matrix did not negatively affect the viability of the cells, as compared to hydrogels with CMHA-S only. This study showed that ELP2cys is a valuable addition to the family of recombinant elastin-like polypeptides, and shows promise as a crosslinking agent in the formation of hyaluronan hydrogels.

La réponse de l’organisme contre un agent pathogène nécessite la mise en jeu d’interactions complexes entre cellules immunitaires et environnement. La matrice extra-cellulaire est remodelée au cours de la réponse immunitaire pour permettre la migration des cellules vers le site infectieux. Les peptides issus de la dégradation de la matrice peuvent influencer les fonctions biologiques des cellules immunitaires. Dans les pathologies impliquant des tissus riches en élastine, les cellules de la réponse inflammatoire et immunitaire, notamment les polynucléaires neutrophiles (PN) et les cellules dendritiques se retrouvent dans un environnement riche en produits issus de la dégradation de l’élastine. Le travail de thèse présenté ici nous a permis de montrer que les peptides d’élastine (PE) régulent les fonctions des PN de sujets sains en augmentant de façon significative leur capacité migratoire, leur capacité à produire des cytokines pro-inflammatoire et à phagocyter les agents pathogènes. Les effets régulateurs des PE sont moindres chez les sujets BPCO et varient en fonction de l’état clinique des patients. Les propriétés biologiques des PN de patients BPCO en exacerbation ne sont pas affectées par les PE. Cette différence de réponse aux PE des PN de patients BPCO à l’état stable ou à l’état exacerbé est proportionnellement liée au niveau d’expression du récepteur S-Gal à la surface des PN. Dans un second travail nous avons montré que les PE sont capables d’attirer les CD au niveau du site infectieux sans influencer la maturation des cellules induite par une activation antigénique. L’effet des PE sur la migration des CD met en jeu le récepteur S-Gal présent à la surface des cellules. Par ailleurs, les PE orientent la réponse cytokinique des CD activées par le LPS vers un profil de type Th-2 et favorisent l’émergence d‟un profil tolérogénique des CD. Ces effets régulateurs des PE sont médiés via l’interaction PE/S-Gal et conduisent au développement d’une réponse adaptative T régulatrice. L’ensemble de ces résultats suggère que les PE participent activement à la régulation de la réponse immunitaire innée et adaptative / Organisme defense against pathogens needs complexe interactions interactions between immune cells and environnement. The extracellular matrix is being remodeled to aloww cells to migrate to infectious site. Peptides generated by this degradation can influence biological fonctions of immune cells. In pathologies involving elastin rich tissue degradation inflammatory and immune cells, especially neutrophils and dendritic cells are in an environnement including numbers of matrix degradation products. This work shows that Elastin Peptides (EP) can regulate the fonctions of neutrophils from healthy subjects, by increasing significantly their migration, their cytokines production and their phagocytic capacity. These EP effects are howeever less important en stable COPD subject and depends on the clinical state of these patients. Indeed the biological properties of neutrophils from COPD patients in exacerbation are not affected by EP. This discrepancy considering the clinical state can be explained by the level of the S-Gal receptor expression. In a second part we have shown the ability of the EP to attract dendritic cell (DC) on the infection site without couetracting DC maturation. This effect involves the S-Gal receptor present on the surface of the cells. EP can also oriente the cytokine production by LPS-induced toward a Th-2 profil and favorise the emergence of DC with tolerogenic features. These effects are mediated throug EP/S-Gal interaction and leads to the developement of a T regulatory response. All of these results suggest that EP are involved in the regulation of innate and adaptative immunity.

