Biological monitoring of a promissory xenogenic pin for biomedical applications: a preliminary intraosseous study in rats

Objectives: Over the last years, it is known that in some cases metal devices for biomedical applications present some disadvantages suggesting absorbable materials (natural or synthetic) as an alternative of choice. Here, our goal was to evaluate the biological response of a xenogenic pin, derived from bovine cortical bone, intraosseously implanted in the femur of rats. Material and methods: After 10, 14, 30 and 60 days from implantation, the animals (n = 5/period) were killed and the femurs carefully collected and dissected out under histological demands. For identifying the osteoclastogenesis level at 60 days, we performed the immunohistochemisty approach using antibody against RANKL. Results: Interestingly, our results showed that the incidence of neutrophils and leukocytes was observed only at the beginning (10 days). Clear evidences of pin degradation by host cells started at 14 days and it was more intensive at 60 days, when we detected the majority of the presence of giant multinucleated cells, which were very similar to osteoclast cells contacting the implanted pin. To check osteoclastogenesis at 60 days, we evaluated RANKL expression and it was positive for those resident multinucleated cells while a new bone deposition was verified surrounding the pins in all evaluated periods. Conclusions: Altogether, our results showed that pins from fully processed bovine bone are biocompatible and absorbable, allowing bone neoformation and it is a promissory device for biomedical applications.

Toxicity of photodynamic therapy with LED associated to Photogem (R): An in vivo study

The aims of this study were to evaluate the effects of PhotogemA (R)-mediated photosensitization on rat palatal mucosa and the biodistribution of the photosensitizer in this tissue. A solution of PhotogemA (R) (500 or 1000 mg/l) was applied to the palatal mucosa for 30 min and the exposure time to blue LED (460 nm) was 20 min (144 J/cm(2)). At 0, 1, 3, and 7 days, palatal mucosa was photographed for macroscopic analysis. After killing, the palate was removed for microscopic analysis. Thermal mapping evaluated temperature change in the tissue during irradiation. All experimental groups revealed intact mucosa in the macroscopic analysis. Tissue alterations were observed microscopically for only four out of 80 animals subjected to PDT. Fluorescence emitted by PhotogemA (R) was identified and was limited to the epithelial layer. A temperature increase from 35 to 41A degrees C was recorded. PhotogemA (R)- mediated PDT was not toxic to the rat palatal mucosa.

Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy

von Walden F, Casagrande V, Ostlund Farrants AK, Nader GA. Mechanical loading induces the expression of a Pol I regulon at the onset of skeletal muscle hypertrophy. Am J Physiol Cell Physiol 302: C1523-C1530, 2012. First published March 7, 2012; doi:10.1152/ajpcell.00460.2011.-The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.

Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (beta-TCP) (60: 40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100 degrees C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/beta-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging

One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture.

Infrared (810-nm) low-level laser therapy on rat experimental knee inflammation

Arthritis of the knee is the most common type of joint inflammatory disorder and it is associated with pain and inflammation of the joint capsule. Few studies address the effects of the 810-nm laser in such conditions. Here we investigated the effects of low-level laser therapy (LLLT; infrared, 810-nm) in experimentally induced rat knee inflammation. Thirty male Wistar rats (230-250 g) were anesthetized and injected with carrageenan by an intra-articular route. After 6 and 12 h, all animals were killed by CO(2) inhalation and the articular cavity was washed for cellular and biochemical analysis. Articular tissue was carefully removed for real-time PCR analysis in order to evaluate COX-1 and COX-2 expression. LLLT was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity with 1, 3, and 6 J (Joules) of energy. This result was corroborated by cell counting showing the reduction of polymorphonuclear cells at the inflammatory site. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Both COX-1 and 2 gene expression were significantly enhanced by laser irradiation while PGE(2) production was inhibited. Low-level laser therapy operating at 810 nm markedly reduced inflammatory signs of inflammation but increased COX-1 and 2 gene expression. Further studies are necessary to investigate the possible production of antiinflammatory mediators by COX enzymes induced by laser irradiation in knee inflammation.

