O objetivo desse estudo foi investigar a participação de integrina &alpha;1&beta;1 e microRNAs (miRs) no potencial osteogênico de superfícies de titânio (Ti) com nanotopografia. Discos de Ti previamente polidos foram tratados quimicamente com H2SO4/H2O2 para obtenção de nanotopografia, que foi observada por microscopia eletrônica de varredura. Para o estudo da participação da integrina &alpha;1&beta;1, células-tronco mesenquimais (CTMs) de ratos foram cultivadas em condições osteogênicas e não osteogênicas sobre superfícies de Ti com nanotopografia e sem tratamento químico (controle). O resultados mostraram que a nanotopografia de Ti aumentou a proliferação celular, a atividade de fosfatase alcalina (Alp) e regulou positivamente a expressão gênica de marcadores da diferenciação osteoblástica em CTMs cultivadas tanto em condições osteogênicas quanto em condições não osteogênicas. Além disso, uma maior expressão gênica para as integrinas &alpha;1 e &beta;1 foi observada em culturas crescidas sobre nanotopografia em condições não osteogênicas em relação ao Ti controle. O uso de obtustatina, um inibidor de integrina &alpha;1&beta;1, reduziu os efeitos da nanotopografia sobre os marcadores osteoblásticos, indicando a participação da via de sinalização dessa integrina nos efeitos da nanotopografia sobre CTMs. Para investigar a participação de miRs no efeito osseoindutor da nanotopografia de Ti, foram utilizadas CTMs humanas e células préosteoblásticas de camundongos da linhagem MC3T3-E1. A análise em larga escala da expressão de miRs revelou que 60 miRs foram regulados positivamente (no mínimo, 2x maior), enquanto 58 miRs foram regulados negativamente (no mínimo, 2x menor) em CTMs crescidas sobre a nanotopografia. Três desses miRs, miR-4448, -4708 e -4773, cuja expressão foi significativamente reduzida pela nanotopografia de Ti (no mínimo, 5x menor), afetaram a diferenciação osteoblástica de CTMs. Esses miRs atuam diretamente sobre SMAD1 e SMAD4, proteínas transdutoras da sinalização da proteína óssea morfogenética 2 (Bmp-2), conhecida por sua capacidade osseoindutora. Além disso, verificou-se que a sobreexpressão de miR-4448, -4708 e -4773 em células pré-osteoblásticas MC3T3-E1 inibiu a expressão gênica e proteica de SMAD1 e SMAD4 e, consequentemente, a expressão gênica de marcadores ósseos. Esses dados sugerem a influência do circuito miR-SMAD-Bmp-2 sobre o efeito osseoindutor da nanotopografia. Conjuntamente, os achados do presente estudo mostraram que o efeito da nanotopografia de Ti sobre a diferenciação osteoblástica resulta de um mecanismo regulatório complexo, do qual fazem parte as vias de sinalização da integrina &alpha;1&beta;1 e da Bmp-2, com a participação de miRs. Esses resultados podem representar um avanço para o desenvolvimento de novas modificações de superfície, com o objetivo de acelerar e/ou melhorar o processo de osseointegração. / The aim of this study was to investigate the role of the &alpha;1&beta;1 integrin and microRNAs (miRs) on the osteogenic potential of titanium (Ti) with nanotopography. Polished Ti discs were chemically treated with H2SO4/H2O2 to generate nanotopography, which was observed under scanning electron microscopy. For the study related to the &alpha;1&beta;1 integrin, rat mesenchymal stem cells (MSCs) were cultured under osteogenic and non-osteogenic conditions on Ti with nanotopography and non-treated Ti discs (control). Nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of bone markers in cells cultured under osteogenic and non-osteogenic conditions. Furthermore, the gene expression of &alpha;1 and &beta;1 integrins was higher in cells cultured on nanotopography under non-osteogenic conditions compared with control. Obtustatin, an inhibitor of &alpha;1&beta;1 integrin, reduced the higher gene expression of the bone markers induced by nanotopography. These results indicate that &alpha;1&beta;1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. The role of miRs in the osteogenic potential of Ti with nanotopography was evaluated using human MSCs and MC3T3-E1 mouse pre-osteoblastic cells. The miR sequencing analysis revealed that 60 miRs were upregulated (> 2 fold), while 58 miRs were downregulated (< 2 fold) in MSCs grown on nanotopography. Three miRs, miR-4448, -4708 and -4773, which were significantly downregulated (< 5 fold) by nanotopography, affected the osteoblast differentiation of MSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (Bmp-2) osteogenic signal, which were upregulated by nanotopography. Overexpression of miR-4448 - 4708 and 4773 in MC3T3-E1 cells noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and by targeting them, these miRs repressed gene expression of key bone markers. These results suggest that a miR-SMAD-Bmp-2 circuit acts in the Ti nanotopography-mediated osteoblast differentiation. Taken together, our data showed that the osteoblast differentiation induced by Ti with nanotopography is governed by a complex regulatory network involving a crosstalk between &alpha;1&beta;1 integrin and Bmp-2 signaling pathways with participation of miRs.

