O objetivo deste estudo foi avaliar os fatores de risco, aspectos clínicos, imagenológicos e terapêuticos da osteonecrose dos maxilares associada ao uso dos bisfosfontos (ONMAB), bem como compará-los com o estado atual da doença, na busca de parâmetros que favoreceram o reparo tecidual. Foram avaliados retrospectivamente 40 casos de ONMAB de pacientes oncológicos encaminhados para avaliação estomatológica. Os dados demográficos, clínicos, imagenológicos, laboratoriais, histopatológicos e terapêuticos foram analisados. Foi realizada análise descritiva dos dados e para associar o estado atual da osteonecrose com as demais variáveis foi utilizado o teste exato de Fisher. O nível de significância estabelecido foi de 5% (p0,05). A ONMAB se mostrou mais comum em mulheres, com idade média de 59 anos e associadas mais freqüentemente ao de câncer de mama, seguido de câncer de próstata e do mieloma múltiplo. O principal bisfosfonato utilizado foi o ácido zoledrônico com média de uso de 23,54 meses. A exodontia, o tratamento quimioterápico e com corticóides foram associados com mais da metade dos casos. O aspecto clínico principal foi a exposição óssea com sintomatologia dolorosa associados ou não à drenagem de secreção, principalmente em mandíbula. Modificações no trabeculado ósseo, erosão da cortical e osteoesclerose foram os principais achados de imagem. Dentre todos os tratamentos realizados, a associação de cirurgia com plasma rico em plaquetas (PRP) e laser de diodo foi a mais utilizada (52,5%) e que mostrou alto índice de sucesso clínico (62,96%). A evolução dos casos tratados mostrou que 67,5% apresentaram reparo tecidual e mesmo aqueles que mantiveram a exposição óssea não mostraram sintomatologia dolorosa. A classificação clínica e o tipo de tratamento empregado influenciaram de forma significante a resolução da ONMAB. Conclui-se que medidas de controle local de infeccção, antibioticoterapia e pequenos debridamentos ósseos devem ser adotadas nos quadros de ONMAB, porém em conjunto com a utilização de bioestimuladores teciduais como o PRP e laser de diodo que promovem de forma mais eficiente o reparo tecidual. / The aim of this study was to evaluate the risk factors, clinical, radiological and treatment aspects of bisphosphonate-associated osteonecrosis of the jaws (BONJ) in cancer patients. Likewise, comparing the current state of the disease with the different variables in the search for parameters that favored the tissue repair. We evaluated retrospectively 40 cases of BONJ of cancer patients referred for stomatology evaluation. Demographic data, clinical, radiographic, laboratory, histopathological and treatment were analyzed. We performed descriptive analysis of data and to associate the current state of the osteonecrosis with the other variables we used Fisher\'s exact test. The level of significance established was 5% (p 0.05). The BONJ was more common in women, the mean age was 59 years and was more frequently associated to the breast cancer followed by prostate and multiple myeloma. Zoledronic acid was the main bisphosphonate used with average use of 23.54 months. The dental tooth, chemotherapy and corticosteroids history were associated with more than half of the cases. The clinical aspect was the main bone exposure associated with painful symptoms or drainage of secretions, especially in mandible. Structural alteration of trabecular bone, cortical bone erosion and osteosclerosis were the main radiological findings. Among all the treatments performed, the therapy combining bone resection, diode laser and platelet-rich plasma (PRP) was the most used (52.5%) which showed high rate of clinical success (62.96%). The evolution of treated cases showed that 67.5% had tissue repair and only 32.5% maintained the bone exposure, but all without painful symptoms. The clinical classification and type of treatment used significantly influence the outcome of BONJ . It is concluded that measures of infection local control, antibiotics and small bone debridement should be adopted in the BONJ but with the use of tissue stimulation using PRP and diode laser helps to promote more effective tissue repair.

A osteonecrose dos maxilares associada ao uso dos Bisfosfonatos é uma exposição óssea que persiste por mais de 8 semanas na cavidade oral em pacientes sob tratamento com bisfosfonatos e que não foram submetidos a radioterapia de cabeça e pescoço. Explicar o motivo do desenvolvimento destas lesões ósseas principalmente nos maxilares e desvendar a sua patofisiologia ainda é necessário. Por isso, o reparo ósseo em um modelo animal de osteonecrose dos maxilares associada ao uso de bisfosfonatos em ratos Wistar (Rattus norvegicus, albinus) foi avaliado através de análise microscópica e molecular. A amostra foi composta por 48 ratos machos, com 12 semanas de vida e peso aproximado de 300 gramas, que foram submetidos a administração de Ácido Zoledrônico, 0,6 mg/kg a cada 28 dias com um total de 5 doses. Os animais foram dividos em quatro grupos, cada um composto por 12 animais; 2 grupos de tratamento AZ e AZ-experimental (AZ-exp) e 2 grupos controles CO e CO-experimental (CO-exp) com administração de cloreto de sódio 0,9% no mesmo volume e frequencia do Ácido Zoledrônico. As soluções foram administradas por via intraperitoneal. O grupo AZ-exp e o CO-exp foram submetidos a exodontias dos molares superiores direitos e a realização de defeito ósseo no fêmur esquerdo 45 dias após a primeira aplicação das soluções. A eutanásia dos animais ocorreu após 150 dias do início do experimento. A avaliação histológica foi realizada através de análises qualitativa e quantitativa, verificou a presença de sequestros ósseos, áreas de osteonecrose e áreas de osso total por meio de estudos de microscopia óptica pela coloração Hematoxilina e Eosina. Análise quantitativa da expressão do RNAm de proteínas envolvidas no processo de reparo ósseo pelo método de reação em cadeia da polimerase em tempo real (RealTimePCR) também foi realizada para avaliação dos osteoclastos (RANK, RANKL e OPG), osteoblastos (ALPL e OCN) osteócitos (DMP-1 e PHEX) e vascularização (VEGF). Apenas o grupo com administração de AZ e realização de exodontias apresentou sequestros ósseos e áreas significativamente maiores de osteonecrose em comparação aos animais com exodontia e sem a administração de AZ. Não foram encontradas áreas de osteonecrose na análise do fêmur. A avaliação do osso total apresentou maior quantidade de osso nos animais submetidos a procedimentos cirúrgicos e administração de AZ, porém não houve significância estatística. Na análise molecular, a administração de AZ e a realização das exodontias e do defeito ósseo, provocou alteração na expressão de marcadores para osteoclastos. A administração do AZ associada à realização dos procedimentos cirúrgicos aumentou a expressão de marcadores iniciais da osteoblastogênese e diminuiu a expressão tardia dos osteoblastos. A expressão de marcadores de osteócitos foi menor após a administração de AZ e exodontias na maxila, no entanto, no fêmur, houve aumento na expressão destes marcadores. A realização das exodontias e de defeitos ósseos no fêmur aumentou a expressão de VEGF nos grupos com e sem administração de AZ. Estes resultados evidenciam a interferência do Ácido Zoledrônico no reparo ósseo dos alvéolos e dos defeitos ósseos nos fêmures, causando incidência de osteonecrose na maxila, atraso na remodelação óssea, e ausência de lesões de osteonecrose no fêmur. A presença de lesões de osteonecrose na maxila pode ser consequência do atraso na remodelação óssea, evidenciado pela alteração na expressão de marcadores para osteclastos e osteoblastos, e as mesmas ocasionam a diminuição da expressão de proteínas pelos osteócitos. / Bisphosphonate-related osteonecrosis of the jaws is a bone exposure persisting for more than 8 weeks in the oral cavity in patients receiving bisphosphonates and who did not undergo radiation therapy for head and neck. Explain the reason for these development bone lesions mainly in the jaws and unveil its pathophysiology is still needed. Therefore, the bone repair in an animal model of Bisphosphonate-related osteonecrosis of the jaws in Wistar rats (Rattus norvegicus, Albinus) was evaluated by microscopic and molecular analysis. The sample was composed of 48 male rats, with 12 weeks old and weighing approximately of 300 grams, who underwent the administration of zoledronic acid, 0.6 mg / kg every 28 days with a total of 5 doses. The animals were divided into four groups, each consisting of 12 animals; 2 treatment groups AZ and AZ-experimental (AZ-exp) and 2 control groups CO and CO-experimental (CO-exp) with sodium chloride administration 0.9% in the same volume and frequency of zoledronic acid. All solutions were administered intraperitoneally. The AZ-exp group and the CO-exp group underwent extractions rights upper molars and the bone defect was performed in the left femur 45 days after the first application of the solutions. Euthanasia of animals occurred after 150 days from the beginning of the experiment. Histological evaluation was performed through quantitative and qualitative analysis checked the presence of bone sequestration, osteonecrosis area and whole bone areas by means of optical microscopy by hematoxylin-eosin staining. Quantitative analysis of mRNA expression of proteins involved in bone repair by polymerase chain reaction method in real time (RealTimePCR) was also performed: osteoclasts (RANK, RANKL and OPG), osteoblasts (ALPL and OCN) osteocytes (PMD-1 and PHEX) and vascularization (VEGF). Only the group with administration of AZ and performing tooth extractions presented bone sequestration and significantly larger areas of osteonecrosis when compared to animals with extraction and without AZ administration. There were no areas of osteonecrosis of the femoral analysis. The evaluation of the whole bone showed more bone in animals undergoing surgeries and AZ administration but there was no statistical significance. Molecular analysis, AZ administration and the performance of tooth extractions and bone defect changed the expression of markers for osteoclasts. The AZ administration associated with surgical procedures increased the expression of early markers of osteoblastogenesis and decreased the late expression of osteoblasts. The expression of osteocytes markers was lower after AZ administration and maxillary tooth extractions; however, there was an increase in the expression of these markers in the femur. The performance of tooth extractions and bone defects in the femur increased VEGF expression in animals with and without administration of AZ. These results show the interference of zoledronic acid on bone repair of the sockets and bone defects in the femurs, causing incidence of osteonecrosis of the upper jaw, delayed bone remodeling, and no femoral osteonecrosis injuries. The presence of osteonecrosis injuries in the upper jaw can be the consequence of the delay on bone remodeling, evidenced by the change in the markers expression for osteoclasts and osteoblasts, and they cause the decreased in protein expression by osteocytes.

