Multifunctional Organometallic Compounds Active against Infective Trypanosomes: Ru(II) Ferrocenyl Derivatives with Two Different Bioactive Ligands.

Human African trypanosomiasis (HAT, sleeping sickness) and American trypanosomiasis (Chagas disease) are endemic zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Trypanosoma cruzi, respectively). Just a few old drugs are available for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. Only fexinidazole has been recently incorporated into the arsenal for the treatment of HAT. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these pathogens by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: pyridine-2-thiolato-1-oxide ligand (mpo) and polypyridyl ligands (NN). Three [Ru(mpo)(dppf)(NN)](PF6) compounds and their derivatives with chloride as a counterion were synthesized and fully characterized in solid state and solution. They showed in vitro activity on bloodstream T. brucei (EC50 = 31-160 nM) and on T. cruzi trypomastigotes (EC50 = 190-410 nM). Compounds showed the lowest EC50 values on T. brucei when compared to the whole set of metal-based compounds previously developed by us. In addition, several of the Ru compounds showed good selectivity toward the parasites, particularly against the highly proliferative bloodstream form of T. brucei. Interaction with DNA and generation of reactive oxygen species (ROS) were ruled out as potential targets and modes of action of the Ru compounds. Biochemical assays and in silico analysis led to the insight that they are able to inhibit the NADH-dependent fumarate reductase from T. cruzi. One representative hit induced a mild oxidation of low molecular weight thiols in T. brucei. The compounds were stable for at least 72 h in two different media and more lipophilic than both bioactive ligands, mpo and NN. An initial assessment of the therapeutic efficacy of one of the most potent and selective candidates, [Ru(mpo)(dppf)(bipy)]Cl, was performed using a murine infection model of acute African trypanosomiasis. This hit compound lacks acute toxicity when applied to animals in the dose/regimen described, but was unable to control parasite proliferation in vivo, probably because of its rapid clearance or low biodistribution in the extracellular fluids. Future studies should investigate the pharmacokinetics of this compound in vivo and involve further research to gain deeper insight into the mechanism of action of the compounds.

Platinum and Palladium Organometallic Compounds: Disrupting the Ergosterol Pathway in Trypanosoma cruzi.

Current treatment for Chagas' disease is based on two drugs, Nifurtimox and Benznidazol, which have limitations that reduce the effectiveness and continuity of treatment. Thus, there is an urgent need to develop new, safe and effective drugs. In previous work, two new metal-based compounds with trypanocidal activity, Pd-dppf-mpo and Pt-dppf-mpo, were fully characterized. To unravel the mechanism of action of these two analogous metal-based drugs, high-throughput omics studies were performed. A multimodal mechanism of action was postulated with several candidates as molecular targets. In this work, we validated the ergosterol biosynthesis pathway as a target for these compounds through the determination of sterol levels by HPLC in treated parasites. To understand the molecular level at which these compounds participate, two enzymes that met eligibility criteria at different levels were selected for further studies: phosphomevalonate kinase (PMK) and lanosterol 14-α demethylase (CYP51). Molecular docking processes were carried out to search for potential sites of interaction for both enzymes. To validate these candidates, a gain-of-function strategy was used through the generation of overexpressing PMK and CYP51 parasites. Results here presented confirm that the mechanism of action of Pd-dppf-mpo and Pt-dppf-mpo compounds involves the inhibition of both enzymes.