A osteoporose, uma doença óssea progressiva, é considerada um grave problema de saúde pública, sendo uma das condições mais importantes associadas ao envelhecimento e que afeta milhões de pessoas no mundo. Esta doença multifatorial é caracterizada pela densidade óssea reduzida e deterioração da microarquitetura óssea. O objetivo deste trabalho foi investigar as mudanças comportamentais em células mesenquimais da medula óssea e células osteoblásticas da calvária de ratas induzidas à osteoporose. Após aprovação da Comissão de Ética no Uso de Animais, 18 ratas Wistar foram utilizadas e divididas em grupos controle e ovariectomizadas. Após 150 dias, as ratas de ambos os grupos foram sacrificadas para coleta dos fêmures e fragmentos da calvária. As células recolhidas a partir da medula óssea e calvária foram cultivadas em placas de 24 poços (n = 5) para avaliação da proliferação e viabilidade celular, atividade de fosfatase alcalina (ALP), detecção e quantificação de nódulos mineralizados e análise da expressão gênica por meio de PCR em tempo real. Os dados foram submetidos ao testes de Kruskal-Wallis e Mann-Whitney, com nível de significância de 5%. As células da medula óssea do grupo controle (MC) mostraram uma diminuição significativa na proliferação quando comparado com as células do grupo controle da calvária (CC) em todos os períodos (p < 0,05). Por outro lado, as células da medula óssea de ratas com osteoporose (MO) revelaram um aumento significativo na taxa de proliferação após 7 e 10 dias (p < 0,01) em comparação às células da calvária de ratas ovariectomizadas (CO). A viabilidade celular foi maior em todos os períodos estudados dos grupos CC e CO em relação aos grupos MC e MO (p < 0,05). A atividade de fosfatase alcalina não foi significativamente diferente após 7 dias de cultura entre os grupos estudados; por outro lado, após 10 e 14 dias, observou-se uma diminuição da sua atividade no grupo MC quando comparado ao grupo CC (p < 0,01). Nas ratas com osteoporose, as células da medula óssea mostraram um aumento desta atividade quando comparada às células de calvária (p < 0,01). A análise dos nódulos mineralizados após 14 e 21 dias revelou que os grupos controle CC e MC não apresentaram diferenças significativas, ao passo que no grupo MO observou-se um aumento da mineralização quando comparado ao grupo CO nos mesmos períodos experimentais. Os resultados obtidos na análise de expressão gênica mostraram que para os genes Runx2, Oc, Alpl, Rank-l e Er&alpha; a expressão no grupo MO foi maior que no grupo MC (p < 0,05), enquanto que para as células da calvária, CC teve maior expressão gênica que CO (p < 0,05). Os genes Opg e Er&beta; apresentaram variações de acordo com o grupo avaliado. Frente aos resultados obtidos, sugere-se que existam alterações no metabolismo das células progenitoras e células diferenciadas após a indução da osteoporose e que as células da medula óssea apresentam um aumento da sua função como uma resposta compensatória neste modelo animal ovariectomizado. / Osteoporosis, a progressive bone disease, is considered a serious public health problem, being one of the most important conditions associated with aging, affecting millions of people worldwide. This multifactorial disease is characterized by reduced bone density and deterioration of bone microarchitecture. The objective of this work was to investigate the behavioral changes in mesenchymal cells from bone marrow and osteoblastic cells from calvaria bone of female rats induced to osteoporosis. After institutional review board approval, 18 Wistar female rats were used and divided into control and ovariectomized groups. After 150 days, the rats of both groups were sacrificed for collection of femurs and calvariae fragments. The cells collected from bone marrow and calvaria were cultured in 24-well plates (n=5) for the assessment of cell proliferation and viability, alkaline phosphatase (ALP) activity and detection and quantification of mineralized nodules. The data were submitted to Kruskal-Wallis and Mann-Whitney tests, with significance level set at 5%. The cells from bone marrow control group (MC) showed a significantly decrease in proliferation when compared to the cells from calvaria bone control group (CC) in all periods evaluated (p < 0,05). On the other hand, bone marrow cells from osteoporotic rats (MO) revealed a significant increase in their proliferation rate after 7 and 10 days (p < 0,01) compared to calvaria cells from ovariectomized rats (CO). Cell viability was higher in all investigated periods of CC and CO groups compared to MC and MO groups (p < 0,05). Alkaline phosphatase activity was not significantly different after 7 days of culture among the studied groups; in spite of that, after 10 and 14 days, there was a decrease in its activity in MC group when compared to CC (p < 0,01). In the osteoporotic rats, bone marrow cells showed an increase in this activity when compared to calvaria cells (p < 0,01). The analysis of mineralized nodules after 14 and 21 days revealed that control groups (CC and MC) did not have significant differences, whereas it was observed in MO group an increase in the mineralization when compared to CO in the same experimental periods. The data obtained in the analysis of gene expression showed that for Runx2, Oc, Alpl, Rank-l and Er&alpha; genes, the expression in MO group was higher than in MC (p < 0,05), whereas for the calvaria cells, CC group had higher gene expression than CO group (p < 0,05). The Opg and Er&beta; genes showed variations according to the evaluated group. Thus, it is suggested that there are changes in the metabolism of progenitor and differentiated cells after osteoporosis induction and that bone marrow cells present an enhancement of their function as a compensatory response in this ovariectomized rat model.

