Expression of antagonists of WNT and BMP signaling after non-rigid fixation of osteotomies

Delayed fracture healing and non-unions represent rare but severe complications in orthopedic surgery. Further knowledge on the mechanisms of the bone repair process and of the development of a pseudoarthrosis is essential to predict and prevent impaired healing of fractures. The present study aimed at elucidating differences in gene expression during the repair of rigidly and non-rigidly fixed osteotomies. For this purpose, the MouseFix™ and the FlexiPlate™ systems (AO Development Institute, Davos, CH), allowing the creation of well defined osteotomies in mouse femora, were employed. A time course following the healing process of the osteotomy was performed and bones and periimplant tissues were analyzed by high-resolution X-ray, MicroCT and by histology. For the assessment of gene expression, Low Density Arrays (LDA) were done. In animals with rigid fixation, X-ray and MicroCT revealed healing of the osteotomy within 3 weeks. Using the FlexiPlate™ system, the osteotomy was still visible by X-ray after 3 weeks and a stabilizing cartilaginous callus was formed. After 4.5 weeks, the callus was remodeled and the osteotomy was, on a histological level, healed. Gene expression studies revealed levels of transcripts encoding proteins associated with inflammatory processes not to be altered in tissues from bones with rigid and non-rigid fixation, respectively. Levels of transcripts encoding proteins of the extracellular matrix and essential for bone cell functions were not increased in the rigidly fixed group when compared to controls without osteotomy. In the FlexiPlate™ group, levels of transcripts encoding the same set of genes were significantly increased 3 weeks after surgery. Expression of transcripts encoding BMPs and BMP antagonists was increased after 3 weeks in repair tissues from bones fixed with FlexiPlate™, as were inhibitors of the WNT signaling pathways. Little changes only were detected in transcript levels of tissues from rigidly fixed bones. The data of the present study suggest that rigid fixation enables accelerated healing of an experimental osteotomy as compared to non-rigid fixation. The changes in the healing process after non-rigid fixation are accompanied by an increase in the levels of transcripts encoding inhibitors of osteogenic pathways and, probably as a consequence, by temporal changes in bone matrix synthesis.

Delivery mode and efficacy of BMP-2 in association with implants

Bone healing may be improved in implant patients by the administration of osteogenic agents, such as bone morphogenetic protein 2 (BMP-2). But the efficacy of BMP-2 depends upon its mode of application. We hypothesized that BMP-2 is capable of a higher osteogenic efficacy when delivered physiologically, viz., when incorporated into a calcium-phosphate carrier that mimics mineralized bone matrix, than when administered via simple pharmacological modes, such as by adsorption onto a carrier surface. Using an ectopic rat model, we compared the osteoinductive efficacies of calcium-phosphate implant-coatings bearing either incorporated, adsorbed, or incorporated and adsorbed BMP-2. When adsorbed directly onto the naked implant surface, BMP-2 was not osteogenic. When adsorbed onto a calcium-phosphate coating, it was osteoinductive, but not highly efficacious. When BMP-2 was incorporated into calcium-phosphate coatings, it was a potent bone-inducer, whose efficacy was compromised, not potentiated, by the additional deposition of an adsorbed pool.

Biomimetic Coatings and Their Biological Functionalization

The biomimetic coating technique can be used to deposit layers of calcium phosphate (CaP) on medical devices to improve their osteoconductivity and osseointegration.The inorganic layer generated is akin to mineralized bone matrix and can be degraded as such. The biomimetic coating technique involves the nucleation and growth of bone-like crystals on a pretreated substrate by immersing it in a supersaturated solution of CaP under physiological conditions of temperature (37°C) and pH (7.4). The method, originally developed by Kokubo and his co-workers in 1990, has since undergone improvement and refinement by several groups of investigators. Biomimetic coatings are valuable in that they can serve as a vehicle for the slow, sustained release of osteogenic agents at the site of implantation. This attribute is rendered possible by the near-physiological conditions under which these coatings are prepared, which permits the incorporation of bioactive agents into the inorganic crystal latticework rather than their superficial adsorption on preformed layers. In addition, the biomimetic coating technique can be applied to implants of an organic as well as of a metallic nature and to those with irregular surface geometries, which is not possible using conventional methodologies.

