Marta Monjo

The effect of hydrofluoric acid treatment of titanium surface on nanostructural and chemical changes and the growth of MC3T3-E1 cells

Fluoride-modification of dental titanium (Ti) implants is used to improve peri-implant bone growth and bone-to-implant contact and adhesion strength. In this study, the surface topography, chemistry and biocompatibility of polished Ti surfaces treated with hydrofluoric acid solution (HF) were studied. Murine osteoblasts (MC3T3-E1) were cultured on the different groups of Ti surfaces. Surfaces treated with HF had higher roughness, lower cytotoxicity level and better biocompatibility than controls. For short treatment times (40 and 90 s), fluorine was detected only within the first 5 nm of the surface layer (X-ray Photoemission Spectroscopy, XPS), whereas longer treatment time (120 and 150 s) caused fluoride ions to penetrate deeper (Secondary Ion Mass Spectrometry, SIMS). These results suggest that submerging Ti implants in a weak HF solution instigate time-dependant specific surface changes that are linked to the improved biocompatibility of these surfaces.

Expression and regulation of resistin in osteoblasts and osteoclasts indicate a role in bone metabolism

The adipose tissue is the site of expression and secretion of a range of biologically active proteins, called adipokines, for example, leptin, adiponectin, and resistin. Leptin has previously been shown to be expressed in osteoblasts and to promote bone mineralization, whereas adiponectin expression is enhanced during osteoblast differentiation. In the present study we explored the possible role of resistin in bone metabolism. We found that resistin is expressed in murine preosteoclasts and preosteoblasts (RAW 264.7, MC3T3‐E1), in primary human bone marrow stem cells and in mature human osteoblasts. The expression of resistin mRNA in RAW 264.7 was increased during differentiation and seemed to be regulated through PKC‐ and PKA‐dependent mechanisms. Recombinant resistin increased the number of differentiated osteoclasts and stimulated NFκB promoter activity, indicating a role in osteoclastogenesis. Resistin also enhanced the proliferation of MC3T3‐E1 cells in a PKA and PKC‐dependent manner, but only weakly interfered with genes known to be upregulated during differentiation of MC3T3‐E1 into osteoblasts. All together, our results indicate that resistin may play a role in bone remodeling. J. Cell. Biochem. 99: 824–834, 2006.

In vivo expression of osteogenic markers and bone mineral density at the surface of fluoride-modified titanium implants.

The aim of the present study was to investigate the biological mechanisms of the functional attachment of fluoride-modified titanium implants to cortical bone by studying the association of the pull-out test results with gene expression of osteoblast (runx2, osteocalcin, collagen-I and IGF-I), osteoclast (TRAP, H(+)-ATPase and calcitonin receptor) and inflammation (TNF-alpha, IL-6 and IL-10) markers from peri-implant bone tissue using real-time RT-PCR, following a 4- and 8-week healing period. After implant detachment, wound fluid from the implant site was collected for LDH and ALP activity analysis. A new method to study volumetric bone mineral density (vBMD) of sub-implant cortical bone was developed using micro-computed tomography. Our results show lower LDH activity and TRAP mRNA levels in fluoride implants after 4 weeks of healing, yet no differences were found either on the pull-out force or expression of bone formation marker genes. After 8 weeks of healing, both pull-out, vBMD and osteocalcin, runx2 and collage type I gene expression were higher in fluoride implants. In conclusion, fluoride-modified implants seem to modulate both inflammation and bone resorption/formation events at the bone-implant interface, suggesting that these biological effects are an intrinsic part of the clinical performance of this surface.

Titanium implant surface modification by cathodic reduction in hydrofluoric acid: surface characterization and in vivo performance.

Etching is used for the surface modification of titanium to improve the implant performance in bone. In this study, pure titanium implants were surface modified by a cathodic reduction process by using hydrofluoric acid (HF) at various concentrations (0.001, 0.01, and 0.1 vol %) and a constant current of 1 mA/cm(2). The resulting surface microtopographies were analyzed by atomic force microscopy, scanning electron microscopy, and profilometry, while the surface chemical contents were evaluated by time of flight secondary ion mass spectrometry. The competitive forces between ionic surface implementation induced by the current direction and the HF etching effect on titanium were highlighted. The implant performance was evaluated in an in vivo rabbit model by using a pull-out test method. The group of implants modified with 0.01% HF showed the highest retention in bone. Fluoride and hydride amounts measured in the surfaces, as well as surface skewness (S(sk)), kurtosis (S(ku)), and core fluid retention (S(ci)) were positively correlated to the implants retention in bone in vivo. Frequently used parameters for characterizing the implant, such as oxide content and the average height deviation from the mean plane (S(a)), were not correlated to implant performance, suggesting that these parameters are not the most important in predicting the implant performance.

