A implantação de materiais vítreos e vitrocerâmicos bioativos representa estratégia terapêutica importante para se promover a formação de matriz extracelular mineralizada em defeitos ósseos críticos. Quando expostos a fluidos biológicos, estes biomateriais sofrem alterações químicas e topográficas de superfície que afetam as interações de células com sua superfície, reduzindo o espraiamento celular e alterando o padrão de marcação de proteínas do citoesqueleto. O objetivo deste estudo foi avaliar se as alterações no padrão de marcação para as proteínas citoesqueléticas actina e tubulina observadas in vitro em células osteogênicas sobre superfícies do vidro Bioglass® 45S5 e da vitrocerâmica Biosilicato®, são decorrentes de redução quantitativa na expressão do RNAm e das proteínas correspondentes. Células osteogênicas foram obtidas a partir da digestão enzimática de calvárias de ratos Wistar recémnascidos e plaqueadas sobre superfícies de Bioglass® 45S5, Biosilicato® e borosilicato (controle bioinerte) para a avaliação dos seguintes parâmetros: 1) detecção de actina e tubulina por microscopia de fluorescência; 2) expressão de RNAm para actina e tubulina por reação em cadeia da polimerase em tempo real (Real time PCR); 3) quantificação de actina e tubulina por ensaio imunoenzimático direto (ELISA), e 4) análise da morfologia celular por microscopia eletrônica de varredura (MEV). Aos 3 e 7 dias, células crescidas sobre borosilicato exibiam padrões de marcação para actina e tubulina típicos de células aderidas e espraiadas sobre substratos planos in vitro, enquanto que sobre Bioglass® 45S5 e Biosilicato® as células apresentavam áreas circulares destituídas de marcação para essas proteínas. Nos mesmos períodos, culturas crescidas sobre os materiais bioativos apresentavam alterações significantes da expressão de RNAm para actina e tubulina, embora fossem observadas apenas discretas variações na quantidade das proteínas correspondentes em relação ao borosilicato. Além disso, apenas para culturas crescidas sobre borosilicato observava-se correlação positiva entre RNAm e proteína e correspondência entre as observações por epifluorescência e os dados quantitativos. Aos 3 dias, imagens de MEV revelaram células aderidas e espraiadas sobre os materiais bioativos, parcial ou totalmente recobertas por acúmulos de material de aspecto semelhante ao da topografia do substrato, por vezes impedindo a visualização dos limites celulares. Com base nos resultados obtidos, conclui-se que as superfícies bioativas de Bioglass® 45S5 e Biosilicato® afetam a expressão de RNAm para actina e tubulina, mas não de proteína. Assim, as alterações nos padrões de marcação por fluorescência para essas proteínas devem ser atribuídas, pelo menos em parte, a acúmulos de material sobre as células, possivelmente decorrentes das reações de superfície a que estão submetidos Bioglass® 45S5 e Biosilicato® quando em contato com fluidos biológicos. / Bioactive glasses and glass-ceramics have been successfully applied in various therapeutic strategies to promote the formation of mineralized matrix in bone defects. The exposure of these materials to biological fluids results in chemical and topographical modifications that may affect the interactions of cells with the biomaterial surface, with potential effects on cytoskeletal protein expression and/or organization and cell spreading. The aim of the present study was to evaluate whether changes in the labelling pattern for the cytoskeletal proteins actin and tubulin in osteogenic cells cultured on bioactive Bioglass® 45S5 and Biosilicate® are due to altered mRNA and protein expression levels. Osteogenic cells were obtained by enzymatic digestion of newborn Wistar rat calvarial bone and plated on Bioglass® 45S5, Biosilicate® and borosilicate (bioinert control) for periods of up to 7 days. The following parameters were assayed: i) qualitative epifluorescence analysis of actin and tubulin distribution; ii) quantitative mRNA expression for actin and tubulin by real time polymerase chain reaction (real time PCR); iii) quantitative actin and tubulin expression by enzymelinked immunoabsorbent assay (ELISA), and iv) qualitative analysis of cell morphology by scanning electron microscopy (SEM). At days 3 and 7, cells grown on borosilicate showed typical actin and tubulin labeling patterns of adherent and spread cells on flat, rigid substrates, whereas those on Bioglass® 45S5 and Biosilicate® showed dark areas devoid of fluorescent signals for the cytoskeletal proteins. At the same time points, cultures grown on the bioactive materials showed significant changes in mRNA expression for actin and tubulin, although only slight differences in the amount of actin and tubulin were detected compared with borosilicate. Moreover, a positive correlation between mRNA and protein expression levels as well as a correspondence between epifluorescence imaging and the quantitative data were only detected for cultures grown on borosilicate. SEM analysis revealed that cells cultured on bioactive surfaces were partly or totally covered with material accumulations, whose characteristics resembled the ones for the substrate topography, and which, in some cases, prevented the visualization of the cell limits. In conclusion, Bioglass® 45S5 and Biosilicate® affect actin and tubulin mRNA levels, but not the corresponding protein expression, in osteogenic cell cultures. Thus, the observed changes in the labeling pattern for these proteins should be attributed, at least in part, to the accumulation of materials on the cell surface, likely due to substrate reactions that take place on Bioglass® 45S5 and Biosilicate® when exposed to the cell culture medium.