Os bisfosfonatos (BFs) têm sido amplamente utilizados para o tratamento de doenças do metabolismo ósseo, principalmente na prevenção de metástases ósseas e na prevenção e tratamento da osteoporose. No entanto, existem efeitos colaterais indesejáveis sendo um deles, a indução da Osteonecrose dos Maxilares (Medication-Related Osteonecrosis of the Jaws - MRONJ), uma complicação de difícil tratamento e solução. Até o presente momento, não foi definida a fisiopatologia da MRONJ e nem estabelecido protocolo de tratamento eficaz para esta doença. Diversas terapias vem sendo descritas na literatura para o tratamento da MRONJ dentre elas a laserfototerapia. Assim sendo, os objetivos deste estudo foram: inicialmente avaliar in vitro o efeito de diferentes concentrações dos BFs mais empregados na atualidade (alendronato e ácido zoledrônico) na viabilidade de células envolvidas na reparação de lesões MRONJ (osteoblastos e fibroblastos); secundariamente estudar o efeito da fototerapia com laser de baixa potência sobre estas células previamente induzidas pelos BFs. Foram utilizados osteoblastos-símile da linhagem OSTEO 1 e fibroblastos de mucosa bucal humana da linhagem FMM1. Após terem sido submetidos aos testes de citotoxicidade expondo as células as concentrações de 1?M, 10?M e 100?M de alendronato sódico e 3?M, 5?M e 10?M de ácido zoledrônico por 24 horas, os grupos testes foram irradiados com laser de diodo no modo contínuo, puntual e de contato (InGaAIP, 660nm, 30mW, spot 0,028cm2 ) com duas densidades de energia diferentes 5J/cm2 (4,5s) e 10J/cm2 (9s). Duas irradiações com intervalo de 6 horas entre cada uma delas foram executadas. A viabilidade celular foi determinada utilizando o ensaio de redução do MTT, e a atividade de fosfatase alcalina dos osteoblastos foi avaliada utilizando ensaio de ponto final. Os resultados obtidos foram submetidos à análise estatística ANOVA 1 critério complementado por Tukey (p<0,05). Foi possível concluir que: as concentrações de 100?M e 10?M do alendronato sódico foram tóxicas para os osteoblastos e fibroblastos em cultura. As concentrações de 3?M, 5?M e 10?M do ácido zoledrônico foram tóxicas para os osteoblastos e fibroblastos a longo prazo (48h e 72h). A atividade da fosfatase alcalina nos osteoblastos foi afetada por todas as concentrações de ácido zoledrônico testadas (3?M, 5?M e 10?M). Nos parâmetros aqui aplicados a LPT não teve efeito sobre a atividade da fosfatase alcalina das células tratadas com o alendronato de sódio ou ácido zoledrônico. E a laserfototerapia de baixa potência nos parâmetros utilizados nesse estudo não foi capaz de reverter a toxicidade dos bisfosfonatos testados, independentemente das concentrações destas substâncias / Bisphosphonates (BPs) have been widely used for treating bone metabolism diseases, especially for prevention of bone metastasis and osteoporosis. However, there are undesirable side effects and one of them, the induction of Osteonecrosis of the Jaw (Medication-Related Osteonecrosis of the Jaws - MRONJ), a complication of difficult treatment and solution. Until now the pathophysiology and an effective treatment protocol for MRONJ have not been established. Various therapies have been described in the literature for the treatment of MRONJ including laserphototherapy. The objectives of this study were to evaluate the effect of different concentrations of two BPs used today (alendronate and zoledronic acid) on the viability of cells involved in the repair of MRONJ lesions (osteoblasts and fibroblasts); and to study the effect of phototherapy with low power laser on these cells previously treated with referred BPs. After being submitted to cytotoxicity testing by exposing the cells to concentrations of 1?M, 10?M and 100?M of sodium alendronate and 3?M, 5?M and 10?M of zoledronic acid for 24 hours, the test groups were irradiated with diode laser in continuous mode, punctual and contact (InGaAIP, 660nm, 30mW, spot 0,028cm2) with two different energy densities 5 J/cm2 (4,5s) and 10J/cm2 (9s). Two irradiations with an interval of 6 hours between each of them were performed. Cell viability was determined using the MTT reduction assay, and the alkaline phosphatase activity of osteoblasts was evaluated using the end point assay. The results were submitted to statistical analysis using ANOVA 1 criteria complemented by Tukey (p <0.05). It was possible to conclude that: concentrations of 100?M and 10?M of alendronate were toxic to osteoblasts and fibroblasts in culture. The concentrations of 3?M, 5?M and 10?M of zoledronic acid was toxic to osteoblasts and fibroblasts in long-term (48h and 72h). The activity of alkaline phosphatase in osteoblasts was affected by all the zoledronic acid concentrations tested (3?M, 5?M and 10?M). In the LPT parameters applied here had no effect on alkaline phosphatase activity of the cells treated with the sodium alendronate or zoledronic acid. And low power laserphototherapy, in the parameters used in this study, was unable to reverse the toxicity of bisphosphonates tested, irrespective of the concentrations of these substances.

