Knochen ist ein multifunktionales Organ und zugleich ein biologisches Material. In dieser Arbeit wird der Heilungsverlauf eines Knochenbruchs (als biologisches Material) näher untersucht mit Hilfe von Computermodellen. Im menschlichen Körper kommt es nach einem Bruch zu einer vollständigen Regeneration des Knochens, ohne dass eine Narbe nach der Heilung zurückbleibt. In grob 10% der Frakturen kommt es jedoch zu Komplikationen bis zu einem Nicht-Heilen des Bruches. Das Ziel von intensiver interdisziplinärer Forschung ist es daher, nicht nur die medikamentöse Behandlung solcher Komplikationen zu verbessern, sondern auch durch externe, biophysikalische Stimulation die Heilung anzuregen. Gewöhnlich heilt ein Knochenbruch nicht direkt (Primäre Knochenheilung), das heißt durch Bildung von neuem Knochen im Knochenspalt, sondern über Sekundäre Knochenheilung. Während der sekundären Heilung bildet sich vorübergehend zusätzliches Gewebe außerhalb des Frakturspaltes, der so genannte Kallus, der die Aufgabe hat, den Bruch zu stabilisieren. Im Kallus werden im Laufe der Heilung verschiedene Gewebearten gebildet (z.B. Bindegewebe, Knorpel und Knochen). Die Gewebe werden von spezialisierten biologischen Zellen gebildet. Die spezialisierten Zellen entwickeln sich aus mesenchymalen Stammzellen (d.h. sie differenzieren), die in den Kallus wandern. Hauptziel der Arbeit ist das bessere Verständnis der mechano-biologischen Regulation der Gewebeformation während der Heilung eines normalen Knochenbruches. Dazu wurden Computersimulationen durchgeführt und mit experimentellen Daten eines Schafmodels verglichen. / Bone is a multifunctional organ, a biological material and is able to fully restore bone fractures without leaving a scar. However, in about 10% of the bone fractures, healing does not lead to a successful reunion of the broken bone ends. Intensive interdisciplinary research therefore looks for new ways to promote healing not only by medication, but also by external biophysical stimulation. Usually, bone fractures do not heal by a direct bridging of the fracture gap with newly formed bone (primary bone healing). Instead, secondary bone healing proceeds indirectly via the formation of an external callus (additional tissue). Within the callus, intricate tissue type patterns are formed, which evolve during the healing progression. Stem cells differentiate into specialized cells, which lay down different tissues such as fibrous tissue, cartilage and bone. This cell differentiation can be biophysically stimulated, e.g. by mechanical deformation of the cytoskeleton. The main aim of this thesis was to connect the microscopic cell response to mechanical stimulation with the macroscopic healing progression. Simple rules for cell behaviour were implemented in a computer model, the progression of healing was simulated and the outcome of the simulations was compared to results from animal experiments. In comparison to existing simulations of bone healing, this study approached the problem from a more physical viewpoint and linked experimental in vivo data and computer modelling.

Master of Science / Department of Clinical Sciences / David E. Anderson / Drug delivery systems for time release of recombinant human bone morphogenetic protein-2 (rhBMP-2) and antibiotics in orthopedic surgeries continue to be developed. Recently, a biodegradable novel polymeric matrix has been developed for this purpose. We hypothesized that impregnation of the matrix with rhBMP-2 would enhance bone healing. The objectives of the study were to characterize elution of rhBMP-2 and two antimicrobials (tigecycline, tobramycin) from the matrix, and bone response to the matrix in the presence or absence of rhBMP-2 and antimicrobials. In vitro elution of tigecycline, tobramycin, and rhBMP-2 from the matrix was investigated. Drug concentration in media were measured on days 1-6, 8, 10, 13, 15, 17, 21, 25, 28, and 30 using high pressure liquid chromatography/mass spectrometry/mass spectrometry (HPLC/MS/MS; antimicrobials) and ELISA (rhBMP-2). In vivo testing was done using a unicortical defect created into each tibia of twenty adult goats. Animals were randomly assigned to one of 5 groups: 1) control (untreated defect); 2) matrix; 3) matrix+ antimicrobials (tigecycline+tobramycin); 4) matrix+rhBMP-2; and 5) matrix+antimicrobials+rhBMP-2. Plasma concentration of tigecycline and tobramycin and serum concentration of rhBMP-2 were measured by the above techniques on days 1-7, 9, 11, 13, 15, 17, 22, 26, and 30. Bone response was assessed on days 0, 14, and 30 using radiographic scoring and dual energy X-ray absorptiometry (bone mineral density [BMD]). After euthanasia on day 30, histomorphologic analyses of the bone defects were done. Categorical variables were analyzed using a generalized linear model, and continuous variables using an ANOVA with P < 0.05 considered significant. In vitro elution was characterized by a rapid release on day 1 followed by a slow release until day 30 for both antimicrobials and rhBMP-2. Plasma antimicrobial concentrations showed continued release throughout the study period. Serum rhBMP-2 concentration, radiographic scores and BMD were not significantly different between groups. Periosteal and endosteal reaction surface areas were significantly greater surrounding the defects in group 4 (matrix+rhBMP-2). There was no significant difference between the groups for the percent of bone filling the defect. The matrix served as an appropriate antimicrobial and rhBMP-2 delivery system and successfully stimulated bone production when rhBMP-2 was present.