O objetivo do atual trabalho, foi correlacionar os eventos celulares e teciduais com a expressão das proteínas VEGF, BMP-7, RANKL e OPG durante a osteogênese ectópica e ortotópica, induzida pela matriz óssea (MO) e dentinária (MD) alogênica. Matrizes alogênicas desmineralizada em HCl a 0,6N, obtidas de fêmur e incisivo de ratos, fori implantada entre as fáscias musculares da coxa e em defeito trans-ósseo de 8mm de diâmetro nos ossos parietais. As análises radiográfica e histomorfométrica da neoformação óssea e, a imunohistoquímica e o western blotting para as proteínas VEGF, BMP, RANKL e OPG, mostraram que: a) o volume da região do enxerto nos sítios ortotópicos reduziu 19% em 42 dias; b) em ambos tipos de enxerto e locais de implantação, ocorreu formação de tecido cartilaginoso e ósseo; c) nos sítios intramusculares, a reabsorção da matriz alogênica e a remodelação do tecido cartilaginoso, ósseo e medular foi mais acelerado, em relação a implantação ortotópico; d) o aumento na densidade de volume dos vasos sanguíneos e no número de osteoblastos/osteócitos e osteoclastos ocorreu simultaneamente e estava associado à maior reabsorção da matriz alogênica e à formação do tecido medular (hematopoiético); e) as proteínas VEGF, BMP-7, RANKL, OPG foram expressas em condrócitos, osteoblastos ativos, osteócitos recém aprisionados na matriz e em células estromais próximas aos osteoblastos ou às áreas da matriz alogênica reabsorvida; e f) a expressão das proteínas VEGF, BMP-7, RANKL e OPG foi maior no grupo MO. O pico de expressão dessas proteínas ocorreu nos períodos de 14 aos 21 dias no grupo da MO e 21 e 28 dias no grupo da MD. Concluímos que, a capacidade osteoindutora da matriz alogênica desmineralizada está relacionado a origem da matriz e ao sítio de implantação e que, as proteínas VEGF, BMP-7, RANKL e OPG estão associadas a maior reabsorção da matriz implantada, promovendo uma rápida e contínua liberação dos morfógenos contidos em seu interior que, induzem temporal e espacialmente a formação óssea/medular. / The aim of the present work was to correlate the cellular and tissue events with the expression of VEGF, BMP-7, RANKL and OPG during ectopic and orthotopic osteogenesis, induced by bone and dentin allogeneic matrix. Allogenic matrices obtained from femur and incisor of rats and demineralized in 0.6 N HCl were implanted into a intramuscular pocket and a 8mm-diameter bone defect in the skull. The radiographic and histomorphometric analysis of new bone formation, and immunohistochemistry and western blotting for VEGF, BMP, RANKL and OPG proteins, showed that: a) the total volume of the graft region in orthotopic site decreased 19% at 42 days b) in both graft types and implantation sites occurred formation of cartilaginous and bone tissues, c) in intramuscular sites, the resorption of allogenic matrix and remodeling of the new formed cartilage and bone were faster, in relation to orthotopic implantation sites; d) the increase in the volume density of blood vessels and in the number of osteoblasts/osteocytes and osteoclasts occurred simultaneously and was associated with greater reabsorption of the allogenic matrix and hematopoietic bone marrow formation; e) VEGF, BMP-7, RANKL, OPG proteins were expressed in chondrocytes, active osteoblasts, newly osteocytes confined and stromal cells located near the osteoblasts or in the surface of the reabsorbed matrix; and f) the VEGF, BMP-7, RANKL and OPG expression was higher in MO grafts than in the MD. The peak of expression of these proteins each occurred at 14 and 21 days in MO and 21 and 28 days in MD. We concluded that, the osteoinductive capacity of allogeneic demineralized matrix is related to matrix origin and implantation site and that the VEGF, BMP-7, RANKL and OPG proteins are associated with greater reabsorption of the implanted matrix, promoting rapid and continuous matrix-release morphogens that induces spatially and temporally the bone and bone marrow formation.