A osteonecrose dos ossos maxilares relacionada ao uso prolongado de bisfosfonatos (OMRB), associada a procedimentos cirúrgicos, constitui uma entidade com menos de dez anos de ocorrência na clínica estomatológica. Os bisfosfonatos (BFs) são medicamentos antireabsortivos altamente efetivos no tratamento de diversas doenças ósseas, além de metástases. Embora a maioria dos casos de OMRB tenha sido relatada após o uso de potentes BFs da terceira geração, o alendronato de sódio (ALN), um bisfosfonato de segunda geração, é amplamente utilizado no tratamento e prevenção de doenças ósseas. O presente estudo experimental in vivo visou analisar a possível presença de OMRB no processo alveolar de ratos tratados com ALN após exodontia do segundo molar inferior. Para tanto, utilizou-se 30 ratos Wistar, machos, com 7 semanas de vida, divididos em dois grupos, ALN (tratado com alendronato) e CTL (controle, tratado com solução salina). O ALN foi administrado diariamente por injeção subcutânea na dose de 2,5 mg/kg peso por 14 dias. Nesse momento, foi realizada a exodontia, havendo sido eutanasiados cinco animais de cada grupo, 7, 14 e 21 dias após a exodontia. Após a eutanásia, a região da mandíbula foi fixada em 2,5% formaldeído e 2% glutaraldeído, em tampão cacodilato 0,1M - pH 7,4 e descalcificada em EDTA a 4,13% por 30 dias. Os espécimes foram analisados em cortes corados em HE, e foi realizada histomorfometria para análise da reabsorção da crista alveolar mesial e distal, além do tecido ósseo neoformado no interior do alvéolo, que também foi analisado através de imuno-histoquímica para as proteínas não colágenas OPN e BSP e para evidenciação de vasos sanguíneos neoformados com o anticorpo anti-CD105. Os osteoclastos foram evidenciados através de histoquímica para fosfatase ácida resistente ao tartarato (TRAP). Além disso, as células e eventos da reparação foram examinados por microscopia eletrônica de transmissão. Os resultados mostraram que os animais tratados com ALN exibiram OMRB de grau zero em todos os períodos estudados, além da presença de osteoclastos latentes TRAP-positivos e histomorfometria, onde a não reabsorção das cristas alveolares ocasionaram a permanência do osso alveolar, em contato com bactérias. Observou-se ainda, um claro atraso na formação óssea, quando comparado ao controle, além da diminuição dos vasos sanguíneos nos estágios iniciais. A localização e o padrão de marcação para as proteínas OPN e BSP foram considerados normais, observados evidente marcação em áreas ósseas imaturas e em linhas cimentantes. Assim, concluiu-se que o ALN administrado neste protocolo provocou OMRB leve, provavelmente causada pela diminuição da angiogênese, presença de osso alveolar não remodelado expostos a colônias bacterianas, além da diminuição da atividade osteoblástica. / The bisphosphonate-related osteonecrosis of the jaw (BRONJ) associated with surgery procedures, is an entity with less than 10 years of occurrence in dental practice. The bisphosphonates (BFs) are anti-resorbing drugs highly effective in the treatment of several bone diseases, including metastasis. Although the majority of cases of BRONJ had been reported with the potent third generation of BFs, sodium alendronate (ALN), a second generation bisphosphonate, is widely used for treatment or prevent bone diseases as osteoporosis. The present experimental study aimed to analyze the possible presence of BRONJ in the alveolar process of rats treated with ALN following extraction of the mandibular second molar. For this, we used thirty 7-week-old male Wistar rats, divided into two groups, ALN (treated with alendronate) and CTL (control, treated with saline solution). The administration of ALN received daily subcutaneous injection at a dose of 2.5 mg/kg for 14 days. At that moment, the extraction was performed. After 7, 14 e 21 days of surgery, five animals of each group were euthanized and the mandibular region fixed in 2.5% formaldehyde + 2% glutaraldehyde in cacodylate buffer 0.1M - pH 7.4 and decalcified in 4.13% EDTA for 30 days. The specimens were morphologically analyzed in HE stained sections, and, histomorphometry was used to analyze the resorption of mesial and distal alveolar crests, as well as the bone formed into the alveolus. Immunohistochemistry for the noncollagenous proteins OPN and BSP and for CD105 to revel neoformed blood vessels was also performed. The osteoclasts were revealed through tartrate-resistant acid phosphatase (TRAP) histochemistry. Moreover, the cells and events of alveolar healing were examined by transmission electron microscopy. The results showed light degree of BRONJ in the ALN treated animals at all the studied periods, and the presence of latent osteoclasts near the non-resorbing crest alveolar. Bacteria were observed in contact with the non-resorbed alveolar bone. An evident delay in bone formation when compared to control group was also observed, as well as the decrease of blood vessels in early stages. The distribution of immature bone and in cement lines of OPN and BSP was considered normal. Thus, we concluded that the present protocol of ALN yielded light BRONJ areas, probably by reduced angiogenesis, lock of remodeling of alveolar, presence of bacterial colonies and evident decreased osteoblastic ativitity.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-07-31T13:31:04Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Leomar Emanuel Almeida Mecca.pdf: 3375518 bytes, checksum: 131b6866218d384538b938f5a1263a7c (MD5) / Made available in DSpace on 2018-07-31T13:31:05Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Leomar Emanuel Almeida Mecca.pdf: 3375518 bytes, checksum: 131b6866218d384538b938f5a1263a7c (MD5) Previous issue date: 2018-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo deste trabalho foi avaliar se o fármaco ranelato de estrôncio causa quadros de osteonecrose sobre o tecido ósseo de ratas ovariectomizadas, tratadas ou não com bifosfonatos. Para a realização da pesquisa, 40 ratas foram ovariectomizadas e divididas em 4 grupos: C (recebendo solução salina), BF (1 aplicação semanal de ácido zoledrônico IP 0,4 mg / kg), RE (625mg/Kg/dia de ranelato de estrôncio via gavagem) e BF+RE (associação de bifosfonato e ranelato de estrôncio nas concentrações indicadas acima). Foram realizadas exodontias dos primeiros molares inferiores. Após o término do tempo experimental, os animais foram eutanasiados, e seguiram para as seguintes análises: radiográfica e histológica. Os resultados apontam que o grupo RE macroscopicamente obteve uma cicatrização gradual assim como o grupo C. Já o grupo BF apresentou áreas de fenestração óssea e deiscência. Quando comparado radiograficamente por densidade de pixel, os grupos experimentais obtiveram diferença estatística (p<0,05) com o C mas não entre si. Com relação a análise histológica, os resultados apontaram que o ranelato de estrôncio não causa osteonecrose, este possui uma tendência ao aumento o número de osteócitos, vasos sanguíneos e reduz a quantidade de osteoclastos. Já o bifosfonato causa osteonecrose, reduz o número de vasos sanguíneos e osteócitos, porém aumentou o número de osteoclastos. O grupo BF+RE, apesar da redução dos quadros de necrose, não foi eficaz na forma profilática para osteonecrose. Assim, pode-se concluir que o Ranelato não causa quadros de osteonecrose, mas também não pode ser utilizado como agente profilático para a doença. / The aim of this study was to evaluate whether the drug strontium ranelate causes osteonecrosis on the bone tissue of ovariectomized rats, treated or not with bisphosphonates. To perform the research 40 rats were ovariectomized and divided into 4 groups: C (receiving saline solution), BF (1 weekly application of zoledronic acid IP 0.4 mg / kg), RE (625 mg / kg / day strontium ranelate via gavage) and BF+RE (association of bisphosphonate and strontium ranelate at the concentrations indicated above). Exodontia of the lower first molars was performed. After the end of the experimental time, the animals were sacrificed, followed by the following radiographic, microtomographic and histological analyzes. The results indicate that the RE group macroscopically obtained a gradual cicatrization as well as the group C. The BF group presented areas of bone fenestration and dehiscence. When compared radiographically by pixel density, the experimental groups obtained statistical difference with C but not with each other. Regarding the histological analysis, the results pointed out that strontium ranelate does not cause osteonecrosis, this has a tendency to increases the number of osteocytes, blood vessels and reduces the amount of osteoclasts. However, bisphosphonate causes osteonecrosis, reduces the number of blood vessels and osteocytes, but increased the number of osteoclasts. The BF+RE group, despite the reduction of necrosis pictures, was not effective prophylactically for osteonecrosis. Thus, it can be concluded that Ranelate does not cause osteonecrosis, nor can it be used as a prophylactic agent for the disease.

