Pesquisas prévias evidenciaram que a desmineralização óssea com ácido cítrico melhora a consolidação de enxertos autógenos em bloco e favorece o espalhamento e a morfologia de pré-osteoblastos em cultura. Os resultados promissores encontrados com o ácido cítrico levantaram a suspeita de que a tetraciclina ácida pudesse suscitar efeitos semelhantes. Assim, este estudo se propôs a avaliar comparativamente o comportamento de células pré-osteoblásticas MC3T3-E1 cultivadas sobre superfícies ósseas de calvária de ratos desmineralizadas com tetraciclina ácida (50mg/mL) e com ácido cítrico (10%, pH1) em diferentes tempos de aplicação. Foram removidas 126 amostras ósseas bicorticais da calvária de 63 ratos Wistar adultos machos empregando broca trefina de 5 milímetros de diâmetro. As amostras foram distribuídas aleatoriamente em um dos seguintes grupos de estudo (n=18): AC 15 no qual as amostras foram desmineralizadas por ácido cítrico durante 15 segundos; AC 30, no qual as amostras foram desmineralizadas por ácido cítrico durante 30 segundos; AC 60, no qual as amostras foram desmineralizadas por ácido cítrico durante 60 segundos; TCN 15 no qual as amostras foram desmineralizadas por tetraciclina durante 15 segundos; TCN 30, no qual as amostras foram desmineralizadas por tetraciclina durante 30 segundos; TCN 60, no qual as amostras foram desmineralizadas por tetraciclina durante 60 segundos e C, grupo controle formado por amostras não desmineralizadas. Os pré-osteoblastos foram cultivados sobre as amostras por 24, 48 e 72 horas (n= 6) para serem examinadas à microscopia eletrônica de varredura. Vinte e uma amostras coletadas de outros 11 animais foram distribuídas entre os mesmos grupos e analisadas com espectroscopia de energia dispersiva (EDS) para análise da composição química superficial (n=3). A área de recobrimento superficial por células foi significantemente maior após 24 e 48 horas de cultura nos grupos AC15 (60,38% e 100% respectivamente), AC30 (99,32% e 100% respectivamente), AC60 (99,22% e 100% respectivamente), TCN15 (96,73% e 70,24% respectivamente) e TCN30 (64,72% e 57,40% respectivamente) do que nos grupos TCN60 (9,67% e 51,45% respectivamente) e C (5,99% e 31,83% respectivamente). Às 72 horas os grupos apresentaram recobrimento praticamente completo das superfícies ósseas por células, com exceção do grupo TCN60 (56,15%). As células apresentaram-se com morfologia compatível com em estágios mais avançados de diferenciação nos grupos que sofreram desmineralização do que no controle. As variações nas porcentagens anatômicas (A%) dos elementos C, O, Na, Mg, P e Ca foram insuficientes para justificar mudanças no comportamento celular. Concluiu-se que a desmineralização quer por ácido cítrico ou tetraciclina de superfícies ósseas são favoráveis para o crescimento e diferenciação de células pré-osteblásticas especialmente quando empregada conforme os grupos AC30 e TCN15. Os mecanismos por trás desses resultados ainda carecem de elucidação. / Previous researches have demonstrated that bone demineralisation by citric acid improves the consolidation of bone autografts and promotes spreading and morphology of pre-osteoblasts in culture. The promising results with citric acid raised the suspicion that tetracycline could elicit similar effects. Thus, the aim of this study was to comparatively evaluate the behavior of pre-osteoblastic MC3T3-E1 cultured on bone surfaces of rat calvaria demineralized with tetracycline (50mg / ml) and citric acid (10%, pH1) at different times of application. 