Uranium deposition in bones of wistar rats associated with skeleton development

Sixty female Wistar rats were submitted to a daily intake of ration doped with uranium from weaning to adulthood. Uranium in bone was quantified by the SSNTD (solid state nuclear track detection) technique, and bone mineral density (BMD) analysis performed. Uranium concentration as a function of age exhibited a sharp rise during the first week of the experiment and a drastic drop of 70% in the following weeks. Data interpretation indicates that uranium mimics calcium. Results from BMD suggest that radiation emitted by the incorporated Uranium could induce death of bone cells. © 2013 Elsevier Ltd.

Influência do treinamento resistido e do raloxifeno no metabolismo ósseo de ratas Wistar

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do cimento de alfa-fosfato tricálcico em artrodeses tarsocrurais experimentais em cães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeitos da intrusão ortodôntica na reparação de lesões de furca grau III em cães, e da presença de TNFα e/ou IL-β 1 na mecanoresposta de células ósseas in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inhibition of osteoblast activity by zoledronic acid

INTRODUCTION: Patients treated with nitrogen-containing bisphosphonates, such as zoledronic acid (ZA), have frequently shown oral bone exposure areas, termed osteonecrosis. In addition, these patients may also present low repair and regeneration potential, mainly after tooth extractions. These side-effects caused by bisphosphonates may be due to their inhibitory effects on oral mucosa and local bone cells. OBJECTIVE: To evaluate the effects of ZA on the mineralization capacity of cultured osteoblasts. MATERIALS AND METHODS: Human immortalized osteoblasts (SaOs-2) were grown in plain culture medium (Dulbecco's Modified Eagle Medium [DMEM] + 10% fetal bovine serum [FBS]) in wells of 24-well plates. After 48-hour incubation, the plain DMEM was replaced by a solution with ZA at 5 µM which was maintained in contact with cells for seven, 14 or 21 days. After these periods, cells were evaluated regarding alkaline phosphatase (ALP) activity and mineral nodule formation (alizarin red). Data were statistically analyzed by Mann-Whitney test, at 5% of significance level. RESULTS: ZA caused significant reduction on ALP activity and mineral nodules formation by cultured osteoblasts in all evaluated periods (p < 0.05). CONCLUSION: These data indicate that ZA causes inhibition on the osteogenic phenotype of cultured human osteoblasts, which, in turn, may reduce bone repair in patients subjected to ZA therapy.

Aspectos biológico-celulares da osseointegração baseados nas superfícies de implantes

Osseointegration involves a cascade of biological events, which can be accelerated by modifying the micro and/or nanometric topography of dental implant surfaces. Considering that different treatment types modify the titanium surface giving it a more pronounced rough topography, and physicochemical changes that appear to positively influence the osseointegration process, a literature review was made on the main types of surface treatments and their influence on the biological and cellular aspects of osseointegration, with publications dating from 1969 until the present moment. Although the precise role of the implant surface on the osseointegration of dental implants is not completely clear, the specific effects of implant surface on bone regeneration, initial kinetics, and evolution of mechanical properties have shown to be quite promising. Thus, based on dental implant surface modifications, osseointegration can be defined as a process by which rigid asymptomatic fixation of an alloplastic material can be achieved and kept in close contact with bone tissue, being resistant to early and late functional loads. This process can be modulated by an appropriate treatment of the alloplastic material surface.