O objetivo deste trabalho foi avaliar o efeito do exercício físico aeróbio sobre alterações morfológicas ocorridas na parede da aorta abdominal de animais hipertensos (SHR) e normotensos (WKY), sedentários (S) e treinados (T). Ratos SHR e WKY foram utilizados, que consistia de quatro grupos divididos em WKYS, WKYT, SHRS, SHRT. O treinamento durou 13 semanas, sendo 5 horas por semana, 1 hora por dia. Os resultados deste estudo mostraram que o exercício físico foi eficaz em reduzir a pressão arterial, a freqüência cardíaca e a razão parede/luz, bem como em aumentar a quantidade de fibras elásticas e a luz do vaso no grupo SHRT, em comparação ao grupo SHRS. Em relação à expressão protéica de colágeno I e III, os SHR apresentaram um significativo aumento em relação ao grupo WKY e o grupo SHRT apresentou significativa redução em relação ao SHRS. A <font face=\"symbol\">a-actina mostrou maior expressão nos grupos WKYS e SHRS, quando comparado com seus respectivos grupos treinados e a elastina mostrou-se significante aumento na expressão nos grupos treinados em relação aos grupos sedentários. / The aim of this study was to evaluate the effect of aerobic exercises on morphological changes on abdominal aorta wall, in hypertensive (SHR) and normotensive (WKY) animals, sedentary (S) and trained (T). SHR and WKY were used on the experimental protocol that consisted in four groups divided in WKYS, WKYT, SHRS and SHRT. Trained groups were submitted to a training protocol that lasted 13 weeks, 5 hours a week, 1 hour a day. Results showed that physical exercises were effective not only in reducing blood pressure, cardiac frequency and wall-to-lumen ratio, but also in increasing the number of elastic fibers and the internal diameter in SHRT, in comparison to SHRS. According to collagen I and III protein expression, in both, SHR presented a bigger expression than WKY group. Moreover, SHRT group showed a significant reduction of protein expression in comparison to SHRS. <font face=\"symbol\">a-actin showed to be more expressed in WKYS and SHRS, in relation to the WKYT and SHRT groups and elastin showed a significant increased in WKYT and SHRT in relation to the sedentary groups.

Para avaliar as fibras elásticas na junção vésico-uretral de ratos, em diferentes faixas etárias, realizou-se estudos histológicos, microscopia elêtronica e histomorfometria. O estudo histológico do sistema de fibras elásticas mostrou a presença dos três tipos de fibras elásticas, em todas as faixas etárias, a microscopia eletrônica de transmissão, mostrou as diferenças ultra-estruturais entre as fibras elásticas. A avaliação histomerfométrica revelou diminuição na densidade linear das fibras elásticas e oxitalânicas e aumento na das elaunínicas. Na junção vésico-uretral de animais velhos, há uma queda nas propriedades de elasticidade, recuo elástico e ancoragem, que são compensadas pelas fibras elaunínicas. O processo de envelhecimento das fibras elásticas na junção vésico-uretral não contribui sozinha e diretamente no estado de incontinência urinária, mas compensa e dá suporte a muscular, devido ao aumento das fibras elaunínicas, o que torna o sistema elástico na junção vésico-uretral indiretamente responsável pela manutenção da continência urinária. / To evaluate the elastic fibers in the junctio vesico-urethral of the rat, in different age groups, was accomplished studies histologics, electron microcopy and histomorphometrics. Study histologic showed the presence of the three types elastic fibers in all the studied age groups, electron microcopy showed the ultra-structural differences amoung elastic fibers. Histomorphometric studies revealed a decrease the linear density of the mature elastic fibers and oxytalans and an increase elaunin. In the junction vesico-urethral of the old animals there is a fall the properties elastic, elastic recoil and achorage, but they are compensated by elaunins. Processs aging of the elastic system in this place doesn?t contribute alone or directly in urinary incontinence, but it is indirectly responsible for the maintenance urinary continence.

