Para testar a hipótese de que a desmineralização in situ das superfícies de contato enxerto-leito, e a forma como o enxerto é estabilizado ao leito, podem influenciar os mecanismos envolvidos na consolidação do enxerto, fragmentos ósseos de 10 mm de diâmetro foram removidos das metáfises proximais tibiais de 36 coelhos (Oryctolagus Cuniculus) e transplantados para uma área adjacente. Na tíbia esquerda dos animais, as superfícies de contato do enxerto e do leito hospedeiro foram desmineralizadas com ácido cítrico pH 1,0 por 3 minutos antes dos enxertos serem fixados ao leito. Na tíbia direita, o transplante do bloco ósseo não foi precedido de desmineralização. Metade dos enxertos foi imobilizada sobre o leito pela superposição de uma membrana não reabsorvível de politetrafluoretileno colada com cianoacrilato ao leito à distância da interface enxerto-leito. A outra metade dos enxertos foi fixada por um parafuso de titânio no centro do enxerto. Assim, foram formados 4 grupos de estudo: membrana (M), membrana + ácido (MA), parafuso (P) e parafuso + ácido (PA). Três animais de cada grupo forneceram espécimes para análise microscópica quantitativa e qualitativa aos 15, 30 e 45 dias de pós-operatório. A análise qualitativa demonstrou que não houve formação óssea na interface em nenhum espécime aos 15 dias e que nos demais períodos, em todos os grupos, a quantidade de tecido ósseo neoformado na interface e seu estágio de maturação aumentaram com o tempo. Ambos os métodos de fixação empregados foram eficientes em manter os enxertos em posição, porém a membrana promoveu menor reabsorção da estrutura do enxerto. A análise quantitativa computadorizada revelou que, aos 30 dias, os grupos MA e PA apresentaram maior área de formação óssea na interface (71,34 ± 12,03%; 56,74 ± 2,15% respectivamente) em relação aos grupos M e P (51,75 ± 11,02%; 43,95 ± 4,05% respectivamente) e superfícies de consolidação óssea mais extensas (93,41 ± 5,95%; 93,73 ± 4,96% respectivamente) do que os grupos sem tratamento ácido (73,49 ± 7,7%; 73,77 ± 11,77% respectivamente para M e P), sendo essas diferenças estatisticamente significantes (p<0,05). Aos 45 dias de pós-operatório, os grupos MA e PA (71,18 ± 8,9%; 58,97 ± 3,97% respectivamente) apresentaram resultados superiores aos grupos M e P (59,78 ± 11,28%; 46,08 ± 3,53% respectivamente) em relação à área de neoformação óssea na interface, porém essa diferença não foi significativa. Concluiu-se que a desmineralização ácida das superfícies contactantes nos enxertos ósseos autógenos em bloco na tíbia de coelhos promoveu a osteogênese na interface enxerto-leito e acelerou a consolidação dos enxertos. Além disso, quando o tratamento ácido foi associado ao uso de membrana como método de fixação, a consolidação e grau de reabsorção óssea foram otimizados. / In order to test the hypothesis that the demineralization \"in situ\" of contacting surfaces of bone graft/bone bed and the fixation method used for graft stabilization can influence the mechanisms involved in the consolidation of the graft, bone fragments of 10 mm in diameter were removed from the proximal tibial metaphysis of thirty-six male rabbits (Oryctolagus Cuniculus) and transplanted to an adjacent area. In the left tibia of the animals, the contacting surfaces of the graft and host bed were demineralized with citric acid pH 1.0 for 3 minutes before the grafts were fixed to the receptor bed. In the right tibia, the bone block transplantation was not preceded by demineralization. Half of the grafts were immobilized on the bone bed by a nonresorbable polytetrafluoroethylene membrane glued with cyanoacrylate adhesive to the host bed distant from the bone graft-bone bed interface. The other half of the grafts were fixed by a titanium screw in the center of the graft. Thus, four groups were formed: membrane (M), membrane + acid (MA), screw (P) and screw + acid (PA). Three animals from each group provided specimens for quantitative and qualitative microscopic analysis at 15, 30 and 45 days postoperatively. Qualitative analysis showed no significant bone formation at the interface in any specimen of the groups at 15 days and on the other periods in all groups, the amount of newly formed bone at the interface as well as the stage of bone maturation increased with time. Both fixation methods were effective in maintaining the graft in position, but the membrane resulted in less resorption of the graft. Quantitative analysis, performed by means of a computer program for image analysis, showed that at day 30, groups MA and PA, showed greater area of bone formation at the interface (71.34 ± 12.03%; 56.74 ± 2 15%) than groups M and P (51.75 ± 11.02%, 43.95 ± 4.05%) and more osseointegrated bone surfaces (93.41 ± 5.95%, 93.73 ± 4.96%) than those without acid treatment (73.49 ± 7.7%, 73.77 ± 11.77%), and these differences were statistically significant. At 45 days postoperatively MA and PA groups (71.18 ± 8.9%, 58.97 ± 3.97%) showed better results than the M and P groups (59.78 ± 11.28%, 46 , 08 ± 3.53%) compared to the area of new bone formation at the interface and osseointegrated surfaces, but these differences were not significant. It was concluded that the acid demineralization of contacting surfaces in autogenous onlay bone grafts in the tibia of rabbits promotes osteogenesis in bone graft-host bed interface and accelerates the consolidation of the grafts. Furthermore, the association of this surface treatment to the use of membrane/cyanoacrylate fixation method, optimizes the results regarding consolidation and degree of bone graft resorption.