Fundamento: Fratura é ocorrência frequente em lesados medulares, em virtude da acentuada osteoporose, o que compromete o esqueleto como um todo e a consolidação óssea da fratura, em particular, o que resulta em calo ósseo de má qualidade, ou mesmo surgirem anomalias de consolidação. Assim, são importantes a busca e a avaliação de intervenções terapêuticas que possam melhorar o reparo ósseo nessa população. Objetivo: Avaliar os resultados da administração de alendronato de sódio, paratormônio e ultrassom de baixa intensidade na qualidade do calo ósseo de fraturas diafisárias do fêmur de ratos previamente submetidos à lesão medular. Material e Métodos: 75 ratos machos foram distribuídos em 05 grupos (n=15): (1) CON: ratos normais submetidos à fratura do fêmur; (2) LM: ratos com lesão medular e fratura; (3) LM+ALE: ratos com lesão medular, fratura e tratamento com alendronato de sódio; (4) LM+PTH: ratos com lesão medular, fratura e tratamento com paratormônio e (5) LM+US: ratos com lesão medular, fratura e tratamento com ultrassom. A medula espinhal foi completamente seccionada no nível da décima vértebra torácica (T10). No grupo controle (Sham) a medula foi apenas exposta, mas não seccionada. A fratura do fêmur foi realizada 10 dias após a produção da lesão medular. Sua localização foi na diáfise femoral, seguida de fixação com fio de aço no canal medular. A terapêutica em cada grupo foi aplicada durante as duas semanas seguintes à fratura. A avaliação da qualidade óssea e do calo foi feita por imagens de DEXA, microtomografia computadorizada (?CT), histologia, histomorfometria, imunoistoquímica (OPG, RANK e RANKL), resistência mecânica, PCR em tempo real para avaliar a expressão gênica e ELISA (OPG e IGF-1). Resultados: A lesão medular afetou gravemente a qualidade óssea com deterioração da microarquitetura óssea e redução da densidade mineral óssea, além de prejuízo da resistência mecânica. Houve redução da expressão dos genes de formação óssea e aumento da reabsorção, com alteração fenotípica. O processo de consolidação ocorreu predominantemente por ossificação intramembranosa nos animais paraplégicos. No entanto, a qualidade do calo foi pobre, em relação aos animais que não sofrem a lesão. Por outro lado, o uso de recursos terapêuticos como o alendronato de sódio, paratormônio e ultrassom de baixa intensidade foi capaz de recuperar muitas das propriedades microarquiteturais, celulares e mecânicas do osso não fraturado. De forma semelhante, osrecursos farmacológicos e mecânico utilizados neste estudo atuaram positivamente no processo de consolidação uma vez que houve aumento na densidade do calo, melhora da sua microarquitetura, e atividade celular, além do aumento na resistência aos esforços mecânicos. Em resumo, a lesão medular causou grave deterioração do tecido ósseo e modificações no processo de consolidação que, foram amenizadas e até normalizadas pela administração de alendronato de sódio, paratormônio e ultrassom. Conclusão: Os recursos terapêuticos acima citados foram benéficos para a recuperação do tecido ósseo intacto e para melhorar a qualidade do calo ósseo, após a deterioração causada pela lesão medular. / Background: Bone fracture is a frequent event in individuals with spinal cord injury (SCI) as a consequence of the marked reduction in bone mass and quality. SCI-induced bone loss may also induce changes in bone healing, leading to a poor callus formation and even non-unions. Therefore, the search for therapeutic interventions that may improve bone healing in this particular population may have a remarkable clinical importance. Purpose: To evaluate the influence of sodium alendronate, parathyroid hormone and low intensity ultrasound on the quality of fracture bone callus formation in SCI rats. Material and Methods: 75 male Wistar rats were randomly divided into five groups (n=15) (1) CON: control rats subjected to fracture, but not to spinal cord injury; (2) SCI: spinal cord injured rats and femur bone fracture; (3) SCI+ALE: spinal cord injured rats with bone fracture and treatment with sodium alendronate; (4) SCI+PTH: spinal cord injured rats with bone fracture and treatment with parathyroid hormone, and (5) SCI+LIPUS: spinal cord injured rats with bone fracture and treatment with low intensity ultrasound. A complete transection of the spinal cord was performed at the T10 level. In the control group the spinal cord was exposed but not sectioned. At 10 days post-injury (or Sham) a bone fracture was produced at the femoral shaft and fixed with an intramedullary pin. The ALE and PTH treatments began on day 1, and US began on day 3, after fracture and on day 14 all rats were euthanized. Non-fractured tibias and femur bone callus were analyzed by DXA, micro-computed tomography (?CT), histology, histomorphometry, immunohistochemistry (OPG, RANK and RANKL), mechanical test, real time PCR and ELISA (OPG and IGF-1). Results: SCI significantly impaired the bone quality, downregulated the osteoblastic-related gene expression and increased bone resorption, resulting in several phenotypic changes. SCI deteriorated the microarchitecture, reduced bone mass and weakened bone strength. Bone healing in SCI occurred predominantly by intramembranous ossification, leading to a poor callus formation. Conversely, the administration of alendronate, PTH and LIPUS were effective at ameliorating and even fully reestablish the bone microarchitecture, cells activity and mechanical strength. Similarly, both the pharmacological and mechanical therapeutics analyzed in this study were potentially efficient to increase bone callus density, to improve callus microstructure and metabolism, and to increase newly bone strength. In short,we revealed that SCI induced severe bone deterioration and changes in bone healing, which were ameliorated and even fully restored by the administration of alendronate, PTH, LIPUS and passive standing. Conclusions: The therapeutic approaches used in this study had great efficacy in restoring non-fractured bone integrity and with improvement of bone callus in paraplegic rats.