Collagen is the most abundant protein in the animal kingdom and, together with elastin, forms extensive fibrous networks that constitute the primary structure of the mammalian extracellular matrix, respectively endowing it with the tensile and elastic properties that fulfil its principal role as the passive framework of the body. The fibrous proteins are distinctly hierarchically organised from the molecular scale upwards; for example, the nanoscale tropocollagen monomer assembles in arrays that form the micrometer scale microfibrils and fibrils, and thence into collections of millimetre scale collagen fibres, that in-turn, constitute functional tissues such as skin, tendon and bone. Much is known about the structure at each of these individual scales – collagen being the most extensively researched – and the macromechanics of the fibres are well established. However, far less is known about the micromechanics of these proteins, in particular how the monomers influence the functional mechanics of the macroscopic fibres. In this thesis, I explore the multiscale mechanics of collagen and elastin fibres over a range of hydrations – with fibres in direct contact with aqueous solution, and progressively dehydrated in humidity-controlled environments. I use quasi-static tensile testing to probe the macroscopic mechanical response (Young’s modulus and stress relaxation) of the fibres, and employ Brillouin and Raman microscopy to assess the longitudinal modulus in the GHz range and corresponding molecular properties of the proteins. Brillouin microscopy is an emerging technique in the biomedical field. It enables the all-optical, contact-free and non-destructive testing of tissue micromechanics through detection of frequency shifted light scattered off thermally excited acoustic waves or “phonons” in the GHz range. As one of the first studies of Brillouin light scattering in these fibres, it sets the basis for further investigation of tissue biomechanics. In particular, I provide the full description of the protein fibre micromechanics by performing angular measurements using a so-called platelet-like configuration with sample mounted onto a reflective substrate at 45° angle to the excitation beam. I derive the high-frequency longitudinal modulus, and discuss the results in comparison to the Young’s modulus, in terms of the different frequency and spatial scale of the measurements. I obtained a full description of elasticity using Brillouin spectroscopy applied to dried fibres; however, obtaining the same description in hydrated fibres is a challenge, as the Brillouin spectrum is dominated by water. An assessment of the mechanical differences between type-I and type-II collagens is also given here. Water is known to be a primary determinant of tissue biomechanics, and I identified for the first time, the critical hydration ranges between 100 and 85% relative humidity (RH) for collagen, and around 85% RH for elastin, at which point each macroscopic fibre switched from viscoelastic to plastic-like behaviour. Dehydration below these critical points was shown to severely diminish collagen fibrillar sliding, and completely rob elastin of its ability to reversibly deform under strain. The Young’s modulus increased markedly below these hydrations, and I observed a parallel increase in the longitudinal modulus at high frequencies in each protein, indicating a concomitant increase in stiffness at the two scales. The major difference observed between the two fibrous proteins is that, in the case of elastin, I observe a two-fold increase in the longitudinal modulus as the hydration is decreased from 100 to 21% RH, whilst the Young’s modulus increases by two orders of magnitude. This discrepancy was not observed in collagen, which confirmed that the protein maintained its long-range order in the form of the triple helix at all hydrations employed in this work, whilst the elastin ultrastructure experiences a liquid-to-solid state change at a critical hydration. I demonstrate through the analysis of the low-wavenumber region (< 500 cm-1) of the Raman spectrum, that the increase in molecular stiffness of both proteins, is reflected in an increase in torsional rigidity of the peptide backbone upon dehydration. Moreover in collagen, I observe a reduction in the number of inter-protein water bridges, which I propose causes a collapse of the lateral spacing between monomers and an increase in direct backbone-backbone hydrogen bonding, that further stiffens the fibre. Small strain induced reorientations of the amide III and C–C stretching modes in dehydrated collagen fibres suggest that macroscopic stresses may be transferred to the triple helix, otherwise left unperturbed in the hydrated state. I postulate that this is a result of the degraded intra- and interfibrillar sliding mechanism below the critical hydration. Hence in its dehydrated state, the collagen whole-fibre mechanics are similar to those at the molecular scale. The role of proteoglycans and glycosaminoglycans and their potential connection to hydration, is also discussed. In agreement with previous work, I found no Raman spectral changes as a result of stretching hydrated elastin fibres, indicating that even large strains e.g. 80%, have no significant effect on the structural scale probed by Raman microscopy, nor in the air-dried state where the brittle fibres break at low strains. I suggest this may imply a limited sensitivity of Raman bands to these changes, possibly an indication of elastin’s dynamic ultrastructure, or that stress is dissipated at a higher level of the fibre structure. On the macroscopic scale, it is the poroelastic nature of elastin which controls the stress relaxation under strain, and the elastic recovery is mediated by an interplay of hydrophobic interactions and hydration forces.