Lai Yau Ming. / "August 2006." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 260-290). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

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.

[Truncated abstract] Intracellular communication between osteoclasts and osteoblasts is imperative to maintain bone integrity. A myriad of molecules are responsible for regulating osteoblast and osteoclast activity. In particular, it is well documented that osteoblast-derived factors are crucial in directly controlling osteoclast formation and function. Since bone formation is coupled to bone resorption, it would be expected that osteoclasts also have some role in regulating the growth and function of osteoblast cells. However, despite extensive research upon osteoclast and osteoblast biology, the mechanisms by which osteoclasts regulate osteoblast growth and function is not well understood. In an attempt to further elucidate the mechanisms by which osteoclasts and osteoblasts communicate, the technique of subtractive hybridisation was used to identify a novel osteoclastderived factor identical to that of mouse Seminal Vesicle Secretion VII (SVS VII). Previous characterisation of the gene in bone demonstrated that SVS VII was abundantly and specifically expressed by mature osteoclasts (Phan, 2004). Additional research hinted that SVS VII acted as a novel osteoclast-derived factor, that by paracrine mechanisms, targeted osteoblast function (Phan, 2004). However, it remained open as to whether the SVS VII molecule did uniquely target the osteoblast, and whether this interaction influenced bone formation in vivo. Therefore, this thesis endeavoured to functionally characterise the role of the SVS VII molecule in the bone environment. ... Further work is needed to identigy a clear consensus binding sequence, to determine the specificity of the interaction between SVS VII protein and each phage clone, and to isolate a specific binding partner for SVS VII. In conclusion, the studies of this thesis sought to characterise the significance of SVS VII expression by mature osteoclasts, relative to its effects on osteoblast behaviour, but failed to conclusively determine a role for SVS VII in bone. Given that the effects of SVS VII on in vitro osteoblast activity and function are minimal, it is doubtful that SVS VII primarily acts as a paracrine factor integral to osteoblast function. Therefore, these findings conflict with those presented previously (Phan, 2004). However, it was demonstrated that SVS VII treatment was associated with in vivo effect on the skeleton, suggesting that SVS VII may target other elements of the bone microenvironment. Via mechanisms not yet understood, which possibly involves additional factors of the bone 11 extracellular matrix, SVS VII may target a subset of osteoprogenitor cells within the bone environment and act to regulate their proliferation. Therefore, SVS VII may enhance osteogenic precursor cell number at sites of bone formation which would increase the pool of cells that can differentiate down the osteoblast linage and contribute to bone formation. In this regard, SVS VII might function in a manner homologous to the Ly-6 molecule Sca-1 and act as an important factor that maintains a balance between the bone formation and resorption process. Clearly, more work focusing on alternative facets of bone biology is needed to identify whether there is a significant role for SVS VII in skeletal tissue.