Transcriptomic analysis of the adaptation to prolonged starvation of the insect-dwelling Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi is a digenetic unicellular parasite that alternates between a blood-sucking insect and a mammalian, host causing Chagas disease or American trypanosomiasis. In the insect gut, the parasite differentiates from the non-replicative trypomastigote forms that arrive upon blood ingestion to the non-infective replicative epimastigote forms. Epimastigotes develop into infective non-replicative metacyclic trypomastigotes in the rectum and are delivered via the feces. In addition to these parasite stages, transitional forms have been reported. The insect-feeding behavior, characterized by few meals of large blood amounts followed by long periods of starvation, impacts the parasite population density and differentiation, increasing the transitional forms while diminishing both epimastigotes and metacyclic trypomastigotes. To understand the molecular changes caused by nutritional restrictions in the insect host, mid-exponentially growing axenic epimastigotes were cultured for more than 30 days without nutrient supplementation (prolonged starvation). We found that the parasite population in the stationary phase maintains a long period characterized by a total RNA content three times smaller than that of exponentially growing epimastigotes and a distinctive transcriptomic profile. Among the transcriptomic changes induced by nutrient restriction, we found differentially expressed genes related to managing protein quality or content, the reported switch from glucose to amino acid consumption, redox challenge, and surface proteins. The contractile vacuole and reservosomes appeared as cellular components enriched when ontology term overrepresentation analysis was carried out, highlighting the roles of these organelles in starving conditions possibly related to their functions in regulating cell volume and osmoregulation as well as metabolic homeostasis. Consistent with the quiescent status derived from nutrient restriction, genes related to DNA metabolism are regulated during the stationary phase. In addition, we observed differentially expressed genes related to the unique parasite mitochondria. Finally, our study identifies gene expression changes that characterize transitional parasite forms enriched by nutrient restriction. The analysis of the here-disclosed regulated genes and metabolic pathways aims to contribute to the understanding of the molecular changes that this unicellular parasite undergoes in the insect vector.

Multifunctional organometallic compounds for the treatment of Chagas disease: Re(I) tricarbonyl compounds with two different bioactive ligands.

Chagas' disease (American Trypanosomiasis) is an ancient and endemic illness in Latin America caused by the protozoan parasite Trypanosoma cruzi. Although there is an urgent need for more efficient and less toxic chemotherapeutics, no new drugs to treat this disease have entered the clinic in the last decades. Searching for metal-based prospective antichagasic drugs, in this work, multifunctional Re(I) tricarbonyl compounds bearing two different bioactive ligands were designed: a polypyridyl NN derivative of 1,10-phenanthroline and a monodentate azole (Clotrimazole CTZ or Ketoconazol KTZ). Five fac-[Re(CO)3(NN)(CTZ)](PF6) compounds and a fac-[Re(CO)3(NN)(KTZ)](PF6) were synthesized and fully characterized. They showed activity against epimastigotes (IC50 3.48-9.42 µM) and trypomastigotes of T. cruzi (IC50 0.61-2.79 µM) and moderate to good selectivity towards the parasite compared to the VERO mammalian cell model. In order to unravel the mechanism of action of our compounds, two potential targets were experimentally and theoretically studied, namely DNA and one of the enzymes involved in the parasite ergosterol biosynthetic pathway, CYP51 (lanosterol 14-α-demethylase). As hypothesized, the multifunctional compounds shared in vitro a similar mode of action as that disclosed for the single bioactive moieties included in the new chemical entities. Additionally, two relevant physicochemical properties of biological interest in prospective drug development, namely lipophilicity and stability in solution in different media, were determined. The whole set of results demonstrates the potentiality of these Re(I) tricarbonyls as promising candidates for further antitrypanosomal drug development.

New multifunctional Ru(II) organometallic compounds show activity against Trypanosoma brucei and Leishmania infantum.

Human African trypanosomiasis (sleeping sickness) and leishmaniasis are prevalent zoonotic diseases caused by genomically related trypanosomatid protozoan parasites (Trypanosoma brucei and Leishmania spp). Additionally, both are co-endemic in certain regions of the world. Only a small number of old drugs exist for their treatment, with most of them sharing poor safety, efficacy, and pharmacokinetic profiles. In this work, new multifunctional Ru(II) ferrocenyl compounds were rationally designed as potential agents against these trypanosomatid parasites by including in a single molecule 1,1'-bis(diphenylphosphino)ferrocene (dppf) and two bioactive bidentate ligands: 8-hydroxyquinoline derivatives (8HQs) and polypyridyl ligands (NN). Three [Ru(8HQs)(dppf)(NN)](PF6) compounds were synthesized and fully characterized. They showed in vitro activity on bloodstream Trypanosoma brucei (IC50 140-310 nM) and on Leishmania infantum promastigotes (IC50 3.0-4.8 µM). The compounds showed good selectivity towards T. brucei in respect to J774 murine macrophages as mammalian cell model (SI 15-38). Changing hexafluorophosphate counterion by chloride led to a three-fold increase in activity on both parasites and to a two to three-fold increase in selectivity towards the pathogens. The compounds affect in vitro at least the targets of the individual bioactive moieties included in the new chemical entities: DNA and generation of ROS. The compounds are stable in solution and are more lipophilic than the free bioactive ligands. No clear correlation between lipophilicity, interaction with DNA or generation of ROS and activity was detected, which agrees with their overall similar anti-trypanosoma potency and selectivity. These compounds are promising candidates for further drug development.