Dental enamel is an excellent example of a highly mineralised tissue, composed of hierarchically organised apatite mineral. This unique organisation gives enamel superior mechanical properties. However, when mature, enamel becomes acellular and unable to repair itself during traumatic or carious damage. The lack of self-repair requires dental intervention, where the common treatment of decayed enamel is to remove the affected and healthy tissue, and replace with restorative materials. The restorative materials, currently used, can cause further complications in the form of secondary caries or failure due to thermal and mechanical property mismatch with enamel. Problems associated with current restorative materials have driven researchers to explore biomimetic enamel treatment routes. To mimic the natural enamel formation, we can explore how proteins can guide mineral growth, in order to form enamel-like ordered mineral structures. In this thesis, the use of a synthetic, recombinant protein called an elastin-like protein (ELP) containing the analogue of the N-terminal of statherin (STNA15) was under investigation. Statherin is a protein present in saliva that is said to aid in the remineralisation of enamel. ELP with STNA15 (STNA15-ELP) has already shown promise in biomimetic mineralisation. This thesis investigated how conformation and structure of STNA15-ELP can be affected and manipulated by different chemical environments, surface constraint and crosslinking. The STNA15-ELP characteristics were related to formation of fluorapatite. STNA15-ELP conformation changed due to presence if salts in solution and whether or not it was constrained. We linked the conformational changes within STNA15-ELP, in solution versus on the surface, to two different routes of mineral formation. FAp formed in an uncontrolled manner with free STNA15-ELP. Ordered FAp formed via a precursor when STNA15-ELP was constrained on a surface. This work leads to an understanding of biomimetic mineralisation using STNA15-ELP. This information can aid in the design of novel biomimetic, enamel-like therapeutics.

La broncho-pneumopathie chronique obstructive (BPCO) est une maladie respiratoire inflammatoire chronique caractérisée par une limitation progressive et irréversible des débits aériens causée par l’inhalation à long terme de particules nocives telles que le tabac. La BPCO présente un remodelage majeur et hétérogène des voies aériennes comportant une grande variabilité inter-individuelle. La réponse inflammatoire et immunitaire au cours de la BPCO est caractérisée par une infiltration du tissu respiratoire par les polynucléaires neutrophiles (PN), les macrophages et par les cellules T. La dégradation des fibres élastiques du poumon en peptides solubles d’élastine (PE) par le biais de la sécrétion de protéases par les cellules de l’immunité innée est une caractéristique constante de la BPCO. Ces PE participent à la physiopathologie de la BPCO comme cela a été démontré dans différents modèles murins de la maladie emphysémateuse. L’orientation de la réponse des cellules T helper (Th) et des cellules T cytotoxiques (Tc) au cours de la BPCO n’est pas élucidée et reste controversée. L’objectif principal de ce travail de thèse a été de définir l’orientation de la réponse Th et Tc ainsi que le rôle des PE dans le remodelage associé à la BPCO. Pour cela, nous avons étudié la signature cytokinique Th-1/Tc-1 (IFN-), Th-2/Tc-2 (IL-4), Th-17/Tc-17 (IL-17) spécifique du remodelage bronchique associé à la BPCO au niveau cellulaire, mais également transcriptionnel et fonctionnel. L’association entre les résultats expérimentaux obtenus et le phénotype des patients inclus a été analysé de manière à déterminer le rôle de ces mécanismes dans l’expression clinique de cette pathologie respiratoire chronique. Les résultats obtenus ont mis en évidence une diminution de l’expression de l’IL-4 (Th2) chez les patients BPCO par rapport aux sujets contrôles ainsi qu’une potentialisation de l’expression de cette cytokine en présence des PE. / Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory pathology characterized by a progressive and irreversible limitation of airflow caused by the long-term inhalation of harmful particles such as tobacco. COPD presents a major and heterogeneous remodeling of the airways with important inter-individual variability. The inflammatory and immune response during COPD is characterized by infiltration of pulmonary tissue by neutrophils (PN), macrophages, and T cells. The degradation of lung elastic fibers into soluble elastin peptides (EP) is caused by the secretion of proteases by innate immunity cells and it is a consistent feature of COPD. These EP participate in the pathophysiology of COPD as demonstrated in different murine models of the emphysematous disease. The T helper (Th) and the T cytotoxic (Tc) orientation during COPD is unclear andremains controversial. The main objective of this work was to define the Th and Tc responses as well as the role ofEP in airways remodeling associated to COPD. For this purpose, we studied the cytokine signature Th-1/Tc-1 (IFN- ), Th-2/Tc-2 (IL-4), Th-17/Tc-17 (IL-17) specific of the airway remodeling associated to COPD both at the cellular, transcriptional and functional level. Association between the experimental results obtained and the phenotype of the patients included in the study was analyzed in order to determine the role of these mechanisms in the clinical expression of this chronic respiratory pathology. The results obtained showed a decrease in the expression of IL-4 (Th2) in COPD patients compared to control subjects as well as a potentiation of this cytokine expression in the presence of EP.