In order to investigate growth modifications of the temporomandibular joint (TMJ) during dentofacial orthopaedic treatment, various functional appliances have been used to prompt the mandible into a protrusive position in various animal experimental models. The general purpose of this project was (i) to test the effectiveness of a functional appliance specially designed for sheep; (ii) to clarify whether or not forward mandibular displacement in sheep is associated with faster and/or redirected condylar growth; (iii) to evaluate the sheep as a model for dentofacial orthopaedic research by comparing the similarities of mandibular condylar growth in sheep and humans; (iv) to detail the position of the mandible during forward mandibular posturing and the effects of mandibular forward displacement on modelling and remodelling of the mandibular condyle. The specific purpose of this project was to reveal whether functional appliance treatment increases the quantity of bone formed during the treatment, or changes the distribution of the bone, or both. Eight, 4-month old, castrated male Merino sheep were randomly assigned to experimental or control groups with 4 in each group. Cast functional appliances were fabricated for the animals in the experimental group. The treatment period was 15 weeks. Calcein (day 1) tetracycline (13 weeks) and alizarin red S (3 days before sacrifice) fluorochromes were administered to all animals. Dental casts, endosseous implant markers and cephalograms were used to analyse the 3-D displacement of the mandible. Undecalcified mid-sagittal sections of TMJ were used to evaluate the tissue responses induced by the appliances. Dynamic parameters of bone formation, static indices of bone-forming and resorbing activity as well as structural indices of trabecular bone were estimated using histomorphometry. The trabecular bone was sampled from two regions: (i) a subchondral region; (determined by 2nd and 3rd labels), believed to comprise bone newly-formed during the experimental period; and (ii) a central region (labelled by all the three fluorochromes), believed to comprise bone which existed before the experiment. The cortical bone was divided into anterior and posterior regions for analysis. The weight of the animals was measured monthly to monitor their growth. Metacarpus growth was also evaluated. During the experimental period, the animals were found to maintain their weight within the normal range and grew normally. The appliance was found to displace the mandible to a downward and forward position with a net condylar displacement of 2.4 mm. The observed adaptive responses in the TMJ induced by the appliances included; the condylar process was less tapered and rounder in the experimental group than in the controls, and anteriorly thickened condylar cartilage and a thickened compact bone layer along the anterior surface of the posterior wall of the glenoid fossa. The mandibular condylar growth vector in sheep was found to be in a postero-superior direction. Condylar growth in the control sheep during the experimental period varied from 8.8 to 11.9 mm, with the mean being 10.6 mm, which is quantitatively similar to two years of condylar growth in human adolescents. In the experimental sheep, the condylar growth varied from 8.5 to 13.3 mm, with the mean being 11.4 mm. When metacarpal growth and weight gain were taken into consideration using multivariant analysis, the coefficients for growth in the postero-superior and posterior direction were found to be high, with adjusted r2 as 0.84 and 0.82 respectively. The induced condylar growth was estimated to be largest in the posterior direction (2.3 mm), which is also similar to previous reports in humans. Regional differences in adaptive response within the mandibular condyle were found in this study. In the experimental group, bone volume fraction (BV/TV) of the subchondral regions decreased, although the specific bone surface and bone formation rates increased. This low BV/TV was associated with decreased trabecular thickness and increased trabecular separation. In the central region of the experimental group's condyle, BV/TV was unchanged. However, an increased osteoid surface (OS/BS) was defined when the eroded surface (ES/BS) was taken into consideration. The sheep were found to cope well with the experimental procedures and the appliance used in this study has been effective in inducing adaptive responses in the TMJ. Consequently, it is believed that the sheep is an appropriate animal model for quantitative histological analysis of the responses to functional appliance treatment. The first null hypothesis, that functional appliance treatment has no effect on bone matrix mineralisation was rejected. The second null hypothesis, functional appliance treatment has no effect on the mineralisation lag time, was rejected. The results indicated that the treatment effects of functional appliances involve reorganisation of the TMJ through bone modelling and remodelling. An important mechanism of functional appliance treatment is, therefore, suggested to be a change in the distribution of bone rather than an increase in the quantity of bone. Posterior rotation of the principle tensile strain angle (Et) suggested an posteriorly altered direction of the condylar growth. Increased new bone formation in the glenoid fossa suggested an anterior re-positioning of the temporomandibular joint. / Thesis (Ph.D.)--Dental School, 2002.