Application of microwave plasma atomic emission spectrometry in bioanalytical chemistry of bioactive rhenium compounds.

Five newly synthetized fac-Re(I) tricarbonyl compounds were explored as prospective antitrypanosomal agents. The biological activity of the whole series was evaluated preliminarily against the epimastigote form of Trypanosoma cruzi. All compounds showed activity against epimastigotes with IC50 values in the low micromolar range. The most active compound [fac-Re(I)(CO)3(tmp)(CTZ)](PF6), with CTZ = clotrimazole and tmp = 3,4,7,8-tetramethyl-1,10-phenantroline, showed good selectivity towards the parasites and thus was selected to carry out further metallomic studies. For this task, a newly bioanalytical method based on microwave plasma atomic emission spectrometry (MP-AES) was developed and validated. The accuracy of the method was ensured by testing a certified reference material. Results of rhenium elemental analysis by MP-AES agreed with the proposed formula of the studied compounds, contributing to the overall validation of the method, which was then applied to evaluate the percentage of rhenium uptaken by the parasites and the association of the compounds with parasite biomacromolecules. Metallomics results showed low total rhenium percentage uptaken by parasites (∼1.2%) and preferential accumulation in the soluble proteins fraction (∼82.8%). Thus, the method based on MP-AES turned out to be an economical and green alternative for metallomics studies involving potential rhenium metallodrugs. Moreover, a comparison against rhenium determination by electrothermal atomic absorption spectrometry (ET-AAS) was included.

Preclinical Studies and Drug Combination of Low-Cost Molecules for Chagas Disease.

Chagas disease is caused by the protozoan Trypanosoma cruzi (T. cruzi). It remains the major parasitic disease in Latin America and is spreading worldwide, affecting over 10 million people. Hundreds of new compounds with trypanosomicidal action have been identified from different sources such as synthetic or natural molecules, but they have been deficient in several stages of drug development (toxicology, scaling-up, and pharmacokinetics). Previously, we described a series of compounds with simple structures, low cost, and environmentally friendly production with potent trypanosomicidal activity in vitro and in vivo. These molecules are from three different families: thiazolidenehydrazines, diarylideneketones, and steroids. From this collection, we explored their capacity to inhibit the triosephosphate isomerase and cruzipain of T. cruzi. Then, the mechanism of action was explored using NMR metabolomics and computational molecular dynamics. Moreover, the mechanism of death was studied by flow cytometry. Consequently, five compounds, 314, 793, 1018, 1019, and 1260, were pre-clinically studied and their pharmacologic profiles indicated low unspecific toxicity. Interestingly, synergetic effects of diarylideneketones 793 plus 1018 and 793 plus 1019 were evidenced in vitro and in vivo. In vivo, the combination of compounds 793 plus 1018 induced a reduction of more than 90% of the peak of parasitemia in the acute murine model of Chagas disease.

Heteroleptic Oxidovanadium(V) Complexes with Activity against Infective and Non-Infective Stages of Trypanosoma cruzi.

Five heteroleptic compounds, [VVO(IN-2H)(L-H)], where L are 8-hydroxyquinoline derivatives and IN is a Schiff base ligand, were synthesized and characterized in both the solid and solution state. The compounds were evaluated on epimastigotes and trypomastigotes of Trypanosoma cruzi as well as on VERO cells, as a mammalian cell model. Compounds showed activity against trypomastigotes with IC50 values of 0.29-3.02 µM. IN ligand and the new [VVO2(IN-H)] complex showed negligible activity. The most active compound [VVO(IN-2H)(L2-H)], with L2 = 5-chloro-7-iodo-8-hydroxyquinoline, showed good selectivity towards the parasite and was selected to carry out further biological studies. Stability studies suggested a partial decomposition in solution. [VVO(IN-2H)(L2-H)] affects the infection potential of cell-derived trypomastigotes. Low total vanadium uptake by parasites and preferential accumulation in the soluble proteins fraction were determined. A trypanocide effect was observed when incubating epimastigotes with 10 × IC50 values of [VVO(IN-2H)(L2-H)] and the generation of ROS after treatments was suggested. Fluorescence competition measurements with DNA:ethidium bromide adduct showed a moderate DNA interaction of the complexes. In vivo toxicity study on C. elegans model showed no toxicity up to a 100 µM concentration of [VVO(IN-2H)(L2-H)]. This compound could be considered a prospective anti-T. cruzi agent that deserves further research.