Devido as limitações relacionadas ao emprego do enxerto autólogo, o uso dos biomateriais poliméricos naturais tornou-se uma opção viável em terapias regenerativas do tecido ósseo. O objetivo desse trabalho é avaliar de forma qualitativa e quantitiva a contribuição da membrana de elastina utilizada isoladamente ou em associação a hidroxiapatita e proteína morfogenética óssea, no reparo de defeitos ósseos no crânio de ratos. Foram utilizados 49 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para a criação do defeito ósseo no osso parietal esquerdo e divididos em 7 grupos com 7 animais cada. Os grupos foram implantados com os seguintes bioamateriais: grupo 1 controle (G1-C) sem implante, grupo 2 (G2-E24h) membrana de elastina 24 h, grupo 3 (G3-E24h/HA) membrana de elastina 24 h com hidroxiapatita, grupo 4 (G4-E24h/BMP) membrana de elastina 24 h com proteína morfogenética óssea, grupo 5 (G5-E96h) membrana de elastina 96 h, grupo 6 (G6-E96h/HA) membrana de elastina 96 h com hidroxiapatita, grupo 7 (G7-E96h/BMP) membrana de elastina 96 h com proteína morfogenética óssea. Após a morte indolor induzida em 6 semanas, as calotas cranianas foram retiradas para análise macroscópica, radiográfica, histológica e morfométrica. As análises macroscópicas, radiográficas e histológicas demonstraram a biocompatibilidade dos biomateriais utilizados. As médias e desvios-padrão do volume percentual relativo de osso neoformado nos defeitos cranianos dos grupos G1 a G7 foram 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectivamente. Os biomateriais mostraram-se biocompatíveis e o grupo 7 (G7-E96h/BMP) resultou na maior neoformação óssea. / Due to the limitations related to the use of autologous grafts, the use of natural polymeric biomaterials has become a viable option in regenerative therapies of bone tissue. The objective of this dissertation is to evaluate in qualitative and quantitative way the contribution of the elastin matrice used alone or in combination with hydroxyapatite and bone morphogenetic protein in the repair of bone defects in the skull of rats. Were use 49 Mices (Rattus norvegicus, Wistar), weighting approximately 330 grams and 4 months of age, were used. The animals were submitted to the surgical procedure to create the bone defect in the left parietal bone and divided into 7 groups with 7 animals each. The groups were implanted with the following biomaterials: group 1 control (G1-C) without biomaterial, group 2 (G2-E24h) 24 h elastin membrane, group 3 (G3-E24h/HA) 24 h elastin membrane with hydroxyapatite, Group 4 (G4-E24h/BMP) elastin membrane 24 h with bone morphogenetic protein, group 5 (G5-E96h) elastin membrane 96 h, group 6 (G6- E96h/HA) elastin membrane 96 h with hydroxyapatite, group 7 (G7-E96h/BMP) 96 h elastin membrane with bone morphogenetic protein. After painless death induced at 6 weeks, the skull caps were removed for macroscopic, radiographic, histological and morphometric analysis. Macroscopic, radiographic and histological analysis demonstrated the biocompatibility of the biomaterials used. The mean and standard deviations of the relative percentage volume of newly formed bone in the cranial defects of the G1 to G7 groups were 7,87±2,53; 24,01±0,55; 9,59±1,27; 31,31±6,37; 19,77±2,62; 7,31±2,43; 43,25±3,72, respectively. The implanted biomaterials were shown to be biocompatible and the group 7 (G7-E96h/BMP) resulted with greater bone neoformation.