Steroid-associated osteonecrosis (SAON) accounts for a major fraction in non-traumatic osteonecosis (ON) and generally has poor prognosis even after surgical interventions. This suggests that prevention is one of the important intervention strategies for SAON. So far, there is lacking of proven prevention modalities for SAON. / Study I was to establish an alternative SAON model. Based on the proposed pathogenesis of SAON that the intravascular thrombosis and extravascular marrow fat deposition are the two important contributors to the development of ON lesion, lipopolysaccharides (LPS) could induce vascular dysfunction and even thrombosis, and methylprednisolone (MPS) could induce the adipogenesis of marrow mesenchymal stem cells (MSCs). They were accordingly used in a combination for ON induction in animals. / Study II was to investigate the effect of Herbal Epimedium-derived formula for prevention of ON using the validated SAON animal model. Efficacy of the herbal Epimedium-derived formula was assessed for prevention of SAON using the animal model. Thirty adult male rabbits were used in this study. The ON incidence was set as the end-point for evaluation of the prevention efficay. For the potential intervention targets, the intravascular thrombosis and extravascular marrow fat formation were evaluated hematologically and histopathologically. The vascular structure and function were evaluated by advanced bioimaging modalities of micro-CT and MRI. / Study III was to investigate the bioactive compound(s) from the herbal Epimedium-derived herbal formula for prevention of SAON. Phytochemical analysis identified seven compounds in this efficacy-proven formula, with icariin as the major compound accounting for more than 80% in weight. Icariin was therefore tested for its prevention efficacy using the SAON animal model. / Study IV was to investigate the underlying mechanism(s) of bioactive compound Icariin in effective prevention of SAON using in vitro cell models. As activation of endothelial cells and adipogenesis of MSCs are suggested to be the two major events involving in vascular dysfunction and marrow fat formation in SAON animal model, Icariin were accordingly hypothesized to be able to prevent activation of endothelial cells and inhibit adipogenesis of MSCs. / Summary. After summarizing the major findings of these four logically interrelated studies, it was able to conclude that Icariin was the identified bioactive compound from the herbal Epimedium-derived formula, which was able to reduce the SAON incidence with inhibition of intravascular thrombosis and extravascular marrow fat formation in an established rabbit model. The underlying mechanisms might be related to its effects on protection of endothelial cells activation and inhibition of MSCs adipogenesis (This can be summarized in the following picture). This study provides a new bioactive agent Icariin for SAON prevention and potential future clinical application. (Abstract shortened by UMI.) / The following research questions were addressed in the present study: (1) Is there an alternative SAON animal model? (Study I); (2) Whether the herbal Epimedium-derived formula is able to prevent SAON in this animal model? (Study II); (3) What is the bioactive compound(s) in this herbal Epimedium-derived formula? (Study III); (4) How does this bioactive compound prevent SAON? (Study IV) / Sheng, Hui. / Adviser: Ling Qin. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3421. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

激素性骨壞死是由於經常使用脈衝性激素處理非骨科性問題引起的一種常見的骨科疾病。在組織病理學上，激素性骨壞死指骨死亡，血管內血栓閉塞和血管外骨髓脂肪沉積會引起缺血導致骨修復不足。上游的分子細胞病理學機制研究表明間充質幹細胞細胞池活性下降，成骨細胞凋亡和骨小梁基質退變導致的不充分修復是激素性骨壞死發生的重要因素。 / 間充質幹細胞是骨髓的基質組成部分，具有分化成多種細胞的潛能。最近的研究表明，激素性骨壞死可能是骨細胞和/或間質幹細胞病變引起的一種疾病。研究發現，在接受類固醇治療而發生骨壞死的病人中，骨髓間充質幹細胞活性下降和分化潛能發生改變。在骨髓細胞中，激素能夠誘導脂肪發生。盛輝等發現來源於激素性骨壞死兔子中的間充質幹細胞成脂分化增強，謝新薈等進一步發現發生激素性骨壞死的兔子，骨缺損修復延遲，這可能是由激素導致的間充質幹細胞潛能發生改變引起的。綜合以上研究表明，間充質幹細胞在骨壞死發生和修復過程中起著重要作用。我們之前報導過淫羊藿黃酮（EFs）的腸代謝產物淫羊藿素Icaritin通過抑制血栓的形成和脂肪沉澱預防激素性骨壞死。最近，我們把Icaritin整合到聚乳酸聚乙醇酸共聚物/磷酸三鈣（PLGA/TCP）支架材料中，形成PLGA/TCP/Icaritin複合支架材料。我們發現PLGA/TCP/Icaritin複合材料可以促進激素性骨壞死骨缺損的修復，肌肉移植發現PLGA/TCP/Icaritin也能促進新生血管的發生。我們也發現單純PLGA/TCP複合材料也能夠促進激素性骨壞死骨缺損的修復，但是潛在的機制尚不清楚。 / 骨是一個高度血管化的組織，依賴於血管和骨細胞密切的時空連結維持骨骼的完整性。因此，血管生成在骨骼發育和骨折修復過程中發揮著舉足輕重的作用。血管為骨的發育和再生提供氧氣，為基質輸送刺激間充質細胞特異性成骨的重要信號，另一方面，骨為血管生成輸送生長因數和細胞。 / 本論文分為以下四個主要部分： / 第一部分: 研究Icaritin對人源間充質幹細胞分化的作用及其機制。流式細胞分選鑒定結果表明我們使用的人源間充質幹細胞能夠特異表達間充質幹細胞表面標誌物。MTT實驗結果顯示Icaritin不影響間充質幹細胞的增殖；分化實驗表明Icaritin在沒有成骨誘導試劑存在的情況下無法影響間充質幹細胞的分化。在成骨誘導試劑存在的情況下，Icaritin促進間充質幹細胞成骨分化，抑制其成脂分化；即時螢光實時定量聚合酶鏈式擴增（RT-PCR）結果顯示Icaritin在間充質幹細胞分化過程中上調成骨基因的表達，下調成脂基因表達。進一步研發發現在成骨分化過程中，Icaritin能夠促進BMP2和beta-catenin 蛋白的表達，而BMP2抑制劑Noggin能夠能夠逆轉Icaritin促進的成骨發生。這些發現表明Icaritin能夠促進而非誘導間充質幹細胞的成骨分化，Icaritin調解間充質幹細胞成骨分化具有BMP2信號通路依賴性。 / 第二部分: 評估激素性骨壞死兔源間充質幹細胞的分化潛能及Icaritin 對異常分化的間充質幹細胞分化潛能的影響。結果表明Icaritin促進正常兔源間充質幹細胞的成骨分化，抑制其成脂分化。激素性骨壞死兔源間充質幹細胞的成骨分化潛能降低，成脂分化升高；而Icaritin能夠劑量依賴性地部分恢復降低的成骨分化潛能，抑制升高的成脂分化活性。激素性骨壞死兔源間充質幹細胞的增殖活性也下降但是不能被Icaritin恢復。Icaritin對激素性骨壞死兔源間充質幹細胞中下降的VEGF的表達無影響。這些發現顯示間充質幹細胞的分化潛能在激素性骨壞死發生過程中遭到破壞，但是能夠被Icaritin部分恢復。 / 第三部分: 評估Icaritin對體外成血管的影響。我們對Icaritin對人臍帶靜脈內皮細胞（HUVECs）的增殖、遷移、管狀結構形成及成血管相關基因的表達的影響進行了檢測。結果表明Icaritin不影響HUVECs的增殖、遷移和管狀結構的形成；RT-PCR結果顯示Icaritin對HUVECs中的VEGF, HIF1a, FGF2 and TGF-beta表達也沒有影響。這些發現表明Icaritin在體外並不能直接作用于血管生成。結果謝新薈和陳詩慧等人的體內研究結果可以推測在骨缺損修復過程中，Icaritin通過促進成骨間接促進血管生成。 / 第四部分: 主要研究Icaritin及複合生物材料在體外體內對間充質幹細胞歸巢的影響。結果表明Iaritin能夠促進間充質幹細胞的遷移並上調血管細胞黏附分子1（VCAM1）的表達。複合材料PLGA/TCP和PLGA/TCP/Icaritin在體外培養的條件下能夠募集間充質幹細胞到材料周圍及進入材料。間充質幹細胞體外用修飾性超順磁性氧化鐵（SPIO@SiO₂-NH₂）納米顆粒標記後，其分化潛能依然保留，增殖和潛能能力稍微下降。兔激素性骨壞死造模完成後，股骨遠端髓芯減壓壞死骨缺損手術，PLGA/TCP和PLGA/TCP/Icaritin複合材料植入缺損孔道，同時把SPIO@SiO₂-NH₂標記的間充質幹細胞注射到距離缺損區20毫米的骨髓腔內。結果顯示只有標記的間充質幹細胞植入而沒有材料植入時，缺損區被脂肪細胞充滿，並沒有標記的間充質幹細胞出現，而在缺損區附近和遠離缺損區的部位有標記的間充質幹細胞出現。同時植入PLGA/TCP複合材料和標記的間充質幹細胞時，標記的間充質幹細胞出現在缺損區的材料中，在缺損區附近沒有標記的間充質幹細胞出現，而在遠離缺損區的部位，有標記的間充質幹細胞出現。同時植入PLGA/TCP/Icaritin和標記的間充質幹細胞時，得到跟植入PLGA/TCP複合材料和標記的間充質幹細胞相似的結果，但是在缺損區域，SPIO陽性的間充質幹細胞數目在PLGA/TCP和PLGA/TCP/Icaritin組別中並未發現有顯著性差異。以上發現表明Icaritin和PLGA/TCP複合材料能夠在體外和體內促進間充質幹細胞的歸巢。 / 綜上所述，複合支架材料PLGA/TCP/Icaritin通過調節間充質幹細胞的歸巢和分化促進激素性骨壞死骨缺損的修復。Icaritin通過BMP2和Wnt/beta-catenin通路調解間充質幹細胞的成骨分化。這是首次研究發現Icaritin及PLGA/TCP支架材料影響骨缺損修復過程中幹細胞歸巢，但是分子細胞生物學機制還需要進一步的研究。 / Steroid-associated osteonecrosis (SAON) is a common orthopaedic problem as the pulsed steroids are frequently prescribed for the treatment of non-orthopaedic medical conditions. Histopathologically, SAON refers to death of bone. Intravascular thrombus occlusion and extravascular marrow lipid deposition cause ischemia, which leads to an inadequate repair of the bone. Recent study revealed upstream pathological mechanism at cellular and molecular level. The decrease in activity of mesenchymal stem cell (MSC) pool, apoptosis of osteocytes, and trabecular bone matrix degeneration may cause bone inadequate repair, a key pathological feature found in SAON. / MSCs are the stromal component of bone marrow (BM) and have the potential to differentiate into several cell types. Recent studies have suggested that SAON may be a disease of bone cells and/or MSCs. With corticosteroid therapy in patients, the MSCs activity decreased and differentiation potential changed. Steroids have been also shown to produce adipogenesis in bone-marrow cells. It has been found adipogenesis of MSCs from SAON rabbits elevated (Sheng et al., 2007a) and bone defect repair was delayed in rabbits with SAON (Xie et al., 2011), this may be caused by altered MSCs potentials. All these findings imply MSCs play a vital role in SAON development and bone defect repair. It had been reported that Icaritin, an intestinal metabolite of Epimedium-derived avonoids (EF) reduced SAON incidence with inhibition of both thrombosis and lipid deposition (Zhang et al., 2009a). More recently, we found integrating Icaritin into PLGA/TCP to form PLGA/TCP/Icaritin composite scaffold could promote SAON bone defect repair and more neovascularization formed in an intramuscular implantation model, and further found PLGA/TCP scaffold only also could promote SAON bone defect repair in rabbits (Wang et al., 2012a). But the underlying mechanism remains unclear. / Bone is a highly vascularized tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate MSC specification to promote bone formation and repair. On the other hand, bone also supplies growth factors and cells for angiogenesis. The content of this thesis is divided into the following four major parts: / Part I: to study the effect and molecular mechanism of Icaritin on the differentiation of human bone marrow-derived MSCs. Human MSC was identified first by flow cytometery and result showed our cultured human MSC expressed standard surface markers of MSCs. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation ability of MSCs was not affected by Icaritin. Differentiation assay showed that without oseteogenic supplements (OS), Icaritin had no effect on osteogenic differentiation of MSCs. With presence of OS, Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs. Real- time polymerase chain reaction (RT-PCR) showed that Icaritin up-regulated osteoblastic marker genes expression during osteogenic differentiation of MSCs and inhibited adipogenic gene expression. Further studies showed that Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis. All these findings indicated Icaritin possessed osteopromotive but not osteoinductive potentials during the differentiation of MSCs. Icaritin regulated osteogenic differentiation of MSCs in BMP2 pathway dependent manner. / Part II: to evaluate the differentiation potential of MSCs derived from rabbit with SAON and the effect of Icaritin on the altered differentiation of MSCs. The results showed that Icaritin promoted osteogenic differentiation while inhibited adipogenic differentiation of MSCs derived from normal rabbit. Osteogenic differentiation potential of mesenchymal stem cells derived from rabbit with SAON declined and Icaritin partly rescued the declined osteogenic differentiation potential in dose-dependent manner. Adipogenic differentiation potential of MSCs derived from rabbit with SAON enhanced while the enhanced adipogenesis could be depressed by Icaritin. The proliferation ability of MSCs derived from rabbit with SAON declined while could not be rescued by Icaritin. VEGF expression decreased in MSCs derived from rabbit with SAON but its expression could not be influenced by Icaritin. These findings showed that the differentiation potential of MSCs destroyed during SAON development and this potential could be partially restored by Icaritin. / Part III: to evaluate the in vitro angiogenic effect of Icaritin. The proliferation, migration and tube formation ability of human umbilical vein cells (HUVECs) were detected. The results showed that Icaritin did not affect HUVECs proliferation, migration and tube-like structure formation of HUVECs. Real time PCR showed that VEGF, HIF1a, FGF2 and TGF-beta expression in HUVECs was not changed when HUVECs were treated by Icaritin. These data indicated Icaritin did not directly impact angiogenesis in vitro. Combined with in vivo findings, we supposed Icaritin promoted angiogenesis through its enhanced osteogenesis during bone defect repair. / Part IV: to study Icaritin and scaffold impact on stem cell homing in vitro and in vivo. It was found Icaritin promoted the migration of rabbit MSCs and increased vascular cell adhesion molecule 1 (VCAM1) expression. Composite scaffolds PLGA/TCP and PLGA/TCP/Icaritin could recruit rabbit MSCs under in vitro culture condition. When labeled with SPIO@SiO₂-NH₂, the differentiation potential of rabbit MSCs retained while proliferation and migration ability of rabbit MSCs declined. Two weeks after SAON establishment, PLGA/TCP and PLGA/TCP/Icaritin scaffolds were implanted into the bone tunnel after core-decompression in initial necrotic bone defect in rabbits with SAON, immediately with SPIO@SiO₂-NH₂ labeled MSCs injected into bone marrow cavity locally. The results showed that without scaffold implantation, the tunnel was filled with fat cells and fibrotic tissues and there was no label MSC in the tunnel while there were more labeled cells appeared in bone marrow near the tunnel than far away the tunnel, with both PLGA/TCP and PLGA/TCP/Icaritin implantation, the labeled MSCs migrated into scaffold after its implantation into the bone tunnel while there was no labeled cell next to the tunnel but some were shown away from the tunnel. No significant difference was found in SPIO positive MSCs in bone tunnel between PLGA/TCP and PLGA/TCP/Icaritin group. The findings indicated that at least PLGA/TCP scaffold itself promoted MSCs homing in vitro and in vivo where the released icaritin could execute its osteopromotive effects. / In summary, the composite scaffold PLGA/TCP/Icaritin enhanced bone defect repair in rabbit with SAON by promoting homing and osteogenesis of MSCs. Icaritin promoted osteogenic differentiation of MSCs through BMP2 mediated signal pathway, such as Wnt/beta-catenin signal pathway. It is first time to report that PLGA/TCP scaffold promoted MSCs homing during bone defect repair, but underlying molecular and cellular mechanism need to be further studied. / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Yao, Dong. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 137-158). / Abstract also in Chinese; some appendixes also in Chinese. / ACKNOWLEDGEMENTS --- p.i / TABLE OF CONTENTS --- p.iii / ABSTRACT (IN ENGLISH) --- p.x / ABSTRACT (IN CHINESE) --- p.xiv / FLOWCHART --- p.xviii / LIST OF PUBLICATIONS --- p.xix / LIST OF ABBREVIATIONS --- p.xxi / LIST OF FIGURES --- p.xxiv / Chapter CHAPTER 1: --- Introduction --- p.1 / Chapter 1 --- Osteonecrosis --- p.2 / Chapter 1.1. --- Etiology --- p.2 / Chapter 1.2. --- Anatomy of femoral head --- p.3 / Chapter 1.3. --- Pathogenesis --- p.4 / Chapter 1.3.1. --- Intraosseous hypertension (Compartment Syndrome of Bone --- p.4 / Chapter 1.3.2. --- Intraosseous hypertension (Compartment Syndrome of Bone) --- p.4 / Chapter 1.3.3. --- Coagulation --- p.5 / Chapter 1.4. --- Development stages of osteonecrosis --- p.5 / Chapter 2. --- Steroids-associated osteonecrosis --- p.11 / Chapter 2.1. --- Epidemiology --- p.12 / Chapter 2.2. --- Histopathology --- p.12 / Chapter 2.3. --- Etiopathogenesis --- p.13 / Chapter 2.3.1. --- Steroid and fat metabolism --- p.14 / Chapter 2.3.2. --- Steroid and endothelial cells --- p.15 / Chapter 2.3.3. --- Steroid and coagulation --- p.16 / Chapter 2.3.4. --- Steroid and angiogenesis --- p.17 / Chapter 2.4. --- Steroid and mesenchymal stem cells (MSCs) --- p.18 / Chapter 2.5. --- Treatment strategies for SAON --- p.18 / Chapter 2.5.1. --- Prevention --- p.19 / Chapter 2.5.2. --- Nonoperative treatment --- p.19 / Chapter 2.5.3. --- Operative treatment --- p.19 / Chapter 2.5.3.1. --- Core decompression strategy --- p.20 / Chapter 2.5.3.2. --- Tissue engineering approach --- p.22 / Chapter 3. --- Epimedium-derived flavonoids (EFs) --- p.22 / Chapter 3.1. --- Icaritin -Intestinal metabolism of EFs --- p.24 / Chapter 3.1.1. --- Anti-tumor activity --- p..25 / Chapter 3.1.2. --- Neuroprotective effects --- p.25 / Chapter 3.1.3. --- Embryonic stem cells differentiation --- p.25 / Chapter 3.1.4. --- Osteogenic differentiation --- p.26 / Chapter 4. --- Poly lactic-co-glycolic acid / tricalcium phosphate (PLGA/TCP) scaffold --- p.26 / Chapter 5. --- PLGA/TCP/Icaritin --- p.28 / Chapter 6. --- Hypothesis of this study --- p.28 / Chapter 7. --- Objective --- p.29 / Chapter CHAPTER 2: --- The effect of phytomolecule Icaritin on differentiation of human mesenchymal stem cells in vitro --- p.30 / Chapter 1. --- Introduction --- p.31 / Chapter 2. --- Material and Methods --- p.33 / Chapter 2.1. --- Ethics --- p.33 / Chapter 2.2. --- Reagents and cell culture --- p.33 / Chapter 2.3. --- Surface phenotypes of human BM-MSCs --- p.33 / Chapter 2.4. --- Osteogenic and adipogenic differentiation of human BM-MSCs treated with Icaritin --- p.34 / Chapter 2.5. --- MTT assay for proliferation of BM-MSCs --- p.34 / Chapter 2.6. --- ALP staining --- p.35 / Chapter 2.7. --- ALP activity assay --- p.35 / Chapter 2.8. --- Alizarin Red S staining --- p.35 / Chapter 2.9. --- Oil Red O staining --- p.35 / Chapter 2.10. --- Ribonucleic acid (RNA) isolation --- p.36 / Chapter 2.11. --- Reverse transcription --- p.36 / Chapter 2.12. --- Real time polymerase chain reaction (RT-PCR) --- p.37 / Chapter 2.13. --- Western blotting --- p.37 / Chapter 2.14. --- Osteogenetic analysis of human MSCs after the addition of BMP2 inhibitor Noggin --- p.39 / Chapter 2.15. --- Statistical analysis --- p.39 / Chapter 3. --- Results --- p.40 / Chapter 3.1. --- Characterization of surface phenotypes of human BM-MSCs --- p.40 / Chapter 3.2. --- Icaritin had no effect on human mesenchymal stem cells (MSCs) proliferation --- p..41 / Chapter 3.3. --- Icaritin promoted osteogenic differentiation of MSCs in presence of osteogenic supplement --- p.42 / Chapter 3.4. --- Icaritin enhanced mineralization in osteogenic differentiation of MSCs only in presence of osteogenic supplement --- p.44 / Chapter 3.5. --- Icaritin upregulated mRNA expression of osteoblastic marker genes during osteogenic differentiation of MSCs --- p.45 / Chapter 3.6. --- Icaritin enhanced the protein expression of BMP2 and beta-catenin, while BMP2 inhibitor Noggin reversed the Icaritin-enhanced osteogenesis --- p..48 / Chapter 3.7. --- Icaritin inhibited fat droplets formation during adipogenic differentiation of MSCs --- p.50 / Chapter 4. --- Discussion --- p.52 / Chapter 5. --- Conclusion --- p.56 / Chapter CHAPTER 3: --- Icaritin rescued abnormal differentiation potential of MSCs derived from rabbit with SAON --- p.57 / Chapter 1. --- Introduction --- p.58 / Chapter 2. --- Methods and materials --- p.59 / Chapter 2.1. --- SAON model establishment --- p.59 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.60 / Chapter 2.3. --- Osteogenic and adipogenic differentiation of rabbit BM-MSCs treated with Icaritin --- p.61 / Chapter 2.4. --- MTT Assay for Proliferation of BM-MSCs --- p.62 / Chapter 2.5. --- ALP Staining --- p.62 / Chapter 2.6. --- ALP Activity Assay --- p.62 / Chapter 2.7. --- Alizarin Red S Staining --- p.62 / Chapter 2.8. --- Oil Red O Staining --- p.63 / Chapter 2.9. --- RNA Isolation --- p.63 / Chapter 2.10. --- Reverse transcription --- p.64 / Chapter 2.11. --- Real time Polymerase chain reaction (RT-PCR) --- p.64 / Chapter 2.12. --- Western blotting performance --- p.65 / Chapter 2.13. --- Statistical analysis --- p.65 / Chapter 3. --- Results --- p.66 / Chapter 3.1. --- The osteogenic differentiation potential declined while adipogenic differentiation ability elevated of MSCs derived from SAON rabbits --- p.66 / Chapter 3.2. --- The dose-dependent effect of Icaritin on osteogenic differentiation enhancement of MSCs from normal and SAON rabbits --- p.68 / Chapter 3.3. --- Icaritin inhibited adipogenic differentiation of MSCs both derived from normal and SAON rabbits --- p..71 / Chapter 3.4. --- PPAR-γ and aP2 proteins expression increased in SAON rabbit while inhibited by Icaritin both in normal and SAON rabbit --- p.74 / Chapter 3.5. --- Proliferation ability of MSCs derived from SAON rabbit declined and Icaritin had no effect on proliferation both derived from normal and SAON rabbit --- p.75 / Chapter 3.6. --- Icaritin had no effect on the expression of VEGF which decreased in MSCs derived SAON --- p.76 / Chapter 4. --- Discussion --- p.76 / Chapter 5. --- Conclusion --- p.81 / Chapter CHAPTER 4: --- The effect of Icaritin on angiogenesis in vitro --- p.82 / Chapter 1. --- Introduction --- p.83 / Chapter 2. --- Material and Methods --- p.85 / Chapter 2.1. --- Cell culture --- p.85 / Chapter 2.2. --- Proliferation assay --- p.85 / Chapter 2.3. --- Scratch-wound healing assay --- p..86 / Chapter 2.4. --- Migration Assay --- p.86 / Chapter 2.5. --- In vitro Angiogenesis Assay --- p.87 / Chapter 2.6. --- RNA Isolation and Real-time