我们的研究是基于发现镁金属的成骨现象。在我们组之前的工作中，我们发现大鼠股骨骨髓腔内植入镁棒后，在很短时间内（一周后）就会在股骨骨膜下部位形成新骨。这种镁导致的成骨现象是怎样发生的，以及我们能否利用镁金属的这种特性去促进骨质疏松骨折的愈合？对这两个问题的解答便构成了本篇论文的主要内容。 / 因为镁诱导的新生骨产生在骨膜下方，并且植入镁棒产生的成骨现象在骨膜剥除的部位消失，所以我们认为骨膜是镁成骨的关键点。骨膜是富含感觉神经纤维和干细胞的组织，而且动物骨的感觉神经分布主要集中在骨膜（约占总的神经数量的99%以上）。骨膜神经末端不仅仅感知痛觉触觉和温度觉，而且在外界刺激下释放感觉神经递质。神经递质包裹于处于神经末梢的囊泡当中，CGRP 是经典的也是分布最广的感觉神经递质。在动物体内，血液中CGRP 的含量随年龄的增加而减少，同时骨内的镁含量也随之流失。这也是老年龄动物骨折愈合较慢的原因之一。所以我们提出本课题的研究假设：镁金属降解产生的镁离子作用于骨膜部位的感觉神经末梢，刺激神经递质CGRP 的释放。骨膜内增多的CGRP 作用于骨膜内的干细胞进行成骨分化，最后形成新骨。我们进一步检测镁的这种成骨作用能否促进骨质疏松鼠骨折的愈合。 / 首先我们用过量的辣椒素破坏大鼠股骨的感觉神经末端之后，镁的成骨显著减少，这说明镁的成骨作用相当程度上依赖于通过骨膜的神经组织。我们通过免疫组化染色及蛋白定量测定发现，植入镁后的骨组织内CGRP（降钙素基因相关肽，一种感觉神经末端分泌的主要神经递质）含量增加了一倍多。我们用CGRP 受体拮抗剂同样发现可以部分抑制镁的成骨作用。我们推测镁降解过程中产生的镁离子在骨膜部位增加了感觉神经递质的释放，骨膜部位增多的神经递质作用于骨膜源性间充质干细胞以及骨髓源性间充质干细胞想成骨方向分化成骨。体外试验结果表明，CGRP 在高浓度下显著促进骨膜及骨髓源性干细胞的成骨分化。我们从大鼠的脊髓腰段L3-5 背根神经节分离出背根神经节神经元，在体外用荧光对神经元内的突触小泡进行染色，发现当培养液中的镁离子浓度升高时（1-2mM），这些富含神经递质CGRP 的突触小泡不但数目增加，而且从胞体中心向轴突末梢迁移。在这个过程中，我们同时记录到显著的镁离子内流。实验结果表明，镁离子可以促进神经元的复极化以及神经递质向轴突末端迁移聚集，从而在下一次刺激中释放出更多的神经递质。同时体外干细胞分化实验结果表明高浓度的镁离子（5-10mM）显著促进干细胞的成骨分化。对干细胞和神经元的胞内镁离子内流检测发现，在胞外镁离子浓度升高的情况下，胞内镁离子内流主要通过一种膜通道MagT1。至此，关于镁成骨的机制可以归纳为：镁金属在降解过程中产生的镁离子作用于骨膜感觉神经末梢，使之释放出更多的神经递质，增加释放的神经递质和镁离子共同促进分布在骨膜和骨髓的干细胞进行成骨分化，从而增加成骨。 / 镁的成骨效应使之有很大的潜力用于骨质疏松骨折的修复。由于镁金属强度不足以直接用来固定大鼠骨折，所以我们设计了一种中空的不锈钢针管作为髓内骨折固定针。针管中部与骨折线对应的部位开出一些小孔，细的镁棒可以插入针管，在体内镁降解产生的镁离子可以从中部的小孔释放出去发挥其成骨效应，进而促进骨折的愈合。我们用卵巢切除大鼠进行闭合性骨折造模，然后用我们设计的髓内针固定。X 射线结果表明，手术后第二、四周镁治疗组骨折愈合组织的面积和宽度显著大于对照组。Micro-CT 扫描结果也同样表明，镁治疗组骨折部位愈合组织的总体积和骨组织体积在术后第四周显著大于对照组。组织学染色表明，在术后第二周，镁治疗组的骨折部位的骨膜内成骨大量增加，并且有大量间充质细胞充塞与骨折部位。第术后第四周，更多的软骨组织形成于镁治疗组的骨折部位。荧光双染色结果也表明，镁治疗组的骨折部位在第四周有更多的新生骨形成。第八周和十二周的偏振光图像表明，镁治疗组的骨折愈合部位形成的胶原纤维比对照组更规则且更多更亮。这说明在骨折愈合后期软骨内成骨以及编制骨向层状骨转化的过程中，镁治疗组的骨重建更加规则。最后在第十二周的力学实验结果证明，镁治疗组的骨干所能承受的最大压力显著高于对照组（大约增强了27%）。这部分体内试验证明镁金属可以加速并优化大鼠骨质疏松骨折的愈合，而且我们设计的中空含镁髓内针可以作为将来临床新型骨折髓内固定针的原型。 / 结论：我们对镁成骨的作用和其机制进行了比较深入全面的研究，并初步证实镁金属可以用于动物骨质疏松骨折的修复。我们的研究结果为将来镁金属在临床尤其是骨科领域的实际应用提供了一些基本的理论依据。 / In the rodent femur, almost 99% of all sensory nerves are distributed densely in the periosteum. Neuropeptides encapsulated in the synaptic vesicles are located at the axon terminals and released through exocytosis after being stimulated at the sensory nerve endings. The neuropeptides released from nerve endings have an osteo-anabolic effect on osteoblasts. Among the many kinds of neuropeptides, which include α-calcitonin gene-related peptide (CGRP), substance P, and other amino molecules, CGRP is the classical and dominantly distributed peptide in sensory nerve endings. In aged animals, decreased serum CGRP and loss of bone Mg content may be the factors inhibiting fracture healing. / In this study, Mg was found to significantly promote new bone formation in the subperiosteal cortical region after it was intramedullarily implanted in the rat femur canal. Histomorphological analysis revealed that the newly formed bone grew from periosteum, a fibrous membrane constituted of blood vessels, sensory serves, and mesenchymal stem cells, and did not form any cartilage-like tissue, the latter of which is a feature of intramembranous ossification. Observation that Mg-induced new