Controllo molecolare del differenziamento osteoblestico per applicazioni nel campo della rigenerazione del tessuto osseo

The aim of the study was to identify expression signatures unique for specific stages of osteoblast differentiation in order to improve our knowledge of the molecular mechanisms underlying bone repair and regeneration. We performed a microarray analysis on the whole transcriptome of human mesenchymal stem cells (hMSCs) obtained from the femoral canal of patients undergoing hip replacement. By defining different time-points within the differentiation and mineralization phases of hMSCs, temporal gene expression changes were visualised. Importantly, the gene expression of adherent bone marrow mononuclear cells, being the undifferentiated progenitors of bone cells, was used as reference. In addition, only the cultures able to form mineral nodules at the final time-point were considered for the gene expression analyses. To obtain the genes of our interest, we only focused on genes: i) whose expression was significantly upregulated; ii) which are involved in pathways or biological processes relevant to proliferation, differentiation and functions of bone cells; iii) which changed considerably during the different steps of differentiation and/or mineralization. Among the 213 genes identified as differentially expressed by microarray analysis, we selected 65 molecular markers related to specific steps of osteogenic differentiation. These markers are grouped into various gene clusters according to their involvement in processes which play a key role in bone cell biology such as angiogenesis, ossification, cell communication, development and in pathways like TGF beta and Wnt signaling pathways. Taken together, these results allow us to monitor hMSC cultures and to distinguish between different stages of differentiation and mineralization. The signatures represent a useful tool to analyse a broad spectrum of functions of hMSCs cultured on scaffolds, especially when the constructs are conceived for releasing growth factors or other signals to promote bone regeneration. Morover, this work will enhance our understanding of bone development and will enable us to recognize molecular defects that compromise normal bone function as occurs in pathological conditions.

Die Rolle des Fibronektins im Knochen und in der diabetischen Nephropathie

Diese Arbeit befasst sich mit der Rolle des Fibronektins im Knochen sowie in der diabetischen Nephropathie. Fibronektin im Knochen: Es war bekannt, dass Osteoblasten für ihre Differenzierung in vitro Fibronektin benötigen, dass Fibronektin für die Ausbildung einer Kollagenmatrix erforderlich ist und für die Matrixintegrität eine kontinuierliche Fibronektin-Versorgung gewährleistet sein muss. Um die Rolle des Fibronektins im Knochen, dessen Matrix zu 90% aus Kollagen besteht, näher zu untersuchen, wurde das Fibronektin der Osteoblasten spezifisch über das Cre/loxP-System in Mäusen ausgeschaltet. Dies führte zu einer erhöhten Anzahl an Osteoblasten, deren Fähigkeit die Matrix zu mineralisieren jedoch beeinträchtigt war. Dennoch zeigte sich kein Einfluss auf die Eigenschaften der Knochenmatrix. Insbesondere war der Fibronektingehalt nicht vermindert, entgegen der allgemeinen Annahme, dass die Osteoblasten die Produzenten des Fibronektins der Knochenmatrix seien. Im Gegensatz dazu stellte sich durch Untersuchungen an anderen genetisch veränderten Mäusen heraus, dass eine Ausschaltung des Plasmafibronektins im Blut zu einer deutlichen Verringerung des Fibronektingehalts des Knochens sowie zu einer Verminderung des Mineralgehalts bezogen auf die Proteinmenge führte. Auch die Komposition des Minerals war verändert. Da es jedoch keinen nennenswerten Effekt auf die Knochenzellen gab, lässt sich schlussfolgern, dass die Osteoblasten-spezifische Fibronektin-Isoform für eine regelgerechte Funktion der Osteoblasten notwendig ist, während das von der Leber produzierte Plasmafibronektin die Zusammensetzung der Knochenmatrix beeinflusst. Fibronektin in der diabetischen Niere: Mit der diabetischen Nephropathie geht eine Ausdehnung des Mesangiums in den Glomeruli einher, die mit dem Ausmaß des Nierenschadens korreliert ist. Fibronektin ist ein Bestandteil dieses expandierten Mesangiums. Vorarbeiten hatten gezeigt, dass injiziertes Fibronektin durch die Blutzirkulation in die Niere gelangt und in der Mesangialmatrix der Glomeruli eingelagert wird. Daher wurden in konditionellen Knockout-Mäusen das Plasmafibronektin bzw. das Fibronektin der Mesangialzellen und das Plasmafibronektin zugleich ausgeschaltet. In diesen Mäusen wurde ein Diabetes mellitus induziert und die Tiere für 22 Wochen mit Diabetes gehalten. Die Ausschaltung des Fibronektins hatte eine geringere Ausbreitung der Mesangialmatrix sowie eine geringere Mortalität der Tiere zur Folge. Interessanterweise schien das Plasmafibronektin alleine bereits grob ein Drittel der Ausdehnung des Mesangiums zu verursachen. Die kombinierte Ausschaltung von zirkulierendem und lokalem Fibronektin vermochte die Expansion der Mesangialmatrix sogar beinahe zu halbieren. Zusammengefasst zeigten sich neue Rollen eines traditionellen Proteins der Extrazellulärmatrix in physiologischen und pathologischen Zuständen. Einige dieser Aspekte demonstrieren die große Bedeutung der Fibronektin-Produktion durch die Leber.