Este estudo analisou a influência de diferentes superfícies de titânio na adesão, proliferação e diferenciação de células semelhantes a osteoblastos de rato (osteo-1) em culturas, na presença ou não da proteína morfogenética óssea recombinante-2 (rhBMP-2). As células osteo-1 foram cultivadas sobre as seguintes superfícies de titânio: 1. superfície lisa, 2. superfície desgastada com partículas de areia e condicionamento ácido (SLA) e 3. superfície desgastada com partículas de areia e condicionamento ácido sob proteção de nitrogênio e armazenadas em solução isotônica de cloreto de sódio (SLActive), na presença ou não de 20 ng/ml de rhBMP-2. Foram analisadas a adesão celular em 24 horas, o conteúdo total de proteínas, o conteúdo de colágeno e a atividade de fosfatase alcalina em 7, 14 e 21 dias e a formação de nódulos calcificados em 21 dias. Os resultados mostraram que a adesão não foi influenciada nem pelo tipo de superfície nem pelo tratamento com rhBMP-2 (p=0,0936). Quando relacionamos o conteúdo total de proteínas ao número total de células, percebemos que a proliferação não foi influenciada pelo tipo de superfície de titânio, porém a adição de rhBMP-2 levou a uma redução estatisticamente significante na superfície SLA aos 21 dias (p=0,0000). Em relação à diferenciação, pudemos observar que o tipo de superfície não influenciou o conteúdo total de proteínas, o conteúdo de colágeno e a formação de nódulos calcificados em quaisquer dos períodos analisados. A atividade de fosfatase alcalina somente foi influenciada pelo tipo de superfície aos 14 dias, onde o grupo C/SLAactive apresentou valores inferiores ao grupo C/Liso (p=0,0000). A adição de rhBMP-2 promoveu uma maior influência sobre o processo de diferenciação, levando a uma redução estatisticamente significante no conteúdo total de proteínas na superfície SLA aos 21 dias (p=0,0000), a um aumento estatisticamente significante no conteúdo de colágeno na superfície SLActive no período de 7 dias (p=0,0005) e a uma diminuição estatisticamente significante na atividade de fosfatase alcalina na superfície lisa nos períodos de 14 e 21 dias, na superfície SLA aos 14 dias e na superfície SLActive aos 21 dias (p=0,0000). Somente a formação de nódulos calcificados não sofreu influência da adição de rhBMP-2. / This study has analyzed the influence of different titanium surfaces in the adhesion, proliferation and differentiation of rat osteoblast-like cells (osteo-1 culture), in the presence or not, of the recombinant bone morphogenetic protein-2 (rhBMP-2). The osteo-1 cells were grown on the following titanium surfaces: 1. smooth surface; 2. coarse grit-blasted and acid-etched surface (SLA); and 3. coarse grit-blasted and acid-etched surface under nitrogen protection, and stored in sodium chloride isotonic solution (SLActive), in the presence or not, of 20 ng/ml of rhBMP-2. It was analyzed the cell adhesion in 24 hours, the total protein content, the collagen content, and the alkaline phosphatase in 7, 14 and 21-day periods, and also the formation of calcified nodules in 21 days. The results showed that the adhesion was neither influenced by the surface type, nor by the treatment with rhBMP-2 (p=0.0936). When we related the total protein content to the total number of cells, we noticed that the proliferation was not influenced by the titanium surface type; however, the addition of rhBMP-2 led to a statistically significant reduction on the SLA surface at 21 days (p=0.0000). Concerning the differentiation, we could observe that the surface type did not influence the total content of proteins, the collagen content and the formation of calcified nodules in any of the analyzed periods. The alkaline phosphatase activity was only influenced by the surface type at 14 days, where the group C/SLActive presented lower values than the group C/Smooth (p=0.0000). The addition of rhBMP- 2 promoted a bigger influence over the differentiation process, thus leading to a statistically significant reduction in the total protein content on the SLA surface at 21 days (p=0.0000), a statistically significant increase in the collagen content on the surface SLActive in the 7-day period (p=0.0005), a statistically significant reduction in the alkaline phosphatase activity on the smooth surface in the 14 and 21-day periods, on the SLA surface at 14 days, and on the SLActive surface at 21 days (p=0.0000). Only the formation of calcified nodules did not undergo influence of the rhBMP-2 addition.