A new model of a critically-sized segmental femoral bone defect in rats was developed to enable in vivo imaging and facilitate post-mortem mechanical testing of samples. The critically-sized nature of the model was assessed and confirmed. The efficacy of sustained co-delivery of osteogenic (BMP-2 and TGF- Ò3) and angiogenic (VEGF) growth factors in promoting functional bone repair was assessed. Effects of scaffold modification in terms of geometry and composition were evaluated. The results indicated that co-delivery of BMP-2 and TGF- Ò3 resulted in a dose-dependent improvement in functional bone repair. Modification of the polylactide scaffold to include an absorbable ceramic component and a cored out geometry enhanced rate of union. Addition of VEGF to the scaffold treatment did not significantly impact revascularization of the defect site or functional repair of the bone defect. These data demonstrate that the complex environment of an acute bone defect requires different treatment strategies than simple ectopic models would suggest. A positive predictive correlation between bone repair parameters measured in vivo and mechanical functionality was established. The novel defect model demonstrated robustness and reproducibility. Implications for further research are discussed.

Non-healing bone defects have a significant socioeconomic impact in the U.S. with approximately 600,000 bone grafting procedures performed annually. Autografts and allografts are clinically the most common treatments; however, autologous donor bone is in limited supply, and allografts often have poor mechanical properties. Therefore, tissue engineering and regenerative medicine strategies are being developed to address issues with clinical bone grafting. The overall objective of this work was to develop bone tissue engineering strategies that enhance healing of orthotopic defects by targeting specific osteogenic cell signaling pathways. The general approach included the investigation of two different tissue engineering strategies, which both focused on directed osteoblastic differentiation to promote bone formation. In the first cell-based strategy, we hypothesized that constitutive overexpression of the osteoblast-specific transcription factor, Runx2, in bone marrow stromal cells (BMSCs) would promote orthotopic bone formation in vivo. We tested this hypothesis by delivering Runx2-modified BMSCs on synthetic scaffolds to critically-sized defects in rats. We found that Runx2-modified BMSCs significantly increased orthotopic bone formation compared to empty defects, cell-free scaffolds and unmodified BMSCs. This gene therapy approach to bone regeneration provides a mineralizing cell source which has clinical relevance. In the second biomaterial-based strategy, we hypothesized that incorporation of the collagen-mimetic peptide, GFOGER, into synthetic bone scaffolds would promote orthotopic bone formation in vivo without the use of cells or growth factors. We tested this hypothesis by passively adsorbing GFOGER onto poly-caprolactone (PCL) scaffolds and implanting them into critically-sized orthotopic defects in rats. We found that GFOGER-coated scaffolds significantly increased bone formation compared to uncoated scaffolds in a dose dependent manner. Development of this cell-free strategy for bone tissue engineering provides an inexpensive therapeutic alternative to clinical bone defect healing, which could be implemented as a point of care application. Both strategies developed in this work take advantage of specific osteoblastic signaling pathways involved in bone healing. Further development of these tissue engineering strategies for bone regeneration will provide clinically-relevant treatment options for healing large bone defects in humans by employing well-controlled signals to promote bone formation and eliminating the need for donor bone.

Multiwall carbon nanotubes (MWNTs) have extraordinary mechanical and thermal transport properties. They significantly improve the static and dynamic mechanical properties of acrylic orthopaedic bone cement when added to the dry cement polymer powder. Understanding the role MWNTs play on bone cement polymerization temperatures will lead to improved mechanical integrity of the cement-bone interface in joint arthroplasties. It was determined through thermal testing that MWNTs increased the polymerization time of the methylmethacrylate by 45-460% and decreased the peak exothermic temperature of bone cement with and without antibiotics. The flow of heat produced during polymerizing cement was reduced 25-85% with the addition of MWNTs to the cement powder. This decreases the probability of thermal necrosis and “hot” spots caused by high exothermic polymerization temperatures that can destroy the bone adjacent to the cement. These high temperatures also affect the potency and range of antibiotics used in arthroplasty. Isothermal and elution studies determined that MWNTs altered the heat flow and amount of antibiotic release from bone cement during polymerization. Antibiotic elution from bone cement containing MWNTs could match the elution seen in pure cement. The alteration of the flow of heat from bone cement leads to new options for heat-labile antibiotics in total joint arthroplasty.

Thesis--København Tandlægehøjskole. / Summary in Danish. Bibliography: p. 179-194.

Thesis--København Tandlægehøjskole. / Summary in Danish. Bibliography: p. 179-194.