As prostaglandinas (PGs) são derivadas do metabolismo do ácido aracdônico pela via das enzimas cicloxigenases (COX-1 e COX-2) e participam do controle do metabolismo ósseo. Os anti-inflamatórios não-esteroidais (AINEs), inibidores das COX, podem interferir negativamente com a formação óssea, atrasando o reparo de fratura de ossos longos, a fusão espinhal e a osseointegração de implantes. O presente trabalho teve por objetivo avaliar, quantitativamente, o efeito de diferentes tipos de AINEs (convencional, preferencial e seletivo para COX-2), assim como de um analgésico com efeito anti-inflamatório fraco, sobre o reparo ósseo alveolar, em ratos machos. Os animais foram divididos em 5 grupos experimentais: a) controle (administração de 1 mL água/dia), b) cetorolaco de trometamina (inibidor não-seletivo COX-1/COX-2; ingestão de 4 mg/kg/dia), c) nimesulida (inibidor preferencial de COX-2; ingestão de 5 mg/kg/dia), d) paracetamol (efeito anti-inflamatório fraco; ingestão de 80 mg/kg/dia), e) etoricoxibe (inibidor seletivo de COX-2; ingestão de 10 mg/kg/dia). As dosagens foram baseadas em trabalhos experimentais da literatura e na equivalência com a terapêutica humana. A administração foi realizada por gavage gástrica, iniciando logo após a extração do incisivo superior direito e seguindo por um periódo de 14 dias, após o que os ratos foram sacrificados, as hemi-maxilas contendo os alvéolos em reparação foram coletadas e processadas para inclusão em parafina, orientada de maneira a permitir cortes semi-seriados longitudinais de 6 &mu;m de espessura (a intervalos de 60 &mu;m), que foram corados pela hematoxilina e eosina. O percentual de tecido ósseo neoformado foi estimado por método de contagem diferencial de pontos, utilizando-se um microscópio óptico munido de câmera digital de vídeo para captura de imagens e um programa para histometria. Foram contados cerca de 1200 pontos (1182,9 ± 47,6 pontos; média ± EPM) no terço cervical alveolar de cada animal, em cerca de 8 secções histológicas intercaladas (8,6 ± 0,3 secções; média ± EPM). Após a aplicação de teste que comprovou a normalidade de todas as distribuições (teste de Kolmogorov-Smirnov, p > 0,10) aplicou-se a Análise de Variância (gl=4; F = 1,52; x2 = 0,13), que comprovou que o tratamento com os diferentes tipos de AINEs não interferiu com a formação óssea reparacional, neste modelo experimental. Os presentes resultados, obtidos em animais, não devem ser diretamente extrapolados para humanos, nem conduzir a uma inferência sobre o uso de AINEs na clínica odontológica. No entanto, as numerosas evidências experimentais e clínicas de que os diferentes tipos de AINEs podem ter efeitos indesejados na clínica ortopédica, somadas ao número reduzido de estudos voltados especificamente para a área odontológica, indicam a necessidade de mais investigações nessa área, com variação dos parâmetros experimentais e das ferramentas de avaliação, antes que se descarte a possibilidade de que os AINEs possam ter efeitos indesejados em procedimentos odontológicos que necessitam de formação óssea, principalmente no caso de utilização prolongada. / The cyclooxygenase enzymes COX-1 and COX-2 catalyze the conversion of arachidonic acid to prostaglandins (PGs), eicosanoids important for control of bone metabolism. The non-steroidal anti-inflammatory drugs (NSAIDs), which are COX inhibitors, may negatively interfere with bone formation and delay long bone fracture healing, spinal fusion and implant osseointegration. The aim of the present study was to investigate quantitatively whether different types of NSAIDs (conventional, preferential and COX-2-selective), as well as an analgesic drug with a weak anti-inflammatory action, can hinder alveolar bone healing, in male rats. The animals were divided in 5 experimental groups: a) control (oral administration of 1 mL water/day), b) cetorolac (non-selective COX-1/COX-2 inhibito - 4 mg/kg/day oral dose), c) nimesulide (preferential COX-2 inhibitor - 5 mg/kg/day oral dose), paracetamol (weak anti-inflammatory - 80 mg/kg/day oral dose), etoricoxib (selective COX-2 inhibitor - 10 mg/kg/day oral dose). The doses of NSAIDs were compatible to human therapy and in the range of investigations carried out in laboratory animals. The drugs were administered by gavage from the day of extraction of the upper right incisors until death 2 weeks later, when the hemi-maxillae containing the alveolar sockets were collected, decalcified and processed for paraffin embedding. Semi-serial longitudinal 6-&mu;m-thick sections were cut at 60-&mu;m intervals and stained with hematoxylin and eosin. The degree of new bone formation inside the alveolar socket was estimated by a differential point-counting method, using an optical microscopy with a digital camera for image capture and a public domain histometry software. A total of 1182,9 ± 47,6 points (mean ± SEM) were counted in the cervical alveolar third, in 8,6 ± 0,3 (mean ± SEM) histological sections per alveolus (final magnification 100x), the percentage of points lying on bone trabeculae being proportional to their volume density. After confirmation