126 bicortical samples were removed from the calvarial bone of 63 adult male Wistar rats using trephine drill of 5 mm in diameter. Samples were randomly assigned to one of the following study groups (n = 18) AC 15 in which the samples were demineralized by citric acid for 15 seconds; AC 30, in which the samples were demineralized by citric acid for 30 seconds; AC 60 wherein the samples were demineralized by citric acid for 60 seconds; TCN 15 in which the samples were demineralized tetracycline for 15 seconds; TCN 30, in which the samples were demineralized tetracycline for 30 seconds; TCN 60 wherein the samples were demineralized tetracycline for 60 seconds, and C, control group of samples not demineralized. The pre-osteoblasts were cultured on the samples for 24, 48 and 72 hours (n = 6) to be examined in the scanning electron microscope. Twenty-one samples were collected from other 11 animals and were distributed among the same groups for analysis of the surface chemical composition by energy dispersive spectroscopy (EDS) (n = 3). The average percentage of the bone surfaces covered by cells was significantly higher after 24 and 48 hours of culture in groups AC15 (60.38% and 100% respectively), AC30 (99.32% and 100% respectively), AC60 (99.22% and 100% respectively) TCN15 (96.73% and 70.24% respectively) and TCN30 (64.72% and 57.40% respectively) than in groups TCN60 (9.67% and 51.45% respectively), and C (5.99% and 31.83% respectively). At 72 hours, all the groups presented almost complete covering of the surfaces by cells, with the exception of TCN60 group (56.15%). Cells presented with morphology compatible with more advanced stages of differentiation in groups undergone to demineralization than control. Variations in the anatomical percentages (A%) of the elements C, O, Na, Mg, Ca and P were insufficient to justify changes in cell behavior. The conclusion was that both demineralization of citric acid or tetracycline are favorable for the growth and differentiation of pre-osteoblasts especially when used according to AC30 and TCN15 groups. The mechanisms behind these results still need elucidation.

Dentre os vários compostos utilizados na pesquisa e na terapia de doenças osteo-degenerativas, a fototerapia com laseres de baixa potência (LLLT) e os diodos emissores de luz (LEDs) vem sendo investigada com o intuito de avaliar seus efeitos no metabolismo ósseo. Estes, que possuem comprimentos de ondas específicos, atuam na biomodulação das células, funcionando como um agente terapêutico, reequilibrando e normalizando a sua atividade. No entanto, pouco se sabe sobre o efeito dos diferentes espectros na proliferação e diferenciação de osteoblastos humanos, bem como seus efeitos no metabolismo celular como a síntese e a ativação de proteínas sinalizadoras envolvidas nesses processos. Diante disso, o objetivo deste trabalho foi avaliar, comparativamente, a influência da fototerapia com LLLT e LED na proliferação e diferenciação de osteoblastos humanos. Além disso, investigamos o envolvimento da ativação da via de sinalização ERK1,2 nestas respostas, utilizando o seu inibidor específico e/ou avaliando a sua ativação durante a proliferação e após fototerapia. Para esse estudo, osteoblastos humanos (HOAL) foram cultivados em meio de cultura DMEM suplementado com 10% de soro fetal bovino (SFB) e incubados em estufa de CO2. As células foram irradiadas pontualmente com os laseres vermelho (660nm), infravermelho (780nm) e LED (637nm), nas doses de 10, 20 e 50 J/cm2 na potência de 40mW, após adesão celular. Após 24, 48, e 72 horas foram realizados os ensaios de redução do MTT (brometo de 3-(4,5-dimetiltiazol-2-yl)-2,5- difeniltetrazólio) e cristal violeta (CV) para avaliar a viabilidade das células e após 72 horas foi realizada a análise da proliferação por citometria de fluxo nos quais os resultados sugerem aumento de células viáveis ou proliferação quando estimuladas pelos diferentes espectros. Após a verificação do efeito positivo dos laseres e LED na viabilidade e/ou proliferação, foi realizada a análise da ativação da proteína intracelular ERK por western blotting usando anticorpo específico após 10 minutos da irradiação pontual. Mostramos que a irradiação das células HOAL com LLLT, na dose de 10 J/cm2, aumentou significativamente a fosforilação