Análise microscópica do osso bovino inorgânico microgranular implantado em subcutâneo de ratos

The tissue response to porous bovine anorganic bone implanted in rat connective tissue was evaluated by subjective light microscopy analysis. Forty rats were divided into two groups: control (empty collagen capsules) and test (collagen capsule filled with 0.1g biomaterial) and killed 10, 20, 30 and 60 days after implantation. At 10 days, intense chronic inflammatory infiltrate consisting mainly of macrophages and inflammatory multinucleated giant cells (IMGC) was observed. Neutrophils, plasma cells and lymphocytes were present in discrete amounts and slowly disappeared along the repair process. Porosity of the material was filled by reaction connective tissue exhibiting IMGC. The fibrosis was more intense after 60 days and clearly higher than the control group. Thus, the material did not cause any severe adverse reactions and did not stimulate the immune system. Based on the results it could be concluded that deproteinized bovine cancelous bone was well tolerated by rat connective tissue.A resposta tecidual ao osso inorgânico bovino medular implantado em subcutâneo de rato foi avaliada por análise subjetiva através de microscopia de luz. Quarenta ratos foram divididos em 2 grupos: controle (cápsulas vazias de colágeno) e teste (cápsulas de colágeno contendo 0,1g do biomaterial) e mortos 10, 20, 30 e 60 dias após a implantação. Histologicamente, aos 10 dias, observou-se infiltrado inflamatório crônico composto por macrófagos e Células Gigantes Multinucleadas Inflamatórias (IMGC). Neutrófilos, plasmócitos e linfócitos estavam presentes de maneira discreta, desaparecendo durante o processo de reparo tecidual. A porosidade do material foi preenchida pelo tecido conjuntivo reacional mostrando as IMGC. A fibrose foi mais intensa aos 60 dias e evidentemente superior ao grupo controle. Entretanto, o material não causou reações adversas severas, não estimulando a resposta imunológica. Baseado nos resultados encontrados, concluímos que o osso inorgânico bovino medular foi bem tolerado pelo tecido conjuntivo de rato

Updating the role of matrix metalloproteinases in mineralized tissue and related diseases

Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of “smart” medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer

Signal transduction activated during differentiation of osteoblsts and osteocytes

Orientadores: Carmen Verissima Ferreira, Jose Mauro Granjeiro, Maikel PeppelenboschTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Este trabalho teve como principal objetivo investigar os mecanismos de transdução de sinal disparados durante a diferenciação de células ósseas. Desta forma, vários aspectos moleculares desse processo foram analisados. A modulação da Src kinase pela proteína tirosina fosfatase de baixo peso molecular (LMWPTP) é essencial para a diferenciação dos pré-osteoblastos induzida pelo ácido ascórbico/glicerofosfato. Outro enfoque dado nesse trabalho foi a avaliação, sob o aspecto molecular e morfológico, da diferenciação de pré-osteoblastos em ¿osteocyte-like cells¿, processo esse induzido quando o Matrigel foi utilizado como substrato. De forma inédita demonstramos que nessa condição as células produziram a proteína sonic hedgehog (Shh), a qual também foi essencial para o processo de diferenciação. A análise do perfil quinômico dessas células apontou uma prevalência das quinases envolvidas com a comunicação celular. Este fato é coerente com a super-expressão de conexina 43 observada. Além disso, observamos que RECK e TIMP-1 modulam a atividade do rearranjo da matriz extracelular durante a diferenciação de osteoblastos, bem como o requerimento das proteínas PP2A e p38 MAPK durante a adesão de osteoblastos. Adicionalmente observamos uma modulação refinada das PTPs (LMWPTP, SHP2 e PTPa) bem como do status redox celular. Os resultados em conjunto demonstram que o estudo de transdução de sinal pode fornecer informações importantes para o entendimento do funcionamento celular bem como definir alvos moleculares que podem servir como ferramentas para diferentes aplicaçõesAbstract: The main goal of this work was to investigate the signal transduction pathways triggered during the bone cells differentiation. In this way, several molecular aspects of this process were analyzed. Src kinase modulation by the low molecular weight protein tyrosine phosphatase (LMWPTP) was essential for the occurrence of the differentiation induced by ascorbic acid and glycerophosphate. We also evaluated, under molecular and morphological patterns, the differentiation of pre-osteoblasts into osteocyte-like cells induced by 3D scaffold (matrigel as substrate). Interestingly, under this condition, the cells produced sonic hedgehog (Shh), which also was essential for stimulating the differentiation signaling pathway. Kinomic profiling of these cells revealed a prevalence of kinases involved in cellular communication, which is in agreement with the overexpression of connexin 43 observed. Besides we observed that RECK and TIMP-1 modulated the extracellular matrix rearrangement and that PP2A and p38 MAPK are required for osteoblasts adhesion. In addition, we observed that during pre-osteoblasts differentiation both PTPs and cellular redox are tightly regulated. Our findings demonstrated that the signal transduction evaluation can provide important information for understanding the cell biology as well as defining molecular targets that can be useful for different applicationsDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Expanding the role of Src and protein-tyrosine phosphatases balance in modulating osteoblast metabolism : lessons from mice