of a normal distribution (Kolmogorov-Smirnov normality test, p>0,10) and comparison among groups by Analysis of Variance (gl = 4; F = 1,52; x2 = 0,13), histometric data confirmed that none of the anti-inflammatory drugs have detrimental effects in the volume fraction of new bone trabeculae filling the tooth extraction socket. Although experimental results obtained in animals may not be directly extrapolated to human neither induce to inferences about the clinical use of NSAIDs, numerous evidences have confirmed the deleterious effects of NSAIDs in the orthopedic clinic. Considering the reduced amount of studies on this subject pertaining to the dental field, more investigations are needed varying the experimental parameters and techniques, before the possibility of deleterious effects of NSAIDs in dental procedures requiring new bone formation are discharged.

La réparation osseuse est assurée par le recrutement constant de cellules souches/progéniteurs ostéo-compétents de nature hématopoïétique (CSH/PH), et mésenchymateuse (CSM). Une approche prometteuse pour le traitement des défauts osseux graves consisterait à favoriser le recrutement et la mobilisation des CSH/PH et des CSM à partir de la moelle osseuse vers le site de lésion. Plusieurs facteurs environnementaux sont connus pour moduler la prolifération, la mobilisation et la différenciation des progéniteurs ostéocompétents, dont l’hypoxie et l’hypodynamie (absence de contraintes mécaniques). Le but de ce travail de thèse a été d’investiguer in vivo l’impact de l’hypoxie respiratoire et de l’absence de contraintes mécaniques, appliquées séparément ou ensemble sur i) la mobilisation des progéniteurs ostéocompétents et sur ii) la réparation d’un défaut osseux cavitaire fémoral chez la souris. Sur un modèle murin dépourvu de défaut osseux, nos données montrent que l’hypoxie respiratoire est un agent mobilisateur des progéniteurs ostéocompétents, et qu’elle pourrait donc potentiellement exercer des effets bénéfiques sur la réparation osseuse. Toutefois, les effets de l’hypoxie sont modulés selon le statut hypodynamique ou non de l’animal. L’absence de contraintes mécaniques limite la mobilisation des progéniteurs érythrocytaires et mésenchymateux initiée par l’hypoxie, suggérant un effet potentiellement délétère de l’hypodynamie en condition hypoxique dans le contexte de la réparation osseuse. Chez les souris opérées, nous confirmons que l’hypoxie respiratoire déclenchée lors des phases de remodelage améliore la réparation du défaut osseux cavitaire. Une mobilisation des progéniteurs mésenchymateux et hématopoïétiques du fémur contra-latéral vers le fémur opéré est noté, mais le mode d’action de l’hypoxie passerait plutôt par une accélération du mécanisme de réparation dans la zone lésée. De façon intéressante, nous montrons que l’hypodynamie ne diminue pas le bénéfice apporté par l’hypoxie respiratoire à la réparation osseuse. En conclusion, ce travail de thèse identifie l’hypoxie respiratoire comme un candidat thérapeutique pertinent pour l’amélioration de la réparation osseuse. Bien que la perte des contraintes mécaniques module la biologie des cellules ostéoprogénitrices en absence de lésion, l’hypodynamie ne semble pas influencer la consolidation osseuse dans le cadre d’une amélioration de la réparation par un épisode hypoxique tardif. / Many environmental factors are known to influence bone cell fate, including proliferation, mobilization and differentiation of osteoprogenitor cells deriving from both hematopoietic and mesenchymal lineages. Among these factors, hypoxia and unloading (lack of mechanical loading / hypodynamia) are of particular interest. This study aims at investigating the impact of short-term hypobaric hypoxia and hindlimb unloading applied alone or in combination i) on the mobilization of osteocompetent progenitor cells on mice and ii) on the healing in a mouse model of surgical metaphyseal bone defect.In mice free of bone defect, our data indicate that respiratory hypobaric hypoxia acts as a mobilizing stimulus for osteoprogenitor cells. However, the effects of hypoxia in the bone marrow depend on whether mice are subjected to hindlimb unloading or not: hypodynamia tends to restrain the mobilization of both mesenchymal and erythroid progenitors under hypoxia. This suggests a potential detrimental influence of hypodynamia in the course of bone healing in hypoxic condition.In mice with surgery, we showed that hypobaric hypoxia during the remodelling process strongly enhances bone healing. A mobilization of both mesenchymal and hematopoietic progenitors is detected from the contralateral femur to the operated femur. In the lesion area, an acceleration of the repair process is evidenced. Interestingly, hindlimb unloading does not exert any negative influence on bone repair in our animal model. In conclusion, this study identifies delayed hypobaric hypoxia as a potent candidate to enhance bone healing. Even if unloading exerts significant effects on the biology of osteoprogenitor cells on mice free of bone defect, its influence is not detrimental for bone repair.