High Throughput Approaches to Unravel the Mechanism of Action of a New Vanadium-Based Compound against Trypanosoma cruzi.

Treatment for Chagas disease, a parasitosis caused by Trypanosoma cruzi, has always been based on two drugs, nifurtimox and benznidazole, despite the toxic side effects described after prolonged prescription. In this work, we study a new prospective antitrypanosomal drug based on vanadium, here named VIVO(5Brsal)(aminophen). We found a good IC50 value, (3.76 ± 0.08) µM, on CL Brener epimastigotes. The analysis of cell death mechanism allowed us to rule out the implication of a mechanism based on early apoptosis or necrosis. Recovery assays revealed a trypanostatic effect, accompanied by cell shape and motility alterations. An uptake mostly associated with the insoluble fraction of the parasites was deduced through vanadium determinations. Concordantly, no drastic changes of the parasite transcriptome were detected after 6 h of treatment. Instead, proteomic analysis uncovered the modulation of proteins involved in different processes such as energy and redox metabolism, transport systems, detoxifying pathways, ribosomal protein synthesis, and proteasome protein degradation. Overall, the results here presented lead us to propose that VIVO(5Brsal)(aminophen) exerts a trypanostatic effect on T. cruzi affecting parasite insoluble proteins.

Can changes in implant macrogeometry accelerate the osseointegration process?: An in vivo experimental biomechanical and histological evaluations.

OBJECTIVES: The propose was to compare this new implant macrogeometry with a control implant with a conventional macrogeometry. MATERIALS AND METHODS: Eighty-six conical implants were divided in two groups (n = 43 per group): group control (group CON) that were used conical implants with a conventional macrogeometry and, group test (group TEST) that were used implants with the new macrogeometry. The new implant macrogeometry show several circular healing cambers between the threads, distributed in the implant body. Three implants of each group were used to scanning electronic microscopy (SEM) analysis and, other eighty samples (n = 40 per group) were inserted the tibia of ten rabbit (n = 2 per tibia), determined by randomization. The animals were sacrificed (n = 5 per time) at 3-weeks (Time 1) and at 4-weeks after the implantations (Time 2). The biomechanical evaluation proposed was the measurement of the implant stability quotient (ISQ) and the removal torque values (RTv). The microscopical analysis was a histomorphometric measurement of the bone to implant contact (%BIC) and the SEM evaluation of the bone adhered on the removed implants. RESULTS: The results showed that the implants of the group TEST produced a significant enhancement in the osseointegration in comparison with the group CON. The ISQ and RTv tests showed superior values for the group TEST in the both measured times (3- and 4-weeks), with significant differences (p < 0.05). More residual bone in quantity and quality was observed in the samples of the group TEST on the surface of the removed implants. Moreover, the %BIC demonstrated an important increasing for the group TEST in both times, with statistical differences (in Time 1 p = 0.0103 and in Time 2 p < 0.0003). CONCLUSIONS: Then, we can conclude that the alterations in the implant macrogeometry promote several benefits on the osseointegration process.

Comparative high-throughput analysis of the Trypanosoma cruzi response to organometallic compounds.