PCR Performance --- p.87 / Chapter 2.7. --- Statistical Analysis --- p.88 / Chapter 3. --- Results --- p.88 / Chapter 3.1. --- Icaritin did not affect HUVECs migration --- p.88 / Chapter 3.2. --- Icaritin had no effect on tube formation on growth factors reduced Matrigel --- p.92 / Chapter 3.3. --- Icaritin had no effect on HUVECs proliferation --- p.94 / Chapter 3.4. --- Icaritin did not change the angiogenesis related gene expression --- p.95 / Chapter 4. --- Discussion --- p.96 / Chapter 5. --- Conclusion --- p.100 / Chapter CHAPTER 5: --- Effect of PLGA/TCP and PLGA/TCP/Icaritin composite scaffolds on stem cell homing during bone defect repair with SAON --- p.101 / Chapter 1. --- Introduction --- p.102 / Chapter 2. --- Material and Methods --- p.106 / Chapter 2.1. --- Preparation of porous PLGA/TCP/Icaritin composite scaffolds --- p.106 / Chapter 2.2. --- Primary bone mesenchymal stem cells (BMSCs) isolation and culture --- p.106 / Chapter 2.3. --- Wound healing assay --- p.107 / Chapter 2.4. --- In vitro MSCs recruitment assay of scaffolds --- p.107 / Chapter 2.5. --- MSCs labeling with SPIO@SiO2-NH2 nanoparticle --- p.108 / Chapter 2.6. --- Prussian blue staining --- p.108 / Chapter 2.7. --- MTT assay for SPIO@SiO2-NH2 labeled MSCs --- p.108 / Chapter 2.8. --- Osteogenic and adipogenic differentiation of SPIO@SiO2-NH2 labeled MSCs --- p.109 / Chapter 2.9. --- Real time PCR --- p.109 / Chapter 2.10. --- Animal model establishment --- p.109 / Chapter 2.11. --- Descriptive histology and histomorphometry --- p.110 / Chapter 2.12. --- In vivo magnetic resonance imaging (MRI) of nanoparticle-labeled MSCs --- p.112 / Chapter 2.13. --- Statistical analysis --- p.112 / Chapter 3. --- Results --- p.112 / Chapter 3.1. --- Icaritin promoted MSCs migration in vitro --- p.112 / Chapter 3.2. --- PLGA/TCP and PLGA/TCP/Icaritin recruited MSCs when incubated in vitro --- p.114 / Chapter 3.3. --- Stem cell potentials of MSC after SPIO@SiO2-NH2 labeling --- p.118 / Chapter 3.4. --- PLGA/TCP and PLGA/TCP/Icaritin promoted MSCs homing in vivo --- p.122 / Chapter 4. --- Discussion --- p.126 / Chapter 5. --- Conclusion --- p.136 / Chapter CHAPTER 6: --- Summary of the study and future research --- p.137 / Chapter 1. --- Summary of the study --- p.138 / Chapter 2. --- Limitations and further studies --- p.139 / APPENDIXES --- p.142 / REFERENCES --- p.147

OBJETIVO. Identificar fatores preditivos para o desenvolvimento da osteonecrose (ONA) em pacientes com Lúpus Eritematoso Sistêmico (LES) de diagnóstico recente. METODOLOGIA. Quarenta e seis pacientes consecutivos, de uma coorte informatizada no ambulatório de LES do serviço de Reumatologia do Hospital das Clínicas de São Paulo, participaram deste protocolo que ocorreu entre julho de 2004 e julho de 2005. Os critérios de inclusão foram: pacientes do sexo feminino; menos de cinco anos de diagnóstico de LES; e idade maior que 18 anos. Todas as pacientes foram submetidas à ressonância nuclear magnética (RNM) dos quadris para o diagnóstico de ONA, independente da sintomatologia. Variáveis clínicas foram obtidas através de prontuários médicos, entrevista e exame clínico. Variáveis laboratoriais incluíram: lipoproteínas séricas, auto-anticorpos, fatores trombofílicos e de hipofibrinólise. Densidade mineral óssea foi medida através da densitometria de dupla emissão de raios-X. Fraturas vertebrais foram investigadas através da realização de radiografias da coluna. RESULTADOS. A ONA foi encontrada em 10 das 46 pacientes. Idade, duração de doença e raça não diferiram entre pacientes lúpicas com e sem ONA. Comparações envolvendo as várias manifestações clínicas do LES, perfil lipoprotéico e de auto-anticorpos, freqüência de trombofilia e hipofibrinólise também não foram estatisticamente diferentes entre os grupos. A freqüência de pacientes com SLEDAI ?8 no ano anterior ao diagnóstico clínico de ONA foi significativamente maior (60%) do que no grupo sem ONA considerando-se o ano anterior à entrada no estudo (19,4%), p=0,011. Corroborando com esse achado, a dose cumulativa de glicocorticóide (GC) utilizada no anterior ao diagnóstico de ONA foi maior quando comparada ao ano anterior à entrada no estudo(p=0,045). Não foram observadas diferenças com relação aos dados densitométricos e radiográficos da coluna. Na análise multivariada somente o SLEDAI permaneceu como fator de risco independente para ONA (OR=6,6, IC=1,07-41,29, p=0,042). CONCLUSÃO. Este estudo revela que a atividade de doença no ano anterior ao diagnóstico clínico de ONA é fator de risco preponderante para o desenvolvimento desta complicação no LES recente. / OBJECTIVE. To evaluate predictive factors for osteonecrosis (ON) development in patients with early Systemic Lupus Erythematosus (SLE). METHODS. Forty-six consecutive SLE patients from an electronic cohort in a Lupus Clinic from the Rheumatology Division in the University of São Paulo were enrolled on this study that occurred between July 2004 and July 2005. Inclusion criteria were female gender, age > 18 years-old and less than 5 years of disease duration. All patients underwent magnetic resonance imaging (MRI) of the hips for ON diagnosis irrespective of symptoms. Clinical variables were obtained through medical records, interview and physical examination. Laboratory variables were: serum lipoproteins, autoantibodies profile, trombophilia and hypofibrinolysis factors. Bone mineral density was acquired through dual energy x-ray absorptiometry. Vertebral fractures were investigated by spine X-rays. RESULTS. ON was found in 10 of 46 patients. Age, disease duration and race did not differ between patients with and without ON. The frequency of clinical features, lipoprotein and auto-antibodies profile and frequency of trombophilia and hypofibrinolysis were also alike in the two groups. Importantly, disease activity (frequency of patients with SLEDAI ?8) in the previous year of ON clinical diagnosis was significantly higher when compared to patients without ON in the previous year of study entrance (60.0% vs. 19.4%, p=0.011). Reinforcing this finding, glucocorticoid cumulative dose used in the previous year of ON diagnosis was also higher compared to SLE without ON in the previous year of study entrance (p=0.045). Differences concerning the densitometric and radiographic data were not observed. Remarkably, in the multivariate analysis only SLEDAI remained as an independent risk factor for ON (OR=6.6, CI=1.07-41.29, p= 0.042). CONCLUSION. This study has clearly revealed that disease activity in the previous year of ON clinical diagnosis is the main predictor factor for the development of this complication in early SLE.