Sex Steroid Receptor Expression Profile in Brown Adipose Tissue. Effects of Hormonal Status

Recent investigations suggest that sex hormones play an important role in the brown adipose tissue (BAT) thermogenic program by acting on several steps of the lipolytic signal cascade and on the UCP1 transcription control. However, the number of studies focusing on steroid receptor status in brown adipose tissue is negligible. In the present study, we analyze steroid receptor mRNA levels in brown adipose tissue in male and female rats and in pregnant and lactating females, all of them models with a different hormonal background. The direct effect of sex hormones on the expression of their receptors was studied in vitro in primary culture of brown adipocytes. Oestrogen receptor (ERα) and androgen receptor (AR) densities were higher in male than in female BAT. PR A+B mRNA expression was downregulated in lactation, suggesting a role of progesterone signalling in thermogenesis impairment at this stage. In vitro studies showed that progesterone decreased PR A+B mRNA and that testosterone downregulated ERα mRNA. The results highlighted in this study demonstrate the presence of steroid receptor mRNA in BAT and in brown cell cultured adipocytes, supporting the idea that changes in steroid receptor expression would be important for the understanding of sex hormone effects on BAT physiology.

Quercitrin and taxifolin stimulate osteoblast differentiation in MC3T3-E1 cells and inhibit osteoclastogenesis in RAW 264.7 cells.

Flavonoids are natural antioxidants that positively influence bone metabolism. The present study screened among different flavonoids to identify biomolecules for potential use in bone regeneration. For this purpose, we used MC3T3-E1 and RAW264.7 cells to evaluate their effect on cell viability and cell differentiation. First, different doses of chrysin, diosmetin, galangin, quercitrin and taxifolin were analyzed to determine the optimum concentration to induce osteoblast differentiation. After 48h of treatment, doses ≥100μM of diosmetin and galangin and also 500μM taxifolin revealed a toxic effect on cells. The same effect was observed in cells treated with doses ≥100μM of chrysin after 14 days of treatment. However, the safe doses of quercitrin (200 and 500μM) and taxifolin (100 and 200μM) induced bone sialoprotein and osteocalcin mRNA expression. Also higher osteocalcin secreted levels were determined in 100μM taxifolin osteoblast treated samples when compared with the control ones. On the other hand, quercitrin and taxifolin decreased Rankl gene expression in osteoblasts, suggesting an inhibition of osteoclast formation. Indeed, osteoclastogenesis suppression by quercitrin and taxifolin treatment was observed in RAW264.7 cells. Based on these findings, the present study demonstrates that quercitrin and taxifolin promote osteoblast differentiation in MC3T3-E1 cells and also inhibit osteoclastogenesis in RAW264.7 cells, showing a positive effect of these flavonoids on bone metabolism.

In vivo performance of absorbable collagen sponges with rosuvastatin in critical-size cortical bone defects.

Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties, but its local effect in bone has yet to be investigated. The aim of this study was to evaluate the potential of absorbable collagen sponge (ACS) as a carrier for RSV to enhance bone formation in critical-size cortical bone defects adjacent to titanium implants. ACS, treated with different concentrations of RSV (R1 = 8.7 + or - 1.8 microg; R2 = 52.0 + or - 4.4 microg; R3 = 259.1 + or - 8.8 microg) or phosphate-buffered saline alone, were placed into the bone marrow through a defect made in the proximal tibial cortical bone of New Zealand White rabbits. One empty defect (SHAM) served as an internal control in each animal. After a healing time of 4 weeks, a concentration-dependent increase of alkaline phosphatase activity in ACS treated with RSV was detected in the bone fluid after removing the implants. In addition, a significant concentration-dependent increase in BMP-2 mRNA levels was found in the cortical bone tissue adjacent to the RSV-treated ACS. The cortical architecture of bone defects analysed by micro-computed tomography showed a trend towards higher bone volume in the ACS+R1 group compared with SHAM, which was accompanied by an increase in the bone mineral density. Evaluation of histological sections showed new bone formation in ACS treated with RSV but not in untreated ACS. These results indicate that RSV, when administered locally in bone, may have a potential effect in stimulating bone formation.