Gap Junctional Communication Modulates Gene Expression in Osteoblastic Cells

Bone-forming cells are organized in a multicellular network interconnected by gap junctions. In these cells, gap junctions are formed by connexin43 (Cx43) and connexin45 (Cx45). Cx43 gap junctions form pores that are more permeable to negatively charged dyes such as Lucifer yellow and calcein than are Cx45 pores. We studied whether altering gap junctional communication by manipulating the relative expression of Cx43 and Cx45 affects the osteoblast phenotype. Transfection of Cx45 in cells that express primarily Cx43 (ROS 17/2.8 and MC3T3-E1) decreased both dye transfer and expression of osteocalcin (OC) and bone sialoprotein (BSP), genes pivotal to bone matrix formation and calcification. Conversely, transfection of Cx43 into cells that express predominantly Cx45 (UMR 106–01) increased both cell coupling and expression of OC and BSP. Transient cotransfection of promoter–luciferase constructs and connexin expression vectors demonstrated that OC and BSP gene transcription was down-regulated by Cx45 cotransfection in ROS 17/2.8 and MC3T3-E1 cells, in association with a decrease in dye coupling. Conversely, cotransfection of Cx43 in UMR 106–01 cells up-regulated OC and BSP gene transcription. Activity of other less specific osteoblast promoters, such as osteopontin and osteonectin, was less sensitive to changes in gap junctional communication. Thus, altering gap junctional permeability by manipulating the expression of Cx43 and Cx45 in osteoblastic cells alters transcriptional activity of osteoblast-specific promoters, presumably via modulation of signals that can diffuse from cell to cell. A communicating intercellular network is required for the full elaboration of a differentiated osteoblastic phenotype.

Linking osteopetrosis and pycnodysostosis: Regulation of cathepsin K expression by the microphthalmia transcription factor family

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix–loop–helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found—and TFE3 has been suggested—to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

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.

Macrophages overexpressing tartrate-resistant acid phosphatase show altered profile of free radical production and enhanced capacity of bacterial killing

Activated macrophages and osteoclasts express high amounts of tartrate-resistant acid phosphatase (TRACP, acp5). TRACP has a binuclear iron center with a redox-active iron that has been shown to catalyze the formation of reactive oxygen species (ROS) by Fenton's reaction. Previous Studies Suggest that ROS generated by TRACP may participate in degradation of endocytosed bone matrix products in resorbing osteoclasts and degradation of foreign Compounds during. antigen presentation in activated macrophages. Here we have compared free radical production in macrophages of TRACP overexpressing (TRACP +) and wild-type (WT) mice. TRACP overexpression increased both ROS levels and Superoxide production. Nitric oxide production was increased in activated macrophages or WT mice, but not in TRACP+ mice, Macrophages from TRACP+ mice showed increased capacity or bacterial killing. Recombinant TRACP enzyme was capable of bacterial killing in the presence of hydrogen peroxide. These results suggest that TRACP has an important biological function in immune defense systern.

Use of GLP-1 mimetic in type 2 diabetes mellitus: is it the end of fragility fractures?

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Capacity of the extracellular matrix of the bone marrow to induce proliferation of myeloid cells in vitro in model of protein malnutrition in mice

The aim of this study was to verify the capacity of the extracellular matrix (ECM) obtained from bone marrow of malnourished mice to sustain survival and to induce the proliferation of myeloid cells. We also verified the capacity of the tests to interact with in vitro hematopoietic cytokines. Male ""Swiss"" mice were submitted to protein malnutrition with a diet contents of 4% casein until they lost 20% of the original weight, while the group-control was kept with a diet content of 14% of casein. The bone marrow was extracted with 1.0 mg of aprotinin/mL in PBS. The proliferation tests were carried out with myeloid cell line FDCP-1, by the colorimetric method of reduction of the MTT. The obtained ECM from nourished and undernourished mice induced cellular proliferation in vitro. Tests performed with Il-3 and GM-CSF cytokines in a concentration of 10 and 500 rho g/mL displayed synergic and regulatory effects respectively. The ECM obtained from the malnourished group submitted to the binding to GM-CSF demonstrated higher cellular proliferation than the ECM obtained from the control group (p<0.05). The results suggest that the alterations in the composition of ECM of bone marrow caused by malnutrition might lead to modification of the GM-CSF activity modulation.