In vitro cytotoxicity of silver nanoparticles on osteoblasts and osteoclasts at antibacterial concentrations

Abstract Nanoparticulate silver coatings for orthopaedic implants promise to decrease postoperative infection rates. However, silver-induced cytotoxicity on bone cells has not been investigated in detail. This study investigated the cytotoxic effects of silver nano- and microparticles and Ag(+) on osteoblasts (OBs) and osteoclasts (OCs) and correlated their effects with the antibacterial efficacy on Staphylococcus epidermidis. Silver nanoparticles (50 nm) exhibited strong cytotoxic effects on OBs and OCs. Weak cytotoxic effects were observed for silver microparticles (3 μm). The cytotoxicity was primarily mediated by a size-dependent release of Ag(+). Antibacterial effects occurred at Ag(+) concentrations that were 2-4 times higher than those inducing cytotoxic effects. Such adverse effects on OB and OC survival may have deleterious effects on the biocompatibility of orthopaedic implants. Our study represents an important step toward the detailed investigation of orthopaedic implant with nanoparticulate silver coatings prior to their widespread clinical usage.

Chondrocytes within osteochondral grafts are more resistant than osteoblasts to tissue culture at 37°C

It is proposed that an ideal osteochondral allograft for cartilage repair consists of a devitalized bone but functional cartilage. The different modes of nutrient supply in vivo for bone (vascular support) and cartilage (diffusion) suggest that a modulation of storage conditions could differentially affect the respective cells, resulting in the proposed allograft. For this purpose, osteochondral tissues from porcine humeral heads were either cultured at 37°C for up to 24 hr or stored at 4°C for 24 hr, the temperature at which osteochondral allografts are routinely stored. Functionality of the cells was assessed by in situ hybridization for transcripts encoding collagen types I and II. At 37°C, a time-dependent significant reduction of the bone surface covered with functional cells was observed with only 5% ± 5% coverage left at 24 hr compared with 41% ± 10% at 0 hr. Similarly, cartilage area containing functional cells was significantly reduced from 84% ± 7% at 0 hr to 70% ± 3% after 24 hr. After 24 hr at 4°C, a significantly reduced amount of functional cells covering bone surfaces was observed (27% ± 5%) but not of cells within the cartilage (79% ± 8%). In the applied experimental setup, bone cells were more affected by tissue culture at 37°C than cartilage cells. Even though chondrocytes appear to be more sensitive to 37°C than to 4°C, the substantially reduced amount of functional bone cells at 37°C warrants further investigation of whether a preincubation of osteochondral allografts at 37°C--prior to regular storage at 4°C--might result in an optimized osteochondral allograft with devitalized bone but viable cartilage.

Osteoblast proliferation and differentiation on a barrier membrane in combination with BMP2 and TGFβ1

Bioresorbable collagen membranes are routinely utilized in guided bone regeneration to selectively direct the growth and repopulation of bone cells in areas of insufficient volume. However, the exact nature by which alveolar osteoblasts react to barrier membranes as well as the effects following the addition of growth factors to the membranes are still poorly understood. The objective of the present study was therefore to investigate the effect of a bioresorbable collagen membrane soak-loaded in growth factors bone morphogenetic protein 2 (BMP2) or transforming growth factor β1 (TGFβ1) on osteoblast adhesion, proliferation, and differentiation.