Titanium surfaces with nanotopography modulate cytokine production in cultured human gingival fibroblasts

Implant topography is an important factor that influences many cell types. To understand the role of topography in the inflammatory events, we evaluated the response of human gingival fibroblasts (HGFs) by the release pattern of cytokines. HGFs were cultured on Ti discs for 24 and 48 h. Four different surface treatments were used: machining method (turned), blasting followed by an acid-etching method (BAE), oxidative nanopatterning (ON) method, and an association of blasting followed by an acid-etching plus oxidative nanopatterning (BAE+ON) method. Extracellular levels of IL-6, IL-8, transforming growth factor beta (TGF-beta), IL-4, and IL-10 were measured by enzyme-linked immunosorbant assay. Increased levels of IL-6 and IL-8 were observed in all surfaces after 24 h which decreased after 48 h. BAE, ON, and BAE+ON surfaces showed a reduction in IL-6 levels compared with the turned after 48 h (p < 0.05). On one hand, IL-8 production was lower in BAE+ON in comparison to the turned surface (p < 0.05). On the other hand, IL-4 showed increased levels with 48 h, which were significantly different between turned, BAE, and ON surfaces, but not with BAE+ON. Additionally, TGF-beta and IL-10 production were not detected. This study indicates that nanotopography might be important in the modulation of the inflammatory response in cultured HGFs. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A:2629-2636, 2012.

Effects of the combination of low-level laser irradiation and recombinant human bone morphogenetic protein-2 in bone repair

Low-level laser irradiation (LLLI) and recombinant human bone morphogenetic protein type 2 (rhBMP-2) have been used to stimulate bone formation. LLLI stimulates proliferation of osteoblast precursor cells and cell differentiation and rhBMP-2 recruits osteoprogenitor cells to the bone healing area. This in vivo study evaluated the effects of LLLI and rhBMP-2 on the bone healing process in rats. Critical bone defects were created in the parietal bone in 42 animals, and the animals were divided into six treatment groups: (1) laser, (2) 7 mu g of rhBMP-2, (3) laser and 7 mu g of rhBMP-2, (4) 7 mu g of rhBMP-2/monoolein gel, (5) laser and 7 mu g rhBMP-2/monoolein gel, and (6) critical bone defect controls. A gallium-aluminum-arsenide diode laser was used (wavelength 780 nm, output power 60 mW, beam area 0.04 cm(2), irradiation time 80 s, energy density 120 J/cm(2), irradiance 1.5 W/cm(2)). After 15 days, the calvarial tissues were removed for histomorphometric analysis. Group 3 defects showed higher amounts of newly formed bone (37.89%) than the defects of all the other groups (P < 0.05). The amounts of new bone in defects of groups 1 and 4 were not significantly different from each other (24.00% and 24.75%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). The amounts of new bone in the defects of groups 2 and 5 were not significantly different from each other (31.42% and 31.96%, respectively), but were significantly different from the amounts in the other groups (P < 0.05). Group 6 defects had 14.10% new bone formation, and this was significantly different from the amounts in the other groups (P < 0.05). It can be concluded that LLLI administered during surgery effectively accelerated healing of critical bone defects filled with pure rhBMP-2, achieving a better result than LLLI alone or the use of rhBMP-2 alone.

Effect of dental tissue conditioners and matrix metalloproteinase inhibitors on type I collagen microstructure analyzed by Fourier transform infrared spectroscopy

This study aimed to evaluate the chemical interaction of collagen with some substances usually applied in dental treatments to increase the durability of adhesive restorations to dentin. Initially, the similarity between human dentin collagen and type I collagen obtained from commercial bovine membranes of Achilles deep tendon was compared by the Attenuated Total Reflectance technique of Fourier Transform Infrared (ATR-FTIR) spectroscopy. Finally, the effects of application of 35% phosphoric acid, 0.1M ethylenediaminetetraacetic acid (EDTA), 2% chlorhexidine, and 6.5% proanthocyanidin solution on microstructure of collagen and in the integrity of its triple helix were also evaluated by ATR-FTIR. It was observed that the commercial type I collagen can be used as an efficient substitute for demineralized human dentin in studies that use spectroscopy analysis. The 35% phosphoric acid significantly altered the organic content of amides, proline and hydroxyproline of type I collagen. The surface treatment with 0.1M EDTA, 2% chlorhexidine, or 6.5% proanthocyanidin did not promote deleterious structural changes to the collagen triple helix. The application of 6.5% proanthocyanidin on collagen promoted hydrogen bond formation. (c) 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2012.

Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress, and telomere length.

Early embryonic exposure to maternal glucocorticoids can broadly impact physiology and behaviour across phylogenetically diverse taxa. The transfer of maternal glucocorticoids to offspring may be an inevitable cost associated with poor environmental conditions, or serve as a maternal effect that alters offspring phenotype in preparation for a stressful environment. Regardless, maternal glucocorticoids are likely to have both costs and benefits that are paid and collected over different developmental time periods. We manipulated yolk corticosterone (cort) in domestic chickens (Gallus domesticus) to examine the potential impacts of embryonic exposure to maternal stress on the juvenile stress response and cellular ageing. Here, we report that juveniles exposed to experimentally increased cort in ovo had a protracted decline in cort during the recovery phase of the stress response. All birds, regardless of treatment group, shifted to oxidative stress during an acute stress response. In addition, embryonic exposure to cort resulted in higher levels of reactive oxygen metabolites and an over-representation of short telomeres compared with the control birds. In many species, individuals with higher levels of oxidative stress and shorter telomeres have the poorest survival prospects. Given this, long-term costs of glucocorticoid-induced phenotypes may include accelerated ageing and increased mortality.

Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Performance Tuning Non-Uniform Sampling for Sensitivity Enhancement of Signal-Limited Biological NMR

Non-uniform sampling (NUS) has been established as a route to obtaining true sensitivity enhancements when recording indirect dimensions of decaying signals in the same total experimental time as traditional uniform incrementation of the indirect evolution period. Theory and experiments have shown that NUS can yield up to two-fold improvements in the intrinsic signal-to-noise ratio (SNR) of each dimension, while even conservative protocols can yield 20-40 % improvements in the intrinsic SNR of NMR data. Applications of biological NMR that can benefit from these improvements are emerging, and in this work we develop some practical aspects of applying NUS nD-NMR to studies that approach the traditional detection limit of nD-NMR spectroscopy. Conditions for obtaining high NUS sensitivity enhancements are considered here in the context of enabling H-1,N-15-HSQC experiments on natural abundance protein samples and H-1,C-13-HMBC experiments on a challenging natural product. Through systematic studies we arrive at more precise guidelines to contrast sensitivity enhancements with reduced line shape constraints, and report an alternative sampling density based on a quarter-wave sinusoidal distribution that returns the highest fidelity we have seen to date in line shapes obtained by maximum entropy processing of non-uniformly sampled data.

NEW TARGET GENES FOR TUMOR SUPPRESSORS p53 AND p73 IN REGENERATING LIVER

The p53-family of proteins regulates expression of target genes during tissue development and differentiation. Within the p53-family, p53 and p73 have hepatic-specific functions in development and tumor suppression. Despite a growing list of p53/p73 target genes, very few of these have been studied in vivo, and the knowledge regarding functions of p53 and p73 in normal tissues remains limited. p53+/-p73+/- mice develop hepatocellular carcinoma (HCC), whereas overexpression of p53 in human HCC leads to tumor regression. However, the mechanism of p53/p73 function in liver remains poorly characterized. Here, the model of mouse liver regeneration is used to identify new target genes for p53/p73 in normal quiescent vs. proliferating cells. In response to surgical removal of ~2/3 of liver mass (partial hepatectomy, PH), the remaining hepatocytes exit G0 of cell cycle and undergo proliferation to reestablish liver mass. The hypothesis tested in this work is that p53/p73 functions in cell cycle arrest, apoptosis and senescence are repressed during liver regeneration, and reactivated at the end of the regenerative response. Chromatin immunoprecipitation (ChIP), with a p73-antibody, was used to probe arrayed genomic sequences (ChIP-chip) and uncover 158 potential targets of p73-regulation in normal liver. Global microarray analysis of mRNA levels, at T=0-48h following PH, revealed sets of genes that change expression during regeneration. Eighteen p73-bound genes changed expression after PH. Four of these genes, Foxo3, Jak1, Pea15, and Tuba1 have p53 response elements (p53REs), identified in silico within the upstream regulatory region. Forkhead transcription factor Foxo3 is the most responsive gene among transcription factors with altered expression during regenerative, cellular proliferation. p53 and p73 bind a Foxo3 p53RE and maintain active expression in quiescent liver. During liver regeneration, binding of p53 and p73, recruitment of acetyltransferase p300, and an active chromatin structure of Foxo3 are disrupted, alongside loss of Foxo3 expression. These parameters of Foxo3 regulation are reestablished at completion of liver growth and regeneration, supporting a temporary suspension of p53 and p73 regulatory functions in normal cells during tissue regeneration.