Vários estudos têm avaliado os efeitos do ultra-som e da radiação laser de baixa intensidade separadamente no reparo ósseo. No entanto, são escassas as comparações entre estas duas modalidades terapêuticas. Este estudo teve por objetivo avaliar e comparar, através de análise histomorfométrica e ensaio mecânico de flexão em três pontos, as conseqüências que estes agentes físicos podem trazer ao reparo do tecido ósseo em osteotomias transversais experimentais em tíbias de ratos. Foram utilizados 48 ratos com fratura cirúrgica unilateral parcial do terço superior de tíbia. Os animais foram divididos em três grupos de 16 animais. Em um grupo, o membro fraturado foi tratado com ultra-som pulsado de baixa intensidade (freqüência de 1,5 MHz, ciclo de trabalho 1:4, intensidade SATA 30 mW/centímetro quadrado) em sessões de 20 minutos, 5 vezes por semana, em 12 dias de tratamento. Em um segundo grupo, o membro fraturado foi tratado com laser As-Ga-Al (112,5 J/centímetro quadrado, 780 nm, 30 mW) em sessões de 2,5 minutos, 5 vezes por semana, em 12 dias de tratamento. O terceiro grupo serviu de controle, sendo submetido à mesma cirurgia na tíbia direita mas não recebeu qualquer tratamento. No ensaio mecânico, a carga no limite máximo suportada pelo grupo tratado com laser foi significantemente maior (p < 0,05) que os grupos tratado com ultra-som e controle. Na análise histomorfométrica, o grupo tratado com laser apresentou significância estatística quanto ao número e superfície de osteoblastos e volume e superfície de osteóide e o grupo ultra-som obteve significância para as superfícies de reabsorção e de osteoclastos. Pode-se concluir que o ultra-som acelerou o reparo ósseo (em relação ao grupo controle) por viabilizar mais rapidamente a fase de reabsorção, enquanto que a terapia laser foi capaz de acelerar ainda mais este processo por já promover predomínio de formação óssea no décimo nono dia pós-cirúrgico no modelo experimental utilizado neste estudo / Many studies have assessed the effects of ultra-sound and low intensity laser therapy separatedly in bone repair. However, the comparison between these two therapeutic modalities is rare. The objective of this study was to verify and compare, through histomorphometrical analysis and a three-point bending test, the consequences of these physical agents on bone healing in animals. 48 male Wistar rats were used with tibial bone partial osteotomy, divided into 3 groups of 16. In one group, rats had their fractured limb treated with GaAIAs laser (780 nm, 30 mW, 112,5 J/square contimeter) in 12 five times-a-week sections. In another group, rats were treated with low intensity pulsed ultra-sound (1,5 MHz, 30 mW/square centimeter) in 12 five times-a-week sections too. In a third group, animals were taken as control, being submitted to the same osteotomy but receiving no treatment. After 19 days the tibias were extracted and half of them were submitted to a three-point bending test and the other half to histomorphometric analysis. During the bending test, the maximum load at failure of the tibia in the laser group was significantly higher (p<0.05). Histomorphometry showed statistical significance in osteoblasts number and surface and osteoid volume and surface for the laser group, and eroded and osteoclasts surfaces for the ultra-sound group. Ultra-sound was able to enhance bone healing (compared to control group) by speeding up the reabsorption phase, while low intensity laser therapy could accelerate this process even more by promoting mostly new bone formation 19 days after osteotomy in this experimental model

As prostaglandinas (PGs) são derivadas do metabolismo do ácido aracdônico pela via das enzimas cicloxigenases (COX-1 e COX-2) e participam do controle do metabolismo ósseo. Os anti-inflamatórios não-esteroidais (AINEs), inibidores das COX, podem interferir negativamente com a formação óssea, atrasando o reparo de fratura de ossos longos, a fusão espinhal e a osseointegração de implantes. O presente trabalho teve por objetivo avaliar, quantitativamente, o efeito de diferentes tipos de AINEs (convencional, preferencial e seletivo para COX-2), assim como de um analgésico com efeito anti-inflamatório fraco, sobre o reparo ósseo alveolar, em ratos machos. Os animais foram divididos em 5 grupos experimentais: a) controle (administração de 1 mL água/dia), b) cetorolaco de trometamina (inibidor não-seletivo COX-1/COX-2; ingestão de 4 mg/kg/dia), c) nimesulida (inibidor preferencial de COX-2; ingestão de 5 mg/kg/dia), d) paracetamol (efeito anti-inflamatório fraco; ingestão de 80 mg/kg/dia), e) etoricoxibe (inibidor seletivo de COX-2; ingestão de 10 mg/kg/dia). As dosagens foram baseadas em trabalhos experimentais da literatura e na equivalência com a terapêutica humana. A administração foi