6 p. : il.The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. Tyrosine phosphorylation of cytoplasmic proteins is a critical event in the regulation of intracellular signaling pathways activated by external stimuli. An adequate balance in protein phosphorylation is a major factor in the regulation of osteoclast and osteoblast activities involved in bone metabolism. However, although phosphorylation is widely recognized as an important regulatory pathway in skeletal development and maintenance, the mechanisms involved are not fully understood. Among the putative protein-tyrosine kinases (ptk) and protein-tyrosine phosphatases (ptp) involved in this phenomenon there is increasing evidence that Src and low molecular weight-ptps play a central role in a range of osteoblast activities, from adhesion to differentiation. A role for Src in bone metabolism was first demonstrated in Src-deficient mice and has since been confirmed using low molecular weight Src inhibitors in animal models of osteoporosis. Several studies have shown that Src is important for cellular proliferation, adhesion and motility. In contrast, few studies have assessed the importance of the ptk/ptp balance in driving osteoblast metabolism. In this review, we summarize our current knowledge of the functional importance of the ptk/ptp balance in osteoblast metabolism, and highlight directions for future research that should improve our understanding of these critical signaling molecules

Biological behavior of pre-osteoblasts on natural hydroxyapatite: A study of signaling molecules from attachment to differentiation

8 p. : il., tab.Several biomaterials have been widely used in bone regeneration in both orthopedic and oral surgeries. However, it is poorly understood how these biomaterials alter osteoblast phenotype. It prompted us to examine the involvement of signaling proteins during preosteoblast adhesion (attachment), proliferation, and differentiation on natural hydroxyapatite (HA) from bovine bone. Our results indicated that natural HA is able to promote osteoblast adhesion, proliferation, and differentiation. The osteoblast/HA interaction requires phosphorylation of tyrosine residues of focal adhesion kinase, Src, and Paxillin upon integrin activation, which culminates in the control of cofilin phosphorylation (at serine 03) via rac-1 activation. In part, these signaling pathways are responsible for actin-rearrangement, responsible to adapt cell-shape on HA particles. In regarding to osteoblast differentiation, we showed that natural HA favored extracellular matrix remodeling by stimulating matrix metalloproteinase activities and alkaline phosphatase activity. Overall, this study demonstrates that osteoblast response toward bovine bone HA is initially mediated by activation of focal adhesion components, culminating on actin-rearrangement executed by cofilin activation via rac-1. Moreover, bovine bone HA provided an excellent microenvironment for osteoblast activity, since adhesion to differentiation

Intracellular signal transduction as a factor in the development of ‘‘smart’’ biomaterials for bone tissue engineering

5 p. : il., tab.Signal transduction involves studying the intracellular mechanisms that govern cellular responses to external stimuli such as hormones, cytokines, and also cell adhesion to biomaterials surfaces. Several events have been shown to be responsible for cellular adhesion and adaptation onto different surfaces. For instance, cytoskeletal rearrangements during cell adhesion require the recruitment of specific protein tyrosine kinases into focal adhesion structures that promote transient focal adhesion kinase and Src phosphorylations, initially modulating cell behavior. In addition, the phosphorylation of tyrosine (Y) residues have been generally accepted as a critical regulator of a wide range of cell-related processes, including cell proliferation, migration, differentiation, survival signalling, and energy metabolism. The understanding of the signaling involved on the mechanisms of osteoblast adhesion, proliferation, and differentiation on implant surfaces is fundamental for the successful design of novel ‘‘smart’’ materials, potentially decreasing the repair time, thereby allowing for faster patient rehabilitation

Periosteal derived cells for bone bioengineering: a promising candidate

5 p. : il., tab.Purpose: Over the last years so many efforts have been made in order to indentify natural sources of osteogenic cells for the success of bone bioengineering. Among them, periosteum tissue has emerged as an interesting candidate. Thus, we decided to evaluate the osteogenic potential of periosteal-derived cells by describing a sequence of biological events since initial morphological changes to mineralization of extracellular matrix (ECM). Methods: Periosteal-derived cells were obtained from calvarial of adult rats. After the primary culture and expansion, the adherent cells were cultured at 7, 14, 21 and 28 days under a classical osteogenic culture medium in order to evaluate the differentiation of those cells in mature osteoblast. It was monitored by evaluating a time-line of alkaline phosphatase (ALP) activity (biomarker of osteoblast differentiation) and afterwards nodules of mineralization (measured by von Kossa staining and calcium content). Results: Analysis from phase-contrast microscopy revealed mainly morphological changes ranging since fibroblast-shaped (7 days, semi-confluent culture at exponential growth) to polyhedralshaped cells (14–28 days, confluent culture during differentiation process). ALP activity was linearly increased since 14–28 days while amount of protein remained unchanged. Interesting, our data from von Kossa staining reveled a highest incidence of mineralization nodules at 28 days. Conclusion: Taken our results together, we can suggest that periosteal-derived cells present an interesting potential to differentiate in mature osteoblast able to promote mineralization in vitro by incorporating to ECM circulating calcium from extracellular compartment. From our point of view, this source of osteogenic cells can be explored by bioengineers in order to advance therapeutic protocols able to solve bone degenerative lesions