bone formation disappeared at the periosteum-stripped region revealed the existence of an interaction between the periosteum and Mg ions. / Based on previous findings, this study examined the following hypotheses: (1) Mg ions from Mg implanted in the rat femur canal act on sensory nerve endings in the periosteum and promote neuropeptide CGRP release, (2) mass CGRP release in the periosteum promotes periosteum-derived stem cells osteoblastogenesis and leads to new bone formation. Mg ions affect synaptic replasticity in dorsal root ganglia neurons, and (3) pure Mg metal affects fracture healing in ovariectomized (OVX) rats. / Neuropeptide CGRP plays a pivotal role in Mg-induced new bone formation. This hypothesis was supported by femur bone analysis showing that CGRP content significantly increased in Mg-implanted femur bone compared to control femur bone. When rat sensory nerves were destroyed by administration of high-dose capsaicin, induction of new bone formation by Mg implantation significantly decreased, proving that sensory nerves play an important role in Mg-induced osteogenesis. Because neuropeptide CGRP from sensory nerve endings may play a pivotal role in Mg’s osteogenic process, the effective CGRP antagonist BIBN4096bs was administered to Mg-implanted rats. Administration of the CGRP antagonist significantly reduced newly formed bone volume after Mg implantation. To examine whether this phenomenon is dependent on the interaction between neuropeptides and MSCs, which are richly distributed in the periosteum, periosteum-derived stem cells (PDSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) were isolated from the periosteum and bone marrow, respectively. It was observed that high concentrations of CGRP significantly promoted osteogenic differentiation in both PDSCs and BMSCs while high concentrations of CGRP had an obvious chemotaxis effect on BMSCs. / Mg increases CGRP release by affecting DRG neurons. The results of immunochemical staining and ELISA CGRP quantification analysis of femur samples showed that femur CGRP content in Mg-implanted samples was almost twice that of controls. Previous studies reported that Mg ions could promote neural synaptic replasticity in hippocampus neurons in vitro. This study examined the hypothesis that Mg ions could promote synaptic replasticity in DRG neurons. The neural synaptic vesicles, which contain neuropeptides of DRG neurons, including CGRP, derived from the L3-5 dorsal root ganglion were stained in vitro. The synaptic vesicles were found to significantly increase in number when their medium was changed from Mg-free medium to Mg-rich medium of 1 mM and 2 mM and to migrate from the neuron body to its axon terminals. These results proved that Mg could facilitate neuron replasticity and prompt synaptic vesicle aggregation at axon terminals, indicating that much neuropeptide release occurs after stimulation. Real-time recording of the intracellular Mg signal revealed that DRG neuron Mg influx significantly increased after Mg medium had been added and that Mg influx into neurons was mainly