realizada por gavage gástrica, iniciando logo após a extração do incisivo superior direito e seguindo por um periódo de 14 dias, após o que os ratos foram sacrificados, as hemi-maxilas contendo os alvéolos em reparação foram coletadas e processadas para inclusão em parafina, orientada de maneira a permitir cortes semi-seriados longitudinais de 6 &mu;m de espessura (a intervalos de 60 &mu;m), que foram corados pela hematoxilina e eosina. O percentual de tecido ósseo neoformado foi estimado por método de contagem diferencial de pontos, utilizando-se um microscópio óptico munido de câmera digital de vídeo para captura de imagens e um programa para histometria. Foram contados cerca de 1200 pontos (1182,9 ± 47,6 pontos; média ± EPM) no terço cervical alveolar de cada animal, em cerca de 8 secções histológicas intercaladas (8,6 ± 0,3 secções; média ± EPM). Após a aplicação de teste que comprovou a normalidade de todas as distribuições (teste de Kolmogorov-Smirnov, p > 0,10) aplicou-se a Análise de Variância (gl=4; F = 1,52; x2 = 0,13), que comprovou que o tratamento com os diferentes tipos de AINEs não interferiu com a formação óssea reparacional, neste modelo experimental. Os presentes resultados, obtidos em animais, não devem ser diretamente extrapolados para humanos, nem conduzir a uma inferência sobre o uso de AINEs na clínica odontológica. No entanto, as numerosas evidências experimentais e clínicas de que os diferentes tipos de AINEs podem ter efeitos indesejados na clínica ortopédica, somadas ao número reduzido de estudos voltados especificamente para a área odontológica, indicam a necessidade de mais investigações nessa área, com variação dos parâmetros experimentais e das ferramentas de avaliação, antes que se descarte a possibilidade de que os AINEs possam ter efeitos indesejados em procedimentos odontológicos que necessitam de formação óssea, principalmente no caso de utilização prolongada. / The cyclooxygenase enzymes COX-1 and COX-2 catalyze the conversion of arachidonic acid to prostaglandins (PGs), eicosanoids important for control of bone metabolism. The non-steroidal anti-inflammatory drugs (NSAIDs), which are COX inhibitors, may negatively interfere with bone formation and delay long bone fracture healing, spinal fusion and implant osseointegration. The aim of the present study was to investigate quantitatively whether different types of NSAIDs (conventional, preferential and COX-2-selective), as well as an analgesic drug with a weak anti-inflammatory action, can hinder alveolar bone healing, in male rats. The animals were divided in 5 experimental groups: a) control (oral administration of 1 mL water/day), b) cetorolac (non-selective COX-1/COX-2 inhibito - 4 mg/kg/day oral dose), c) nimesulide (preferential COX-2 inhibitor - 5 mg/kg/day oral dose), paracetamol (weak anti-inflammatory - 80 mg/kg/day oral dose), etoricoxib (selective COX-2 inhibitor - 10 mg/kg/day oral dose). The doses of NSAIDs were compatible to human therapy and in the range of investigations carried out in laboratory animals. The drugs were administered by gavage from the day of extraction of the upper right incisors until death 2 weeks later, when the hemi-maxillae containing the alveolar sockets were collected, decalcified and processed for paraffin embedding. Semi-serial longitudinal 6-&mu;m-thick sections were cut at 60-&mu;m intervals and stained with hematoxylin and eosin. The degree of new bone formation inside the alveolar socket was estimated by a differential point-counting method, using an optical microscopy with a digital camera for image capture and a public domain histometry software. A total of 1182,9 ± 47,6 points (mean ± SEM) were counted in the cervical alveolar third, in 8,6 ± 0,3 (mean ± SEM) histological sections per alveolus (final magnification 100x), the percentage of points lying on bone trabeculae being proportional to their volume density. After confirmation of a normal distribution (Kolmogorov-Smirnov normality test, p>0,10) and comparison among groups by Analysis of Variance (gl = 4; F = 1,52; x2 = 0,13), histometric data confirmed that none of the anti-inflammatory drugs have detrimental effects in the volume fraction of new bone trabeculae filling the tooth extraction socket. Although experimental results obtained in animals may not be directly extrapolated to human neither induce to inferences about the clinical use of NSAIDs, numerous evidences have confirmed the deleterious effects of NSAIDs in the orthopedic clinic. Considering the reduced amount of studies on this subject pertaining to the dental field, more investigations are needed varying the experimental parameters and techniques, before the possibility of deleterious effects of NSAIDs in dental procedures requiring new bone formation are discharged.