Expression of matrix metalloproteinases-2 and-9 and RECK during alveolar bone regeneration in rat

MMPs are endopeptidases that play a pivotal role in ECM turnover. RECK is a single membrane-anchored MMP-regulator. Here, we evaluated the temporal and spatial expression of MMP-2, MMP-9, and RECK during alveolar bone regeneration. The maxillary central incisor of Wistar rats was extracted and the animals were killed at 1, 3, 7, 10, 14, 21, 28, and 42 days post-operatively (n = 3/period). The hemimaxillae were collected, demineralized and embedded in paraffin. Immunohistochemical analysis was performed by the immunoperoxidase technique with polyclonal antibodies. On day 1, polymorphonuclear cells in the blood clot presented mild immunolabeling for MMPs. During bone remodeling, osteoblasts facing new bone showed positive staining for gelatinases and RECK in all experimental periods. MMPs were also found in the connective tissue and endothelial cells. Our results show for the first time that inactive and/or active forms of MMP-2, MMP-9 and RECK are differentially expressed by osteogenic and connective cells during several events of alveolar bone regeneration. This may be important for the replacement of the blood clot by connective tissue, and in the formation, maturation and remodeling of new bone.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

Alterations in proteins of bone marrow extracellular matrix in undernourished mice

The objective of the present study was to determine the effect of protein malnutrition on the glycoprotein content of bone marrow extracellular matrix (ECM). Two-month-old male Swiss mice were submitted to protein malnutrition with a low-protein diet containing 4% casein as compared to 20% casein in the control diet. When the experimental group had attained a 20% loss of their original body weight, we extracted the ECM proteins from bone marrow with PBS buffer, and analyzed ECM samples by SDS-PAGE (7.5%) and ECL Western blotting. Quantitative differences were observed between control and experimental groups. Bone marrow ECM from undernourished mice had greater amounts of extractable fibronectin (1.6-fold increase) and laminin (4.8-fold increase) when compared to the control group. These results suggest an association between fluctuations in the composition of the hematopoietic microenvironment and altered hematopoiesis observed in undernourished mice.

Co-culture of chondrocytes and bone marrow mesenchymal stem cells in vitro enhances the expression of cartilaginous extracellular matrix components

Chondrocytes and bone marrow mesenchymal stem cells (BMSCs) are frequently used as seed cells in cartilage tissue engineering. In the present study, we determined if the co-culture of rabbit articular chondrocytes and BMSCs in vitro promotes the expression of cartilaginous extracellular matrix and, if so, what is the optimal ratio of the two cell types. Cultures of rabbit articular chondrocytes and BMSCs were expanded in vitro and then cultured individually or at a chondrocyte:BMSC ratio of 4:1, 2:1, 1:1, 1:2, 1:4 for 21 days and cultured in DMEM/F12. BMSCs were cultured in chondrogenic induction medium. Quantitative real-time RT-PCR and Western blot were used to evaluate gene expression. In the co-cultures, type II collagen and aggrecan expression increased on days 14 and 21. At the mRNA level, the expression of type II collagen and aggrecan on day 21 was much higher in the 4:1, 2:1, and 1:1 groups than in either the articular chondrocyte group or the induced BMSC group, and the best ratio of co-culture groups seems to be 2:1. Also on day 21, the expression of type II collagen and aggrecan proteins in the 2:1 group was much higher than in all other groups. The results demonstrate that the co-culture of rabbit chondrocytes and rabbit BMSCs at defined ratios can promote the expression of cartilaginous extracellular matrix. The optimal cell ratio appears to be 2:1 (chondrocytes:BMSCs). This approach has potential applications in cartilage tissue engineering since it provides a protocol for maintaining and promoting seed-cell differentiation and function.