In cases of compromised bone remodeling like osteoporosis, insufficient osseointegration occurs and results in implant failure. Implant retention relies on proper secondary fixation, which is developed during bone remodeling. This process is disrupted in metastatic bone diseases like osteoporosis. Osteoporosis is characterized low bone mass and bone strength resulting from either accelerated osteoclast-mediated bone resorption or impaired osteoblast-mediated bone formation. These two processes are not independent phenomena. In fact, osteoporosis can be viewed as a breakdown of the cellular communication connecting bone resorption to bone formation. Because bone remodeling occurs at temporally generated specific anatomical sites and at different times, local regulators that control cross-talk among the cells of the BRU are important. Previous studies show Ti implant surface characteristics like roughness, hydrophilicity, and chemistry influence the osteoblastic differentiation of human MSCs and maturation of OBs. Furthermore, microstructured Ti surfaces modulate the production of factors shown to be important in the reciprocal communication necessary for the maintenance of healthy bone remodeling. Semaphorin signaling proteins are known to couple the communication of osteoblasts to osteoclasts and are capable of stimulating bone formation or bone resorption depending on certain cues. Implant surface properties can be optimized to exploit these effects to favor rapid osseointegration in patients with osteoporosis.

Paget’s disease is characterised by focal regions of accelerated bone turnover. The aetiology is unknown, but genetic and environmental factors have been implicated. Pagetic lesions contain increased numbers of osteoclasts with abnormal morphology, so an osteoclast defect has been considered central to the pathogenesis. However, given osteoblasts regulate osteoclast differentiation and activity; osteoblast abnormalities may be important in the disease. This study aimed to identify features of pagetic osteoblasts that could clarify their role in Paget’s disease. Gene expression in osteoblasts and bone marrow cultured from pagetic lesions of 23 patients was compared to cells from unaffected tissue using both microarrays and real time RT-PCR. The results indicated global changes in gene expression in pagetic osteoblasts. A number of genes that can stimulate osteoclastogenesis, including interleukins 6 and 1β, and monocyte chemotactic factor 1 were up-regulated, but the RANKL/OPG ratio tended to be decreased. Genes involved in osteoblast differentiation were down-regulated, including the transcription factors RUNX2, DLX5 and SATB2, the osteogenic factor BMP2, and the matrix proteins osteocalcin and bone sialoprotein. Markers of less mature osteoblastic cells, alkaline phosphatase and matrix gla protein were up-regulated. The intermediate filament, keratin 18, was very significantly up-regulated in pagetic cells. Over-expression of this protein in osteoblasts using an adenoviral vector produced some changes in gene expression, but did not produce an overtly pagetic phenotype. Over-expression of SQSTM1 mutants found in some patients with Paget’s disease also produced only minor changes in osteoblast phenotype. The RNA from the primary cell cultures was also used to investigate the presence of measles virus and somatic mutations in SQSTM1 in the disease, but neither were identified in any of the patients. These results suggest that there are important changes in pagetic osteoblasts that are maintained when the cells are removed from the affected bone microenvironment. These include enhanced production of factors to stimulate osteoclastogenesis, while osteoblast differentiation and activity may be impaired. We were unable to identify genetic or environmental factors that could trigger these changes. The pagetic osteoblast is distinct from control cells, and is likely to contribute to the development of Paget’s disease.