Nas doenças que provocam perdas ósseas, destaca-se a utilização de enxerto ósseo autólogo como tratamento padrão-ouro. Entretanto, devido a comorbidades e limitação quantitativa, apresentam-se como alternativa o uso de biomateriais de elastina e hidroxiapatita, bem como a associação com a proteína morfogenética óssea. O objetivo desta pesquisa foi avaliar de forma qualitativa e quantitativa a contribuição da membrana de elastina utilizada isoladamente ou em combinação com a hidroxiapatita e a proteína morfogenética óssea no reparo de defeitos ósseos provocados experimentalmente no fêmur de ratos. Foram utilizados 77 ratos (Rattus norvegicus, Wistar), machos, com peso aproximado de 330 gramas e 4 meses de idade. Os animais foram submetidos ao procedimento cirúrgico para criação do defeito ósseo na superfície anterior da região supracondiliana do fêmur direito e separados em 7 grupos com 11 animais cada. Os grupos foram divididos da seguinte forma: Grupo controle (G1-C), ratos com defeito crítico induzido no osso femoral, sem preenchimento com implante. Grupo 2 (G2-E24/50), ratos com defeito crítico induzido no osso femoral preenchido com membrana de elastina hidrolisada durante 24h a 50°C; Grupo 3 (G3-E24/50/HA), defeito preenchido com membrana de elastina hidrolisada durante 24h a 50°C e hidroxiapatita. Grupo 4 (G4 E24/50/BMP), defeito preenchido com membrana de elastina hidrolisada durante 24h a 50°C e BMP. Grupo 5 (G5-E96/37), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C. Grupo 6 (G6-E96/37/HA), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C e hidroxiapatita. Grupo 7 (G7-E96/37/BMP), defeito preenchido com membrana de elastina hidrolisada durante 96 h a 37°C e BMP. Após a morte indolor induzida após 6 semanas, as peças anatômicas femorais foram retiradas para análise macroscópica, radiográfica, histológica, morfométrica e biomecânica. As médias e desvios-padrão do volume percentual relativo de osso neoformado no defeito femoral foram respectivamente 36,39±3,86 (G1); 66,40±3,69 (G2); 55,12±2,23 (G3); 58,46±1,79 (G4); 51,36±3,15 (G5); 71,28±4,26 (G6); 45,54±3,98 (G7). Os achados demonstraram biocompatibilidade, biodegradabilidade e osteorregeneração maior que o grupo controle nos biomateriais estudados. Os grupos com maior volume ósseo neoformado foram G2-E24/50 e G6-E96/37/HA. / In diseases that cause bone loss, the use of autologous bone graft is still a goldstandard treatment. However, due to comorbidities and quantitative limitation, the use of biomaterials of elastin, hydroxyapatite as well as the association with the bone morphogenetic protein are presented as alternatives. The objective of this research is to evaluate in a qualitative and quantitative way the contribution of the elastin membrane used alone or in combination with the hydroxyapatite and the bone morphogenetic protein in bone defects experimentally created in femur of rats. A total of 77 male rats (Rattus norvegicus, Wistar) weighing approximately 330 grams and 4 months of age were used. The animals were submitted to the surgical procedure to create the bone defect on the anterior surface of the supracondylar region of the right femur and were separated into 7 groups with 11 animals each. The groups were divided as follows: Control group (G1-C), rats with critical defect induced in the femoral bone without implant filling. Group 2 (G2-E24/50), defect filled with elastin membrane prepared for 24 h at 50°C. Group 3 (G3-E24/50/HA), defect filled with elastin membrane prepared for 24 h at 50°C and hydroxyapatite. Group 4 (G4-E24/50/BMP), defect filled with elastin membrane prepared for 24 h at 50°C and BMP. Group 5 (G5- E96/37), defect filled with elastin membrane prepared for 96 h at 37°C. Group 6 (G6- E96/37/HA), defect filled with elastin membrane prepared for 96 h at 37°C and hydroxyapatite. Group 7 (G7-E96/37/BMP) defect filled with elastin membrane prepared for 96 h at 37°C and BMP. After painless death induced after 6 weeks, the femoral anatomical pieces were removed for macroscopic, radiographic, histological, morphometric and biomechanical analysis. The mean and standard deviations of the relative percentage volume of newly formed bone in the femoral defect were respectively 36,39±3,86 (G1); 66,40±3,69 (G2); 55,12±2,23 (G3); 58,46±1,79 (G4); 51,36±3,15 (G5); 71,28±4,26 (G6); 45,54±3,98 (G7). The findings demonstrated biocompatibility, biodegradability and osseous regeneration greater than the control group in the studied biomaterials. The groups with the highest newly formed bone volume were G2-E24/50 and G6-E96/37/HA.