Myogenin modulates exercise endurance by altering skeletal muscle metabolism

The function of myogenic regulatory factors (MRFs) during adult life is not well understood. The requirement of one of these MRFs, myogenin (Myog), during embryonic muscle development suggests an equally important role in adult muscle. In this study, we have determined the function of myogenin during adult life using a conditional allele of Myog. In contrast to embryonic development, myogenin is not required for adult viability, and Myog-deleted mice exhibited no remarkable phenotypic changes during sedentary life. Remarkably, sedentary Myog-deleted mice demonstrated enhanced exercise endurance during involuntary treadmill running. Altered blood glucose and lactate levels in sedentary Myog-deleted mice after exhaustion suggest an enhanced glycolytic metabolism and an ability to excessively deplete muscle and liver glycogen stores. Traditional changes associated with enhanced exercise endurance, such as fiber type switching, and increased oxidative potential, were not detected in sedentary Myog-deleted mice. After long-term voluntary exercise, trained Myog-deleted mice demonstrated an enhanced adaptive response to exercise. Trained Myog-deleted mice exhibited superior exercise endurance associated with an increased proportion of slow-twitch fibers and increased oxidative capacity. In a parallel experiment, dystrophin-deficient young adult mice showed attenuated muscle fatigue following the deletion of Myog. These results demonstrate a novel and unexpected role for myogenin in modulating skeletal muscle metabolism.

CIP4 and Src in Promoting the Migration and Invasion of Breast Cancers

Cellular invasion represents a critical early step in the metastatic cascade, and many proteins have been identified as part of an “invasive signature.” The non-receptor tyrosine kinase Src is commonly upregulated in breast cancers, often in conjunction with overexpression of EGFR. Signaling from this pathway stimulates cell proliferation, migration, and invasion and frequently involves proteins that regulate the cytoskeleton. My data demonstrates that inhibition of Src, using the small-molecule inhibitor dasatinib, impairs cellular migration and invasion. Furthermore, Src inhibition sensitizes the cells to the effects of the chemotherapeutic doxorubicin resulting in dramatic, synergistic inhibition of proliferation with combination treatments. The Src-targeted protein CIP4 (Cdc42-interacting protein 4) associates with curved plasma membranes to scaffold complexes of Cdc42 and N-WASp. In these experiments, I show that CIP4 overexpression correlates with triple-negative biomarker status, cellular migration, and invasion of (breast cancer cells. Inhibition of CIP4 expression significantly decreases migration and invasion. Furthermore, I demonstrate the novel finding that CIP4 localizes to invadopodia, which are finger-like projections of the actin cytoskeleton that are associated with matrix degradation and cellular invasion. Depletion of CIP4 in invasive cells impairs the formation of invadopodia and the degradation of gelatin. Therefore, CIP4 is a critical component of the invasive phenotype acquired by human breast cancer cells. In this body of work, I propose a model in which CIP4 promotes actin polymerization by stabilizing the active conformation of N-WASp. CIP4 and N-WASp are both phosphorylated by Src, implicating this pathway in Src-dependent cytoskeletal rearragement. This represents a novel role for F-BAR proteins in migration and invasion.