There is an urgent need to develop new drugs against Chagas' disease. In addition, the mechanisms of action of existing drugs have not been completely worked out at the molecular level. High throughput approaches have been demonstrated to be powerful tools not only for understanding the basic biology of Trypanosoma cruzi, but also for the identification of drug targets such as proteins or pathways that are essential for parasite infection and survival within the mammalian host. Here, we have applied these tools towards the discovery of the effects of two organometallic compounds with trypanocidal activity, Pd-dppf-mpo and Pt-dppf-mpo, on the transcriptome and proteome of T. cruzi epimastigotes. These approaches have not yet been reported for any other prospective metal-based anti T. cruzi drug. We found differentially expressed transcripts and proteins in treated parasites. Pd-dppf-mpo treatment resulted in more modulated transcripts (2327 of 10 785 identified transcripts) than Pt-dppf-mpo treatment (201 of 10 773 identified transcripts) suggesting a mechanism of action for Pd-dppf-mpo at the transcriptome level. Similar numbers of differentially expressed proteins (342 and 411 for Pd-dppf-mpo and Pt-dppf-mpo respectively) were also observed. We further functionally categorized differentially expressed transcripts and identified cellular processes and pathways significantly impacted by treatment with the compounds. Transcripts involved in DNA binding, protein metabolism, transmembrane transport, oxidative defense, and the ergosterol pathways were found to be modulated by the presence of the compounds. Our transcriptomic dataset also contained previously validated essential genes. These data allowed us to hypothesize a multimodal mechanism of action for the trypanocidal activity of Pd-dppf-mpo and Pt-dppf-mpo, and a differential contribution of the metal moiety of each compound.

Biomechanical Effects of a New Macrogeometry Design of Dental Implants: An In Vitro Experimental Analysis.

The purpose of the present study was to measure and compare the insertion torque, removal torque, and the implant stability quotient by resonance frequency analysis in different polyurethane block densities of two implant macrogeometries. Four different polyurethane synthetic bone blocks were used with three cortical thickness: Bone 1 with a cortical thickness of 1 mm, Bone 2 with a cortical thickness of 2 mm, Bone 3 with a cortical thickness of 3 mm, and Bone 4, which was totally cortical. Four groups were created in accordance with the implant macrogeometry (n = 10 per group) and surface treatment: G1-regular implant design without surface treatment; G2-regular implant design with surface treatment; G3-new implant design without surface treatment; G4-new implant design with surface treatment. All implants used were 4 mm in diameter and 10 mm in length and manufactured in commercially pure titanium (grade IV) by Implacil De Bortoli (São Paulo, Brazil). The implants were installed using a computed torque machine, and following installation of the implant, the stability quotient (implant stability quotient, ISQ) values were measured in two directions using Osstell devices. The data were analyzed by considering the 5% level of significance. All implant groups showed similar mean ISQ values without statistical differences (p > 0.05), for the same synthetic bone block: for Bone 1, the value was 57.7 ± 3.0; for Bone 2, it was 58.6 ± 2.2; for Bone 3, it was 60.6 ± 2.3; and for Bone 4, it was 68.5 ± 2.8. However, the insertion torque showed similar higher values for the regular macrogeometry (G1 and G2 groups) in comparison with the new implant macrogeometry (G3 and G4 groups). The analysis of the results found that primary stability does not simply depend on the insertion torque but also on the bone quality. In comparison with the regular implant macrogeometry, the new implant macrogeometry decreased the insertion torque without affecting the implant stability quotient values.

Impact of Different Titanium Implant Thread Designs on Bone Healing: A Biomechanical and Histometric Study with an Animal Model.

Threads of dental implants with healing chamber configurations have become a target to improve osseointegration. This biomechanical and histometric study aimed to evaluate the influence of implant healing chamber configurations on the torque removal value (RTv), percentage of bone-to-implant contact (BIC%), bone fraction occupancy inside the thread area (BAFO%), and bone and osteocyte density (Ost) in the rabbit tibia after two months of healing. Titanium implants with three different thread configurations were evaluated: Group 1 (G1), with a conventional "v" thread-shaped implant design; Group 2 (G2), with square threads; and Group 3 (G3), the experimental group with longer threads (healing chamber). Ten rabbits (4.5 ± 0.5 kg) received three implants in each tibia (one per group), distributed in a randomized manner. After a period of two months, the tibia blocks (implants and the surrounding tissue) were removed and processed for ground sectioning to evaluate BIC%, BAFO%, and osteocyte density. The ANOVA one-way statistical test was used followed by the Bonferoni's multiple comparison test to determine individual difference among groups, considering a statistical difference when p < 0.05. Histometric evaluation showed a higher BAFO% values and Ost density for G3 in comparison with the other two groups (G1 and G2), with p < 0.05. However, the RTv and BIC% parameters were not significantly different between groups (p > 0.05). The histological data suggest that the healing chambers in the implant macrogeometry can improve the bone reaction in comparison with the conventional thread design.