A osteonecrose dos ossos maxilares relacionada ao uso prolongado de bisfosfonatos (OMRB), associada a procedimentos cirúrgicos, constitui uma entidade com menos de dez anos de ocorrência na clínica estomatológica. Os bisfosfonatos (BFs) são medicamentos antireabsortivos altamente efetivos no tratamento de diversas doenças ósseas, além de metástases. Embora a maioria dos casos de OMRB tenha sido relatada após o uso de potentes BFs da terceira geração, o alendronato de sódio (ALN), um bisfosfonato de segunda geração, é amplamente utilizado no tratamento e prevenção de doenças ósseas. O presente estudo experimental in vivo visou analisar a possível presença de OMRB no processo alveolar de ratos tratados com ALN após exodontia do segundo molar inferior. Para tanto, utilizou-se 30 ratos Wistar, machos, com 7 semanas de vida, divididos em dois grupos, ALN (tratado com alendronato) e CTL (controle, tratado com solução salina). O ALN foi administrado diariamente por injeção subcutânea na dose de 2,5 mg/kg peso por 14 dias. Nesse momento, foi realizada a exodontia, havendo sido eutanasiados cinco animais de cada grupo, 7, 14 e 21 dias após a exodontia. Após a eutanásia, a região da mandíbula foi fixada em 2,5% formaldeído e 2% glutaraldeído, em tampão cacodilato 0,1M - pH 7,4 e descalcificada em EDTA a 4,13% por 30 dias. Os espécimes foram analisados em cortes corados em HE, e foi realizada histomorfometria para análise da reabsorção da crista alveolar mesial e distal, além do tecido ósseo neoformado no interior do alvéolo, que também foi analisado através de imuno-histoquímica para as proteínas não colágenas OPN e BSP e para evidenciação de vasos sanguíneos neoformados com o anticorpo anti-CD105. Os osteoclastos foram evidenciados através de histoquímica para fosfatase ácida resistente ao tartarato (TRAP). Além disso, as células e eventos da reparação foram examinados por microscopia eletrônica de transmissão. Os resultados mostraram que os animais tratados com ALN exibiram OMRB de grau zero em todos os períodos estudados, além da presença de osteoclastos latentes TRAP-positivos e histomorfometria, onde a não reabsorção das cristas alveolares ocasionaram a permanência do osso alveolar, em contato com bactérias. Observou-se ainda, um claro atraso na formação óssea, quando comparado ao controle, além da diminuição dos vasos sanguíneos nos estágios iniciais. A localização e o padrão de marcação para as proteínas OPN e BSP foram considerados normais, observados evidente marcação em áreas ósseas imaturas e em linhas cimentantes. Assim, concluiu-se que o ALN administrado neste protocolo provocou OMRB leve, provavelmente causada pela diminuição da angiogênese, presença de osso alveolar não remodelado expostos a colônias bacterianas, além da diminuição da atividade osteoblástica. / The bisphosphonate-related osteonecrosis of the jaw (BRONJ) associated with surgery procedures, is an entity with less than 10 years of occurrence in dental practice. The bisphosphonates (BFs) are anti-resorbing drugs highly effective in the treatment of several bone diseases, including metastasis. Although the majority of cases of BRONJ had been reported with the potent third generation of BFs, sodium alendronate (ALN), a second generation bisphosphonate, is widely used for treatment or prevent bone diseases as osteoporosis. The present experimental study aimed to analyze the possible presence of BRONJ in the alveolar process of rats treated with ALN following extraction of the mandibular second molar. For this, we used thirty 7-week-old male Wistar rats, divided into two groups, ALN (treated with alendronate) and CTL (control, treated with saline solution). The administration of ALN received daily subcutaneous injection at a dose of 2.5 mg/kg for 14 days. At that moment, the extraction was performed. After 7, 14 e 21 days of surgery, five animals of each group were euthanized and the mandibular region fixed in 2.5% formaldehyde + 2% glutaraldehyde in cacodylate buffer 0.1M - pH 7.4 and decalcified in 4.13% EDTA for 30 days. The specimens were morphologically analyzed in HE stained sections, and, histomorphometry was used to analyze the resorption of mesial and distal alveolar crests, as well as the bone formed into the alveolus. Immunohistochemistry for the noncollagenous proteins OPN and BSP and for CD105 to revel neoformed blood vessels was also performed. The osteoclasts were revealed through tartrate-resistant acid phosphatase (TRAP) histochemistry. Moreover, the cells and events of alveolar healing were examined by transmission electron microscopy. The results showed light degree of BRONJ in the ALN treated animals at all the studied periods, and the presence of latent osteoclasts near the non-resorbing crest alveolar. Bacteria were observed in contact with the non-resorbed alveolar bone. An evident delay in bone formation when compared to control group was also observed, as well as the decrease of blood vessels in early stages. The distribution of immature bone and in cement lines of OPN and BSP was considered normal. Thus, we concluded that the present protocol of ALN yielded light BRONJ areas, probably by reduced angiogenesis, lock of remodeling of alveolar, presence of bacterial colonies and evident decreased osteoblastic ativitity.

Die Durchblutung der Patella beruht auf 2 Zuflüssen. Einerseits Gefäße, welche im mittleren Drittel von ventral über Foramina in die Patella eintreten und außerdem Gefäße, welche am distalen Pol zwischen dem Lig. Patellae und der Gelenkfläche in die Patella eintreten und nach proximal verlaufen. Patellafrakturen können in Längs-, Quer- und Trümmerfrakturen unterteilt werden. Bei Frakturen der Patella kann im Bereich des proximalen Pols eine arterielle Minderversorgung resultieren, wohingegen der distale Pol doppelt versorgt ist und nicht affektiert erscheint. Die arterielle Hypoperfusion kann zu lokalisierten Osteonekrosen führen. Diese lokalen osteonekrotischen Areale können sich radiographisch als hyperdense Areale manifestieren. Im Rahmen dieser Studie sollte das klinische und radiologische Ergebnis nach operativ versorgter Patellafraktur untersucht werden. Dabei wurden 100 Patienten untersucht, die im Zeitraum von 1998 – 2008 operativ versorgt wurden. Davon konnten 60 Patienten im Mittel nach 60.61 ( ± 33.88) Monaten mit einem durchschnittlichen Alter 45.48 ( ± 18.51) Jahren im Rahmen eines Follow-Ups untersucht werden. Dabei wurden mehrere klinische Scores, die Patientenzufriedenheit und die visuelle Analogskala Schmerz erhoben und die vorher angefertigten radiologischen Aufnahmen ausgewertet. Diese wurden auf eventuelle Stufenbildungen, Defekte oder hyperdense Areale hin untersucht. Es zeigte sich bei 9 Patienten (9%) ein hyperdenses Areal im Bereich des proximalen Pols welches durchschnittlich 1 – 2 Monate nach der operativen Versorgung nachgewiesen wurde. Bei 7 Patienten war dieses Areal nach 6 Monaten nicht mehr nachweisbar. Bei 2 Patienten mit bestehenden hyperdensen Arealen konnten diese im Verlauf mittels MRT respektive einer histologischen Untersuchung gesichert werden. Es zeigte sich insgesamt kein signifikanter Unterschied zwischen Patienten ohne radiologische Auffälligkeiten und mit hyperdensen Arealen. Jedoch wurden für die Gruppe mit hyperdensen Arealen im Vergleich tendentiell schlechtere Werte notiert. Radiologisch hyperdense Areale im Bereich des proximalen Pols bei Zustand nach operativer Versorgung einer Patellafraktur können eine Osteonekrose repräsentieren. Eine frühere operative Versorgung bzw. schonendere Operationstechniken könnten die Entstehung von osteonekrotischen Arealen verhindern. Zur Bestätigung dieser Hypothese sind weitere Untersuchungen notwendig.