The aim of the thesis was development of an innovative treatment of bone defects. Human multipotent mesenchymal stromal cells (MSC) play a crucial role in bone healing. Clinical applications of MSC require large amount of cells, which could be obtained by autologous expansion of MSC harvested from bone marrow. As a first step, the standard protocol of MSC expansion based on αMEM medium and fetal bovine serum (FBS) was used. Experiments replacing FBS by pooled human serum (HS) in the culture medium concluded in patenting of a new MSC cultivation protocol (EU 1999250, CR 301141). This one-step cultivation protocol and xenogeneic protein-free cultivation medium is based on CellGro® for Hematopoietic Cells' Medium, HS, human recombinant growth factors, dexamethasone, insulin and ascorbic acid. The preclinical in vitro and in vivo experiments with MSC from both expansion protocols were carried out. Fibrillar polylactic scaffolds were seeded with MSC, cultured, differentiated and implanted in immunodeficient mice (NOD/LtSz-Rag1-). Bone-like mineralized tissue containing vessels was observed. The MSC cultured according to patented method were classified as Advanced-therapy Medicinal Product and has to fulfil the European Medicines Agency regulations to enter the clinical trials. Nevertheless the use of MSC seems...

Vários estudos têm avaliado os efeitos do ultra-som e da radiação laser de baixa intensidade separadamente no reparo ósseo. No entanto, são escassas as comparações entre estas duas modalidades terapêuticas. Este estudo teve por objetivo avaliar e comparar, através de análise histomorfométrica e ensaio mecânico de flexão em três pontos, as conseqüências que estes agentes físicos podem trazer ao reparo do tecido ósseo em osteotomias transversais experimentais em tíbias de ratos. Foram utilizados 48 ratos com fratura cirúrgica unilateral parcial do terço superior de tíbia. Os animais foram divididos em três grupos de 16 animais. Em um grupo, o membro fraturado foi tratado com ultra-som pulsado de baixa intensidade (freqüência de 1,5 MHz, ciclo de trabalho 1:4, intensidade SATA 30 mW/centímetro quadrado) em sessões de 20 minutos, 5 vezes por semana, em 12 dias de tratamento. Em um segundo grupo, o membro fraturado foi tratado com laser As-Ga-Al (112,5 J/centímetro quadrado, 780 nm, 30 mW) em sessões de 2,5 minutos, 5 vezes por semana, em 12 dias de tratamento. O terceiro grupo serviu de controle, sendo submetido à mesma cirurgia na tíbia direita mas não recebeu qualquer tratamento. No ensaio mecânico, a carga no limite máximo suportada pelo grupo tratado com laser foi significantemente maior (p < 0,05) que os grupos tratado com ultra-som e controle. Na análise histomorfométrica, o grupo tratado com laser apresentou significância estatística quanto ao número e superfície de osteoblastos e volume e superfície de osteóide e o grupo ultra-som obteve significância para as superfícies de reabsorção e de osteoclastos. Pode-se concluir que o ultra-som acelerou o reparo ósseo (em relação ao grupo controle) por viabilizar mais rapidamente a fase de reabsorção, enquanto que a terapia laser foi capaz de acelerar ainda mais este processo por já promover predomínio de formação óssea no décimo nono dia pós-cirúrgico no modelo experimental utilizado neste estudo / Many studies have assessed the effects of ultra-sound and low intensity laser therapy separatedly in bone repair. However, the comparison between these two therapeutic modalities is rare. The objective of this study was to verify and compare, through histomorphometrical analysis and a three-point bending test, the consequences of these physical agents on bone healing in animals. 48 male Wistar rats were used with tibial bone partial osteotomy, divided into 3 groups of 16. In one group, rats had their fractured limb treated with GaAIAs laser (780 nm, 30 mW, 112,5 J/square contimeter) in 12 five times-a-week sections. In another group, rats were treated with low intensity pulsed ultra-sound (1,5 MHz, 30 mW/square centimeter) in 12 five times-a-week sections too. In a third group, animals were taken as control, being submitted to the same osteotomy but receiving no treatment. After 19 days the tibias were extracted and half of them were submitted to a three-point bending test and the other half to histomorphometric analysis. During the bending test, the maximum load at failure of the tibia in the laser group was significantly higher (p<0.05). Histomorphometry showed statistical significance in osteoblasts number and surface and osteoid volume and surface for the laser group, and eroded and osteoclasts surfaces for the ultra-sound group. Ultra-sound was able to enhance bone healing (compared to control group) by speeding up the reabsorption phase, while low intensity laser therapy could accelerate this process even more by promoting mostly new bone formation 19 days after osteotomy in this experimental model