Degeneration of the lumbar intervertebral disc, a condition widely implicated in the cause of low back pain among adult humans, is typically characterised by progressive biochemical and structural changes to the extracellular matrix. Comprehensive descriptions of the structural and functional inter-relationships within the extracellular matrix are therefore critical to understanding the degenerative process and developing effective treatments. In the anulus fibrosus, this matrix has a complex, hierarchical architecture comprised of collagens, proteoglycans, and elastic fibres. Elastic fibres are critical constituents of dynamic biological structures that functionally require elasticity and resilience. Studies to date of elastic fibre network structure in the anulus fibrosus have been qualitative and limited in scope. Additionally, there is poor understanding of the structural and functional associations between elastic fibres and other matrix constituents such as collagen, and, critically, there have been no studies directly examining the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical behaviour. In this thesis, multiple experimental studies are described that specifically examine each of these areas. Novel imaging techniques were developed and combined with histochemistry and light microscopy to facilitate the visualisation of elastic fibres at a level of detail not previously achieved. Examination of elastic fibre network structure revealed architectural differences between the intralamellar and interlamellar regions, suggesting that elastic fibres perform functional roles at distinct levels of the anulus fibrosus structural hierarchy. The density of elastic fibres within lamellae was found to be significantly higher in the lamellae of the posterolateral region of the anulus than the anterolateral, and significantly higher in the outer regions than the inner, suggesting it may be commensurate with the magnitude of the tensile strains experienced by each region of the disc in bending and torsion. The nature of the structure-function associations between elastic fibres and collagen was then examined with respect to the reported structural mechanisms of collagen matrix tensile deformation. Histological assessment of collagen crimp morphology in specimens from which elastic fibres had been enzymatically removed revealed no observable differences when compared with controls, suggesting that any contribution made by elastic fibres to maintaining crimp is minimal. Elastic fibres in anulus fibrosus specimens subjected to radial tensile deformations exhibited complex patterns of re-arrangement, suggesting that they maintain cross-collagen fibre connectivity. Elastic fibres were also observed to maintain physical connections between consecutive lamellae undergoing relative separation. Finally, the nature and magnitude of the contribution made by elastic fibres to anulus fibrosus mechanical properties at the tissue level was investigated using a combination of biochemically verified enzymatic treatments and biomechanical tests. Targeted degradation of elastic fibres resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. These results suggest that elastic fibres enhance the mechanical integrity of the anulus fibrosus extracellular matrix in the direction perpendicular to the plane containing the collagen fibres. In summary, the results of the studies presented in this thesis provide important new insights into the structure and function of the anulus fibrosus elastic fibre network, and highlight its potential importance as a contributing or ameliorating factor in the progression of the structural and mechanical changes associated with intervertebral disc degeneration. Additionally, these results establish an improved framework for the development of more accurate analytical and finite element models to describe disc behaviour. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317006 / Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2008