da ERK1/2 em relação ao controle. Para os ensaios de diferenciação, as células receberam pontualmente o estimulo a cada 6 dias e após os períodos de 07, 14, 21 e 28 dias foram realizados os ensaios da atividade de fosfatase alcalina (ALP), expressão gênica do colágeno I (COL1A1) e SPARC (osteonectina) por RT-PCR em tempo real e ensaio de mineralização com vermelho de alizarina. De um modo geral, não foram observados diferenças na atividade da ALP entre os grupos tratados em relação ao controle. A expressão gênica do COL1A1 e SPARC foi aumentada, no qual o LED em ambas doses apresentou maior eficácia em aumentar a expressão desses genes. No que se refere à mineralização, foram observadas pequenas diferenças quanto a deposição de cálcio. Dessa forma, a LLLT e LED, nesse regime de aplicação modulou positivamente o metabolismo dos osteoblastos humanos em relação à viabilidade, porém, no processo de mineralização foram observadas poucas diferenças. / Among the various compounds used in research and bone degenerative diseases therapy, phototherapy with low level laser (LLLT) and light emitting diodes (LEDs) has been investigated in order to evaluate its effects on bone metabolism. Those, who have specific wavelengths, act in biomodulation cells functioning as a therapeutic agent, rebalancing and normalizing their activity. However, little is known about the effect of the different spectra in the proliferation and differentiation of human osteoblasts and their effects on cellular metabolism as well as the synthesis and activation of signaling proteins involved in these processes. Therefore, the aim of this study was to compare the influence of LLLT and LED phototherapy in the proliferation and differentiation of human osteoblasts. In addition, we investigated the involvement of activation of ERK1,2 signaling pathway these responses using its specific inhibitor and/or evaluating their activation during the proliferation and after phototherapy. For this study, human osteoblasts (HOAL) were cultured in DMEM culture medium supplemented with 10 % fetal bovine serum (FBS) and incubated in CO2 incubator . Cells were irradiated with punctual red lasers (660nm), infrared (780nm) and LED (637nm) at doses of 10, 20 and 50 J/cm2 in power 40mW, after cell adhesion. After 24, 48, and 72 hours, MTT assay (- (4,5- dimethylthiazol-2- yl) -2,5 - diphenyltetrazolium bromide 3 ) and violet crystal (CV) were performed to assess the viability of cells and after 72 hours, was performed of proliferation analysis by flow cytometry. The results suggest an increase in viable and proliferation of cells when stimulated by different spectra. After checking the positive effect of lasers and LED viability and/or proliferation, analysis of ERK activation of intracellular protein by western blotting using a specific antibody was performed 10 minutes after the spot irradiation. We show that irradiation of HOAL cells with LLLT at a dose of 10 J/cm2, significantly increased the phosphorylation of ERK1/2 compared to control. For differentiation assays, cells promptly received stimulation every 6 days and after periods of 07, 14, 21 and 28 days testing the activity of alkaline phosphatase (ALP), gene expression of type I collagen (COL1A1) and SPARC (osteonectin) were performed by Real Time RT-PCR and mineralization experiment with alizarine red. In general, no differences in the activity of ALP between the treated groups compared to the control were observed. The gene expression was increased, and SPARC COL1A1, where in the LED in both doses showed greater effectiveness in increasing the expression of these genes. With respect to mineralization, small differences in the deposition of calcium were observed. Thus, LLLT and LED, this enforcement regime, positively modulated the metabolism of human osteoblasts during the cell viability, but in the process of mineralization few differences were observed.