through the membrane Mg ion channel MagT1. Implantation of Mg ions (MgCl₂) of high concentration was found to promote stem cell (PDSCs and BMSCs) osteogenic differentiation. Although the mechanism of Mg’s osteogenic effect on stem cells was not thoroughly studied, cellular Mg influx was found to increase in high-Mg medium through the membrane ion channel MagT1. / Mg accelerated bone fracture in ovariectomized rats. Mg metal is too soft to repair bone fracture in animal models. To overcome this challenge, we designed a novel intramedullary nail containing Mg to accelerate osteoporotic bone fracture healing in ovariectomized (OVX) rats. The novel nail is a hollow stainless steel needle with several interlacing arranged holes drilled midway through the needle. The Mg pin is inserted into the needle canal and Mg ions released through the holes on the needle reach the fracture line during degradation in vivo. Our findings indicate that use of this Mg-containing intramedullary nail could accelerate bone fracture healing in OVX rats. Review of post-surgery X-ray results showed that the fracture callus of the Mg-treated group was significantly larger than that of the control group at weeks 2 and 4. Review of micro-computed tomography (micro-CT) scanning images indicated that both the total volume and area of callus bone in the Mg-treated group exceeded those of the control group at week 4. However, no significant difference was found between the two groups regarding callus area and volume at week 12. / Histomorphological analysis showed a wider intramembranous ossification area and woven bone area in the Mg-treated group at weeks 2 and 4 and more cartilage tissue at the callus site in the Mg-treated group at week 4. Double fluorescence labeling staining revealed more densely stained newly formed bone in the Mg-treated group than the control group at week 4, indicating accelerated callus bone formation in the Mg-treated group. The callus was observed to be undergoing endochondral ossification and woven bone remodeling at weeks 8 and 12. Review of polarized light images showed brighter and more regularly arranged collagen fibers in the Mg-treated group compared to the control group. Biomechanical testing at week 12 revealed that the ultimate load of shaft bone in the Mg-treated group had increased 30% more than that of the control group. These results indicate that the novel Mg-containing intramedullary nail designed in this study could significantly accelerate and optimize osteoporotic fracture healing in OVX rat model. / Significance: The results of this study contribute to a thorough understanding of the osteogenic effect of Mg by explicating its bioeffect on neurons and stem cells. The novel Mg-containing intramedullary nail designed in this study appears promising in osteoporotic fracture healing and to have many potential clinical applications. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Yifeng. / Thesis (Ph.D.) Chinese University of Hong Kong, 2015. / Includes bibliographical references (leaves 170-180). / Abstracts also in Chinese.