Porous ceramic titanium dioxide scaffolds promote bone formation in rabbit peri-implant cortical defect model.

Titanium oxide (TiO₂) scaffolds have previously been reported to exhibit very low mechanical strength. However, we have been able to produce a scaffold that features a high interconnectivity, a porosity of 91% and a compressive strength above 1.2 MPa. This study analyzed the in vivo performance of the porous TiO₂ scaffolds in a peri-implant cortical defect model in the rabbit. After 8 weeks of healing, morphological microcomputed tomography analyses of the defects treated with the TiO₂ scaffolds had significantly higher bone volume, bone surface and bone surface-to-volume ratio when compared to sham, both in the cortical and bone marrow compartment. No adverse effects, i.e. tissue necrosis or inflammation as measured by lactate dehydrogenase activity and real-time reverse transcription polymerase chain reaction analysis, were observed. Moreover, the scaffold did not hinder bone growth onto the adjacent cortical titanium implant. Histology clearly demonstrated new bone formation in the cortical sections of the defects and the presence of newly formed bone in close proximity to the scaffold surface and the surface of the adjacent Ti implant. Bone-to-material contact between the newly formed bone and the scaffold was observed in the histological sections. Islets of new bone were also present in the marrow compartment albeit in small amounts. In conclusion, the present investigation demonstrates that TiO₂ scaffolds osseointegrate well and are a suitable scaffold for peri-implant bone healing and growth.

Controlled electro-implementation of fluoride in titanium implant surfaces enhances cortical bone formation and mineralization.

Previous studies have shown that bone-to-implant attachment of titanium implants to cortical bone is improved when the surface is modified with hydrofluoric acid. The aim of this study was to investigate if biological factors are involved in the improved retention of these implants. Fluoride was implemented in implant surfaces by cathodic reduction with increasing concentrations of HF in the electrolyte. The modified implants were placed in the cortical bone in the tibias of New Zealand white rabbits. After 4 weeks of healing, wound fluid collected from the implant site showed lower lactate dehydrogenase activity and less bleeding in fluoride-modified implants compared to control. A significant increase in gene expression levels of osteocalcin and tartrate-resistant acid phosphatase (TRAP) was found in the cortical bone attached to Ti implants modified with 0.001 and 0.01 vol.% HF, while Ti implants modified with 0.1% HF showed only induced TRAP mRNA levels. These results were supported by the performed micro-CT analyses. The volumetric bone mineral density of the cortical bone hosting Ti implants modified with 0.001% and 0.01% HF was higher both in the newly woven bone (<100 microm from the interface) and in the older Haversian bone (>100 microm). In conclusion, the modulation of these biological factors by surface modification of titanium implants with low concentrations of HF using cathodic reduction may explain their improved osseointegration properties.

Porous titanium granules promote bone healing and growth in rabbit tibia peri-implant osseous defects

OBJECTIVES The aim of this study was to investigate the osteoconductive properties and biological performance of porous titanium granules used in osseous defects adjacent to titanium implants. MATERIAL AND METHODS In this animal experimental study, calibrated defects were prepared in the tibias of 24 New Zealand rabbits. The defects were randomized into two tests and one control group. The test defects were grafted with either metallic or oxidized porous titanium granules (PTG or WPTG, respectively), whereas control defects were left empty (sham). The defects were closed with a submerged coin shaped titanium implant. Defects were left for healing for 4 weeks. After healing, the implants were removed and the new bone tissue formed onto the implant surface was analyzed for run x 2, osteocalcin, collagen-I, tartrate-resistant acid phosphatase, H(+)-ATPase, tumor necrosis factor-alpha, interleukin (IL)-6 and IL-10 gene expression using reverse transcriptase polymerase chain reaction. Wound fluid from the healed defects was analyzed for lactate dehydrogenase and alkaline phosphatase activity. Finally osteoconductivity was analyzed by micro-computed tomography and histology. RESULTS Significantly more new bone formed in PTG and WPTG grafted defects compared with sham. The new bone grew both through the porosities of the granules and onto the implant surfaces. The WPTG group showed significantly less expression of key inflammation markers, but with no significant difference in a marker for necrosis. The WPTG also showed a significant increase in collagen-I mRNA expression compared with PTG. CONCLUSION The results suggest that PTG and WPTG are both osteoconductive materials that can be used to promote bone formation in osseous defects adjacent to titanium implants without hampering implant osseointegration.