<p>Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. </p><p>Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. </p><p>Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. </p><p>The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation.</p><p>The skeletal phenotype of IL-13^-/-, IL-4^-/-IL-13^-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4^-/-IL-13^-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. </p><p>Callus formation in IL-4^-/-IL-13^-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4^-/-IL-13^-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4^-/-IL-13^-/- mice. </p><p>In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis <i>in vitro</i>. They also selectively influence cortical bone formation <i>in vivo</i>. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation. </p>

Cytokines play important roles in bone metabolism, participating in the complex interplay necessary for normal bone formation and turnover. The aim of the present thesis was to investigate the effects of two anti-inflammatory cytokines, interleukin-4 (IL-4) and interleukin-13 (IL-13) on bone. Influence of pro- and anti-inflammatory cytokines on interleukin-6 (IL-6) formation in cultured human osteoblasts (hOBs) was investigated. IL-4 and IL-13 as well as interleukin-1 (IL-1) and tumour necrosis factor alpha and beta (TNF-α/β) stimulated IL-6 secretion in hOBs. Also, IL-4 and IL-13 synergistically potentiated the effect of IL-1 and TNFs on IL-6 secretion. Effects of IL-4 and IL-13 on markers of osteoblastic activity in hOBs were investigated. IL-4 and IL-13 induced a dose-dependent increase in the formation of alkaline phosphatase (ALP) and pro-collagen type I carboxy-peptide (PICP) together with enhanced mineralization rate in hOBs. Formation of osteocalcin (OC) was unaffected. The mechanism behind inhibited proliferation by IL-4 and IL-13 in hOBs was investigated. IL-4 and IL-13 caused a dose-dependent increase in DNA-fragmentation together with escalating Caspase-3 activity in hOBs, reflecting induced apoptosis. Osteoblast apoptosis was also confirmed by TNF-α, dexamethasone and by serum starvation. The skeletal phenotype of IL-13-/-, IL-4-/-IL-13-/- and WT mice was compared. An altered cortical bone mass was detected in adult male IL-4-/-IL-13-/- mice. They displayed a reduction in cortical bone mineral content (BMC) secondary to reduced cortical thickness. Mechanical strength of the cortical bone was reduced in level with the reduction detected in BMC. Trabecular bone mineral density (tvBMD) was unaffected. Callus formation in IL-4-/-IL-13-/- and WT male mice was compared. No differences were found concerning radiological healing, biomechanical properties, callus parameters or histology. Heterotopic bone formation in IL-4-/-IL-13-/- and WT mice was compared using DXBM implants. No differences were found concerning mineralization of implants. Immuno-histology showed inhibition of autonomic nerves and lack of implant vascularization in IL-4-/-IL-13-/- mice. In summery, the two anti-inflammatory cytokines IL-4 and IL-13 influence osteoblast activity and apoptosis in vitro. They also selectively influence cortical bone formation in vivo. These findings suggest a role for IL-4 and IL-13 in osteoblast differentiation, in bone metabolism and in bone formation.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

Skeletal development is a complex event that requires a delicate balance between bone formation and bone resorption. Multiple transcription factors expressed in the bone-forming cells, osteoblasts, play crucial roles during the process of bone formation. Among them, ATF4 (Activating Transcription Factor 4) is a basic domain-leucine zipper transcriptional activator that is responsible for osteoblast differentiation, osteoblast-specific genes expression, synthesis of type I collagen, and osteoclast differentiation. Mice deficient for ATF4 are runted and exhibit severe skeletal dysplasia. Our laboratory has discovered Factor Inhibiting ATF4-mediated Transcription (FIAT), whose name was coined for its interaction with ATF4 and subsequent repression of ATF4-mediated osteocalcin gene transcription. FIAT is a leucine zipper nuclear molecule lacking a basic domain for DNA binding. We hypothesize that FIAT suppresses the bone-forming activities of osteoblasts by interacting with ATF4 and thereby blocking ATF4 attachment to the DNA to mediate downstream signalling pathways. To prove this hypothesis, we monitored the expression profiles of FIAT in parallel with ATF4 during osteoblastogenesis. Mechanism of FIAT repression of ATF4 was investigated through structure-function and mutation analysis. The physiological significance of FIAT expression in osteoblasts was studied through silencing FIAT in osteoblasts by RNA interference, as well as through characterization of two genetic mouse models: FIAT transgenic mice which overexpress FIAT in osteoblasts, and osteoblast-specific FIAT knockout mice. These studies showed that FIAT and ATF4 are co-expressed in osteoblasts, and that FIAT inhibition of matrix mineralization requires dimerization with ATF4 through the second leucine zipper. Furthermore, transgenic mice overexpressing FIAT exhibited osteopenia whereas FIAT knockout mice showed enhanced bone formation. These results support our hypothesis and demonstrate that FIAT is a key transcriptional repressor that modulates osteoblast function.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Umeå universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.