Un os long est composé de tissus osseux cortical et spongieux. Ces tissus ont des structures et des caractéristiques physiques différentes mais ont tous deux la capacité de se régénérer de façon naturelle suite à une lésion. Cette régénération ou réparation implique une séquence bien caractérisée d’événements contrôlés par l’interaction étroite entre des facteurs de croissance, des cellules, l’environnement chimique et dynamique, ainsi que par la matrice extracellulaire. L’ostéopontine (OPN) et la sialoprotéine osseuse (BSP) sont des protéines de la matrice extracellulaire exerçant des fonctions importantes dans le tissu osseux. Le but de ce travail a été d’étudier le rôle de l’OPN et de la BSP dans la réparation osseuse par génétique expérimentale. Les modèles utilisés dans cette études consistent en des lésions, l’un diaphysaire et purement cortical, l’autre région épi-métaphysaire mêlant destruction de l’os cortical, trabéculaire et de la plaque de croissance. La réparation de ces lésions a été analysée par microtomographie haute résolution aux rayons X et par histomorphométrie. Dans un premier temps, la réparation d’une perforation épi-métaphysaire dans le fémur chez la souris, a été caractérisée et comparée à celle de même diamètre réalisée dans la diaphyse. Dans cette étude comparative, des profils distincts de réparation ont été mis en évidence bien que tous deux mettent en place un mécanisme d’ossification intramembranaire. Ainsi, le défect cortical diaphysaire est comblé par une formation osseuse centripète restreinte à la zone corticale. Dans le modèle épi-métaphysaire, la formation osseuse est initiée au fond du défect et se propager vers le cortex. Ce processus aboutit à une restauration des travées mais à une réparation incompléte du cortex. Ainsi, le premier modèle apparaît comme pertinent pour l’étude de la réparation corticale alors que le modèle épi-métaphysaire se présente plus adapté à l’étude de la réparation de l’os trabéculaire. L’OPN et la BSP n’ont pas de fonctions redondantes dans la réparation de ces lésions. En effet, l’OPN intervient principalement dans la réparation de l’os trabéculaire, son absence entraîne un retard lié à un défaut de progression de l’os au sein de la cavité. L’absence de BSP quant à elle, semble intervenir uniquement dans le processus de réparation de l’os cortical diaphysaire, provoquant un retard de réparation dû à un défaut de minéralisation de l’ostéoïde. Les travaux réalisés au cours de cette thèse ont permis de caractériser des modèles de lésions osseuses pertinents pour l’étude de la réparation de l’os cortical et spongieux. L’utilisation de ces modèles a permis d’améliorer la compréhension du rôle de deux protéines de la matrice extracellulaire osseuse dans la réparation de cortical et trabéculaire grâce aux modèles de génétique expérimentale. / Long bones consist of cortical and spongious bone tissue, which have different structures and physiological characteristics. Both can heal spontaneously. Bone healing is a complex multi-step process which depends on cells, soluble factors, mechanical environment and bone matrix. Osteopontin (OPN) and Bone Sialoportein (BSP) are extracellular matrix proteins, which have been shown to exert important functions in bone. The aim of this study is to address the role of OPN and BSP in bone repair using experimental genetic strategies. Injured bone models are drilled-hole defects performed in diaphyseal cortical bone or in the epi-metaphyseal region. Bone healing was analyzed by micro-tomography and histomorphometry. Epi-metaphyseal defect healing was characterized and compared to cortical bone repair. In this comparative study, distinct patterns of bone repair have been shown while in both models repair occurs through intramembranous ossification. Diaphyseal defect was rapidly filled with newly bone formed in a centripetal manner within the cortical gap. In contrast, bone formation within the epi-metaphyseal defect was initiated from the depth of the cavity and spread towards the cortical edges, regenerating cancellous bone and albeit not completely cortical wall. Therefore, diaphyseal drill defects appear pertinent for the study of spontaneous cortical healing whereas epi-metaphyseal drill defects appear as appropriate models to investigate spongy bone regeneration. OPN and BSP do not show redundancy in the bone repair process of these two models. Indeed, OPN is mainly involved in trabecular bone repair; its deficiency induced a delay due to impaired bone progression within the epi-metaphyseal cavity. The lack of BSP only delayed cortical bone repair due to an impaired mineralization of the bone matrix. This study permits to characterize pertinent models of cortical and trabecular bone repair. Application of these models added new insights on the involvement of matrix proteins in cortical defect healing and trabecular bone repair using experimental genetic models.