Novel surgical strategies for spinal disc repair are currently being developed that require materials that (1) possess the appropriate mechanical properties to mimic the tissue the material is replacing or repairing and (2) maintain their mechanical function for long durations without negatively affecting the tissue response of adjacent tissue (i.e. bone). Polymers formed through photopolymerization have emerged as candidate biomaterials for many biomedical applications, but these materials possess limited toughness in vivo due to the presence of water inherent in most tissues. Therefore, the overall objective of this research was to develop photopolymerizable (meth)acrylate networks that are both mechanically and biologically compatible under physiological conditions to be implemented in spinal repair procedures. The fundamental approach was to determine structure-property relationships between toughness and network structure in the presence of phosphate buffered saline (PBS) using several model copolymer networks in order to facilitate the design of photopolymerizable networks that are tough in physiological solution. It was demonstrated that networks toughness could be optimized in PBS by tailoring the Tg of the copolymer network close to body temperature and incorporating the appropriate "tough" chemical structures. The ability to maintain toughness up to 9 months in PBS was dependent upon the viscoelastic state and overall hydrophobicity of the network. In tandem, the effect of network chemistry and stiffness on the response of MG63 pre-osteoblast cells was assessed in vitro. The ability of MG63 cells to differentiate on (meth)acrylate network surfaces was found to be primarily dependent on surface chemistry with PEG-based materials promoting a more mature osteoblast phenotype than 2HEMA surfaces. Amongst each copolymer group, copolymer stiffness was found to regulate osteoblast differentiation in a manner dependent upon the surface chemistry. In general, photopolymerizable (meth)acrylate networks that were deemed "tough" were able to promote osteoblast differentiation in a manner comparable if not exceeding that on tissue culture polystyrene (TCPS). This research will impact the field of biomaterials by elucidating the interrelationships between materials science, mechanics, and biology.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

The trend for designing of a titanium implant explored using different chemical compositions and crystallinity materials until people realized that the implant surface character was another important factor affecting the rate and extent of osseointegartion. Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5μm. An additional mesoporous titania top layer was created that followed the contour of the macropores and having 100–200 nm thickness and a pore diameter of 10 nm. Thus, a coherent laminar titania surface layer was obtained producing a hierarchical macro- and mesoporous surface. The interfacial bonding between the surface layers and the titanium matrix was characterized by a scratch test that confirmed a stable and strong bonding of the laminar titania surface layers upon titanium. The wettability to water and the effects on the osteosarcoma cell line (SaOS-2) proliferation and mineralization of the formed titania surface layers were studied systematically by cell culture and scanning electron microscopy (SEM). A synergistic role of the hierarchical macro- and mesoporosities was revealed in terms of enhancing cell adhesion, proliferation and mineralization, when compared with the titania surface with solo porosity scale topography. For the in vivo results of the evaluation of osseointegration, an argon ion beam polishing technique was applied to prepare the cross sections of implants feasible for the high resolution SEM investigation. The interfacial microstructure between newly formed bone and implants with four modified surfaces including the new hierarchical macro- and mesoporous implant surface retrieved after in vivo tests were characterized. By this approach it has become possible to directly observe early bone formation, the increase of bone density, and the evolution of bone structure. The two bone growth mechanisms, distant osteogenesis and contact osteogenesis, can also be distinguished. These direct observations give, at microscopic level, a better view of osseointegration and explain the functional mechanisms of various implant surfaces for osseointegration. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: Manuscript.</p>

Distraction Osteogenesis (DO) is a technique for bone lengthening and filling of bone defects following trauma, infection or resection of tumors. DO consists of an osteotomy of the bone to be lengthened, followed by controlled distraction of the bone segments with an external fixator until the desired lengthening is obtained (distraction phase). This is followed by the consolidation phase, during which the external fixator is kept in place until the newly formed bone in the distracted zone consolidates. This phase is long and may cause numerous problems. Ongoing research aims at finding a method to accelerate the consolidation of the newly formed bone. / Fibroblast Growth Factors (FGF) play a significant role in bone development and repair. FGF 18 has been shown to be the only FGF member to be expressed throughout both the distraction and the consolidation phases of DO. It was also reported that FGF18 is the physiological ligand of FGFR3. Therefore, we hypothesized that FGF18 and FGFR3 may have an important role in DO. / To test this hypothesis, we investigated DO in FGFR3 deficient mice (FGFR3-/-). (FGF18 deficient mice are not viable). A miniaturized DO apparatus was applied to the tibia followed by an osteotomy. Distraction began after a 5-day latency period at a rate of 0.2 mm/12 hours for 12 days. / Samples were collected at 3 time points comparing the mutants (FGFR3-/-) to their wild type litter' sates: end of distraction (17 days post-surgery), mid-consolidation (34 days post-surgery), and end of consolidation (51 days post surgery). The samples were analyzed using X-ray, DEXA, microCT, histology, biomechanical testing and Real-Time PCR. / Our results revealed that FGFR3 deficient mice showed accelerated bone formation compared to the W.T. littermates at mid-consolidation where the parameters measured revealed increased bone mineral density, bone mineral content and trabecular number in the mutant tibial samples. The newly regenerated bone consolidated faster in the FGFR3 knock-out mice and the bone was of better quality as revealed by biomechanical tests in which more force was needed to break the mutant bone because it exhibited higher resistance than the age matched wild-type sample. The marker gene expression patterns revealed an up-regulation of chondrogenic markers that suggest that the knock-out mice follow the endochondral ossification pathway during DO. All results were statistically significant. / These results show that signaling through FGFR3 acts to decrease bone formation during DO. Consequently, blocking FGFR3 may lead to accelerated bone formation in DO. This may have important clinical implications in attempts to improve the functional outcome of DO by decreasing the long duration that the external fixator has to be kept on.