Time course of biological activity in fresh murine osteochondral allografts paralleled to the recipient's immune response

The use of fresh osteochondral allografts is a popular approach to treat articular cartilage lesions. Immunological reactions of the recipient elicited by the allograft's osseous portion, however, frequently result in their deterioration. So far, little emphasis has been put on describing morphology and biological activity in fresh allografts and paralleling these to the immunological processes triggered in the host. Therefore, in the present study murine neonatal femora, serving as osteochondral grafts, were transplanted as fresh isografts (controls) or allografts (the latter in non- or presensitized mice) and retrieved after 2, 5, 10, and 20 days. It was shown that (1) in isografts active bone cells (osteoblasts, osteoclasts) were present, the bone marrow was repopulated with hematopoietic cells, the diaphysis increased in length, and no specific immunological reaction by the recipient was evoked. (2) Allografts transplanted into nonsensitized hosts initially appeared similar as isografts, but activated T lymphocytes at the transplantation site preceded loss of active bone cells within the graft and development of fibrosis within the marrow cavity. (3) In allografts transplanted into presensitized recipients, severe deterioration of the graft was observed with very few active bone cells, accompanied by an invasion of T lymphocytes and fibrosis in the marrow cavity already in early stages. Similar to vital organ transplantation, the function of cells within osteochondral allografts is severely impaired after being recognized by the immune system. Therefore, emphasis has to be placed on the development of procedures preserving cartilage biology while reducing the antigenicity of the allograft's osseous portion.

Endogenous Msx1 antisense transcript: In vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontoblastic cell line (MO6-G3) showed that the balance between the levels of the two Msx1 RNAs is related to the expression of Msx1 protein. To analyze the impact of this balance in the Msx-Dlx homeoprotein pathway, we analyzed the effect of Msx1, Msx2, and Dlx5 overexpression on proteins involved in skeletal differentiation. We showed that the Msx1-AS RNA is involved in crosstalk between the Msx-Dlx pathways because its expression was abolished by Dlx5. Msx1 was shown to down-regulate a master gene of skeletal cells differentiation, Cbfa1. All these data strongly suggest that the ratio between Msx1 sense and antisense RNAs is a very important factor in the control of skeletal terminal differentiation. Finally, the initiation site for Msx1-AS RNA transcription was located by primer extension in both mouse and human in an identical region, including a consensus TATA box, suggesting an evolutionary conservation of the AS RNA-mediated regulation of Msx1 gene expression.

Modelo numérico mecanobiológico para a obtenção da matriz de rigidez estrutural e da densidade mineral na remodelagem óssea