Bone tissue is continuously degraded and rebuilt to respond to the needs of the body. Cells of the osteoblast lineage are responsible for the formation of bone, whereas the resorption of bone tissue is carried out by osteoclasts. To prevent imbalance between bone formation and resorption, these processes are delicately regulated by a complex network of both systemic factors and factors produced locally in the bone microenvironment, including members of the IL-6 family of cytokines. During the last decades, the presence of nerve fibers in skeletal tissue and presence of receptors for several neurotransmitters on both osteoblasts and osteoclasts, have suggested a possible role for neuropeptides in the regulation of skeletal metabolism. The overall aim of this study was to investigate the roles of cytokines in the IL-6 family and the neuropeptide VIP in regulation of osteotropic cytokine expression and bone metabolism in vitro. In Paper I, stimulation of bone resorption by the cytokine IL-6, in the presence of its soluble receptor sIL-6R, was demonstrated in mouse calvarial bones. OSM and LIF, other members of the IL-6 family of cytokines, were also shown to increase bone resorption. Furthermore, IL-6+sIL-6R, LIF, and OSM increased the expression of RANKL, which by binding to its receptor RANK functions as a crucial inducer of osteoclast formation and activation. In Paper II-IV, the effects of the neuropeptide VIP and related peptides on expression of osteotropic cytokines by osteoblasts and bone resorption in vitro have been studied. VIP and PACAP-38 both increased IL-6 production in osteoblasts in a time- and concentration-dependent manner. In contrast, no effect was seen with the related peptide secretin, indicating that the effects were mediated by the VPAC2 receptor. VIP and PACAP, in contrast to secretin, also induced IL-6 promoter activity in osteoblastic MC3T3-E1 cells transfected with an IL-6 promoter/luciferase construct. The effects of VIP on IL-6 were shown to be mediated by several intracellular pathways, including cAMP/PKA/CREB, AP-1, and C/EBP, but not NF-kB or the cAMP-activated Epac pathway. The release of IL-6 from osteoblasts was increased by several pro-inflammatory osteotropic cytokines, including interleukin-1b, an effect that was further potentiated by VIP, indicating a possible neuro-immunomodulatory interaction in the regulation of bone metabolism. VIP and PACAP-38 also increased the osteoblastic expression of RANKL and decreased the expression of OPG and M-CSF, factors crucial in regulation of differentiation and activation of osteoclasts. Although this indicated a possible bone resorptive effect, VIP was found to decrease osteoclast formation and bone resorption by directly targeting osteoclast progenitor cells through an inhibitory mechanism. In conclusion, the results in this thesis indicate that several cytokines in the IL-6 family stimulate bone resorption in calvarial bones in vitro, most likely through the RANKL-RANK interaction. Furthermore, expression of the osteotropic cytokine IL-6 in osteoblasts is stimulated by the neuropeptide VIP through VPAC2 receptors via several intracellular pathways, further strengthening the role of neuropeptides as local regulators of bone metabolism.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.

Introduction: The objective of this study is to investigate the role of osteoblasts in the pathophysiology of premature suture fusion in infants. Methods: Bone and periosteal tissue from fused and patent cranial sutures and adjacent bone were harvested from infants undergoing surgery for craniosynostosis and used to develop primary osteoblast cell cultures. Dural tissue was obtained from neurosurgical procedures in order to generate an osteoblast-dural co-culture. Osteoblast proliferation, differentiation, mineralization, protein expression (Noggin, BMP3 and Runx2) and response to exogenous FGF2 stimulation were assessed. Results: Cell cultures demonstrated significant (p<0.05) regional variations in osteoblast proliferation, differentiation markers and in vitro bone nodule formation. The expression of anti-osteogenic molecules (Noggin and BMP3) was decreased in osteoblasts from fused suture regions. Conclusion: The creation of a pro-osteogenic environment through the decreased expression of anti-osteogenic signalling molecules and increased expression of osteogenic factors may be responsible for premature suture fusion in infants.

Fracture repair is a regenerative event that involves the precise coordination of a variety of cells for successful healing process. Within the microstructure hierarchy of bone repair, the predominant cells involved include the chondrocytes, osteocytes, osteoblasts, and osteoclasts. Although the role of osteoblasts during fracture healing has been previously shown, their role during the initiation phase of endochondral fracture repair remains unclear. In order to study the role of osteoblasts during fracture repair, we used a transgenic mouse model expressing the herpes simplex virus thymidine kinase gene in early differentiating osteoblasts, which allows conditional ablation of cells in osteoblastic lineage upon treatment with the Gancicolvir drug. Results from this study suggest that not only are osteoblasts required in later stages of fracture repair as the medium for bone synthesis, and osteoclast activation during bone remodelling, but could also be required for the initiation and advancement of the endochondral ossification process.