Alternative bone repair strategies are frequently sought after in orthopaedic surgery to address the growing need for improved morbidity and healing rates. This thesis sought to initiate and validate such an alternative, harnessing the flexible nature of a biomaterial substrate and the unique potential of glycosaminoglycan sugars. A novel, biodegradable biomaterial polymer, PHBV, has previously been identified to have the potential to mimic the characteristics of bone necessary for tissue repair and in this study, it was hypothesized that PHBV would be able to support bone formation. When tested in vitro, PHBV was found to support osteoblast cell attachment, proliferation and differentiation, despite its rougher, more hydrophobic surface characteristics compared to tissue culture plastic (TCP). However, unlike the progression of cells on TCP, PHBV caused a developmental delay at each stage of osteogenesis, suggesting a sub-optimal cell-substrate interaction. The expression profiles of genes involved in the maintenance of the extracellular matrix were monitored to investigate this phenomenon further. The results suggested that cells cultured on PHBV appeared to preference 7 against a collagen-based ECM and, instead, trigger an increase in the expression of other factors, such as osteopontin, presumably to modify the biomaterial microenvironment to optimise continued growth and differentiation. This finding led to the next hypothesis that functionalisation of PHBV with suitable compounds could optimise and enhance the osteogenic development at the implant site by facilitating the desired and appropriate cell-substrate interactions. Non-protein factors are often preferred for functionalisation to material scaffolds over proteins, as they are relatively robust and can survive many of the processes used in the manufacture of biomaterials. Glycosaminoglycan (GAG) sugars were appropriate candidates for this purpose, as they are not only abundantly expressed in bone, but more importantly, they are capable of binding and facilitating the activity of growth factors. Furthermore, they are resistant to several environmental influences including changes in pH, heat and desiccation. To identify a GAG that could be integrated with PHBV or any other biomaterial substrate, GAGs were extracted from phenotypically-distinct stages of MG-63 osteosarcoma cells. These GAGs were identified to display gross structural differences, as well as differences in the enzymes synthesising them, between immature and mature osteoblastic cells, with the increased production of a larger GAG species observed as the cells differentiated. Unexpectedly, however, when these GAGs were subsequently dosed back into the media of growing MG-63 cells, their bioactivity did not match the stage at which they had been harvested: all GAG species were able to influence cell survival and growth to varying degrees but were not capable of affecting cell differentiation. However, if these same GAGs were exposed to cells by first being attached to the growth substrate, they induced varying degrees of aggregation in human mesenchymal stem cells (hMSCs), with more mature GAGs producing the most profound effects. Interestingly, a similar phenomenon was not observed when MG-63 cells where cultured in a similar manner. A direct correlation between the GAGs expressed by osteoblasts and the specific cellular processes they functionally influence has yet to be identified. While the experiments presented here demonstrate an effect of GAGs in osteoblastic cell survival, a role for GAGs in the progression of bone formation was not revealed. Loss-of-function studies were therefore necessary to determine the role of GAGs in bone, but this was hampered by the limited availability of procedures that allow the alteration of GAGs and the subsequent detection of these effects. Therefore, a tool to screen the efficacy of a loss of GAG function was developed. TAT-EGFP, a purpose-designed fluorescent GAG-binding peptide, was able to confirm that treatment with sodium chlorate was an effective 8 strategy to hinder GAG expression in MG-63 cells with minimal cytotoxicity to the cells. Following more extensive studies with chlorate treatment, it was found that a recoverable disruption to both proliferation and mineralisation could be induced in MG-63 cells. This suggested a role for GAGs in osteogenesis. A series of experiments then carried out following gene expression microarray analysis indicated that GAG de-sulfation by chlorate gives rise to an S-phase block in the cell cycle and a disruption to the actin cytoskeleton, which appeared to be associated with a change in the activity of cell-surface proteoglycans, most likely syndecan 4. It was also found that cells up-regulated plasma membrane ALP activity and cholesterol synthesis, presumably in an attempt to recover from a chlorate-induced loss in GAG function. Cholesterol is known to be important in establishing connections between membrane elements and the actin cytoskeleton, and its up-regulation here may reflect dysfunctions in these units and a dysfunction in syndecan 4 activity. With further confirmation, this would suggest that syndecan 4 plays a pivotal role in maintaining osteogenesis, in at least MG-63 cells, and that sulfated GAGs function principally to facilitate this role. The effective use of GAGs in bone repair strategies will require further understanding of GAG/syndecan 4/osteogenesis relationship.

Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 31, 2007). Includes bibliographical references (p. 42-45).

Orientador: Rosemary Adriana Chierici Marcantonio / Resumo: Trabalhos in vivo têm utilizado a microtomografia computadorizada para avaliar a qualidade e quantidade de tecido ósseo presente durante o desenvolvimento de doença periodontal induzida. No entanto, ainda não foram estudados quais os melhores e mais confiáveis padrões estabelecidos para o adequado uso da técnica de Micro CT. O objetivo deste estudo foi avaliar a geração de artefatos e ruídos durante o escaneamento de biópsias ósseas bem como, analisar a influência de diferentes parâmetros estabelecidos durante a mensuração de área e volume ósseo, em modelo experimental de doença periodontal induzida em ratos. Para análise dos diferentes parâmetros de escaneamento com o Micro CT foram utilizados 05 animais aleatoriamente selecionados e submetidos a inserção de ligaduras na região subgengival dos segundos molares superiores. Após o período de 7 dias os animais foram eutanasiados e as biópsias ósseas escaneadas utilizando resoluções de 9 µm e 18 µm, com diferentes filtros: alumínio (0,2 mm, 0,5 mm e 1 mm) e cobre+alumínio (Cu 0,04 mm + Al 0,5 mm). Posteriormente foram definidas duas regiões de interesse (ROI's) ao redor dos segundos molares, de modo que foi somente analisado o tecido ósseo. Após determinação da área de interesse, a quantidade de tecido ósseo foi avaliada e quantificada por meio do software CTAn, a partir de 5 valores de thresholds: 255-75, 255-80, 255-85, 255-70, 255-65. Quando diferentes tresholds foram analisados dentro de um mesmo filtro, somente o filtro de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In vivo studies have used a computerized microtomography (Micro CT) to assess a quality and amount of tissue present in the development of induced periodontal disease. However, have not yet studied which are the most reliable standards established for the proper use of the Micro CT technique. The objective of this study was to evaluate the generation of artifacts during biopsy scanning, as well as the analysis of different parameters defined in an area and bone volume measurement in an experimental model of periodontal disease induced in rats. To analyze the different detection parameters of microcomputers with selected animals and submitted to insertion of ligatures in the subgingival region of the upper second molars. After the 7-day period the animals were used as bone biopsies scanned using resolutions of 9 μm and 18 μm, with different filters: aluminum (0.2 mm, 0.5 mm and 1 mm) and copper + aluminum (Cu 0 04 mm + 0.5 mm). Subsequently, two regions of interest (ROI's) were defined around the second molars, in the way the bone tissue was analyzed. After determining the area of interest, the amount of bone tissue was evaluated and quantified using the CTAn software, from 5 threshold values: 255-75, 255-80, 255-85, 255-70, 255-65. When different thresholds were analyzed within the same filter, only the 0.2 mm Al filter presented statistical difference for all comparisons (9 μm and 18 μm). Already when the different filters with the same threshold were compared, it is only one comparison Al 0.2 mm x Cu + Al presented statistical difference for all ...(Complete abstract electronic access below) / Mestre