Neste trabalho é proposto um modelo mecanobiológico de remodelagem óssea para a estimativa de variações, provocadas por perturbações mecânicas ou biológicas, na matriz de rigidez estrutural da escala macroscópica e na densidade mineral em uma região do osso. Na cooperação entre as áreas da saúde e da engenharia, como nos estudos estruturais de biomecânica no sistema esquelético, as propriedades mecânicas dos materiais devem ser conhecidas, entretanto os ossos possuem uma constituição material altamente complexa, dinâmica e variante entre indivíduos. Sua dinâmica decorre dos ciclos de absorção e deposição de matriz óssea na remodelagem óssea, a qual ocorre para manter a integridade estrutural do esqueleto e adaptá-lo aos estímulos do ambiente, sejam eles biológicos, químicos ou mecânicos. Como a remodelagem óssea pode provocar alterações no material do osso, espera-se que suas propriedades mecânicas também sejam alteradas. Na literatura científica há modelos matemáticos que preveem a variação da matriz de rigidez estrutural a partir do estímulo mecânico, porém somente os modelos mais recentes incluíram explicitamente processos biológicos e químicos da remodelagem óssea. A densidade mineral óssea é um importante parâmetro utilizado no diagnóstico de doenças ósseas na área médica. Desse modo, para a obtenção da variação da rigidez estrutural e da densidade mineral óssea, propõe-se um modelo numérico mecanobiológico composto por cinco submodelos: da dinâmica da população de células ósseas, da resposta das células ao estímulo mecânico, da porosidade óssea, da densidade mineral óssea e, baseado na Lei de Voigt para materiais compósitos, da rigidez estrutural. Os valores das constantes das equações dos submodelos foram obtidos de literatura. Para a solução das equações do modelo, propõe-se uma implementação numérica e computacional escrita em linguagem C. O método de Runge-Kutta-Dorman-Prince, cuja vantagem consiste no uso de um passo de solução variável, é utilizado no modelo para controlar o erro numérico do resultado do sistema de equações diferenciais. Foi realizada uma avaliação comparativa entre os resultados obtidos com o modelo proposto e os da literatura dos modelos de remodelagem óssea recentes. Conclui-se que o modelo e a implementação propostos são capazes de obter variações da matriz de rigidez estrutural macroscópica e da densidade mineral óssea decorrentes da perturbação nos parâmetros mecânicos ou biológicos do processo de remodelagem óssea.

Efeito da oxigenação hiperbárica no reparo ósseo de ratos diabéticos

The chronic state of hyperglycemia due to diabetes mellitus affects multiples organs impairing life quality. In bone, diabetes alters strength and mineral density and also suppresses the osteoblast activity, leading to an unbalanced bone healing process. Hyperbaric oxygen therapy (HBO) is suggested as an adjuvant treatment to accelerate bone repair. This study evaluated the effects of HBO in the number of mast cells and in new bone formation at the initial stage of bone repair in normoglycemic and diabetic rats. It was hypothesized that HBO treatment may improve bone repair in diabetic bone. The rats were equally divided in four groups: Control (C); Control + HBO (CH); Diabetes (D) and Diabetes + HBO (DH). Diabetes was induced by streptozotocin (65mg/kg) and femoral bone defects were created thirty days after diabetes induction in all groups. HBO initiated immediately after surgery procedure and was performed daily, for 7 days, in the CH e DH groups. Seven days after surgery, all animals were euthanized. The femur diaphyses were removed, fixated, decalcified and processed for paraffin embedding. The semi-serial histological sections obtained were stained with Hematoxylin-Eosin (HE), Mallory Trichrome and Toluidine Blue. The qualitative analysis was conducted in the histology slides stained with HE, where it was evaluated the morphological aspects of bone repair in the lesion area, observing the presence of clot, inflammatory cells, granulation tissue, type of bone tissue, morphology of bone cells, and thickness and organization of bone trabeculae. In the slides stained with Mallory Trichrome and Toluidine Blue were evaluated the percentage of new bone formation and number of mast cells, respectively. The qualitative analysis showed that the CH group presented a more advanced stage of bone repair compared to the C group, showing thicker trabeculae and greater bone filling of the lesion area. In D and DH group, the lesion area was partially filled with new bone formation tissue and presented thinner trabeculae and fewer areas associated to osteoclasts compared to control group. The histomorphometric analysis showed a significant improvement in new bone formation (p<0.001) comparing CH (38.08 ± 4.05) and C (32.05 ± 5.51); C and D (24.62 ± 2.28 and CH and DH (27.14 ± 4.21) groups. In the normoglycemic rats there was a significant increasing in the number of mast cells (p<0.05) comparing C (8.06 ± 5.15) and CH (21.06 ± 4.91) groups. In conclusion, this study showed that diabetes impaired bone repair and HBO was only able to increase new bone formation and the number of mast cells in the normoglycemic animals.