Microgrooves and Microrugosities in Titanium Implant Surfaces: An In Vitro and In Vivo Evaluation.

The physical characteristics of an implant surface can determine and/or facilitate osseointegration processes. In this sense, a new implant surface with microgrooves associated with plus double acid treatment to generate roughness was evaluated and compared in vitro and in vivo with a non-treated (smooth) and double acid surface treatment. Thirty disks and thirty-six conical implants manufactured from commercially pure titanium (grade IV) were prepared for this study. Three groups were determined, as described below: Group 1 (G1), where the samples were only machined; group 2 (G2), where the samples were machined and had their surface treated to generate roughness; and test group 3 (G3), where the samples were machined with microgrooves and the surface was treated to generate the roughness. For the in vitro analysis, the samples were submitted to scanning microscopy (SEM), surface profilometry, the atomic force microscope (MFA) and the surface energy test. For the in vivo analyses, thirty-six implants were placed in the tibia of 9 New Zealand rabbits in a randomized manner, after histological and histomorphometric analysis, to determine the level of contact between the bone and implant (BIC%) and the bone area fraction occupancy (BAFO%) inside of the threads. The data collected were statistically analyzed between groups (p < 0.05). The in vitro evaluations showed different roughness patterns between the groups, and the G3 group had the highest values. In vivo evaluations of the BIC% showed 50.45 ± 9.57% for the G1 group, 55.32 ± 10.31% for the G2 group and 68.65 ± 9.98% for the G3 group, with significant statistical difference between the groups (p < 0.0001). In the BAFO% values, the G1 group presented 54.97 ± 9.56%, the G2 group 59.09 ± 10.13% and the G3 group 70.12 ± 11.07%, with statistical difference between the groups (p < 0.001). The results obtained in the evaluations show that the surface with microgrooves stimulates the process of osseointegration, accelerating the healing process, increasing the contact between the bone and the implant and the area of new bone formation.

Biomechanical and Histological Analysis of Titanium (Machined and Treated Surface) Versus Zirconia Implant Materials: An In Vivo Animal Study.

OBJECTIVES: The aim of this study was to perform an in vivo histological comparative evaluation of bone formation around titanium (machined and treated surface) and zirconia implants. For the present study were used 50 commercially pure titanium implants grade IV, being that 25 implants with a machined surface (TiM group), 25 implants with a treated surface (TiT group) and, 25 implants were manufactured in pure zirconia (Zr group). The implants (n = 20 per group) were installed in the tibia of 10 rabbits. The implants distribution was randomized (n = 3 implants per tibia). Five implants of each group were analyzed by scanning electron microscopy and an optical laser profilometer for surface roughness characterization. Six weeks after the implantation, 10 implants for each group were removed in counter-torque for analysis of maximum torque value. The remaining samples were processed, included in historesin and cut to obtain non-decalcified slides for histomorphological analyses and histomorphometric measurement of the percentage of bone-implant contact (BIC%). Comparisons were made between the groups using a 5% level of significance (p < 0.05) to assess statistical differences. The results of removal torque values (mean ± standard deviation) showed for the TiM group 15.9 ± 4.18 N cm, for TiT group 27.9 ± 5.15 N cm and for Zr group 11.5 ± 2.92 N cm, with significant statistical difference between the groups (p < 0.0001). However, the BIC% presented similar values for all groups (35.4 ± 4.54 for TiM group, 37.8 ± 4.84 for TiT group and 34.0 ± 6.82 for Zr group), with no statistical differences (p = 0.2171). Within the limitations of the present study, the findings suggest that the quality of the new bone tissue formed around the titanium implants present a superior density (maturation) in comparison to the zirconia implants.

In Vitro Assessment of the Functional Dynamics of Titanium with Surface Coating of Hydroxyapatite Nanoparticles.

Manipulation of implant surface characteristics constitutes a promising strategy for improving cell growth and tissue response on a variety of materials with different surface topographies. Mesenchymal progenitor cells with a capacity to respond to titanium surface stimuli and differentiate into osteoblasts were used to perform comparative tests between two different implant topographies, including their functional interaction with pre-osteoblasts directly seeded onto the implants. Functional analysis of nanostructured implant surfaces was performed by in vitro assay analysis. The machined surface of titanium implants (mach group) was used as a control and compared with a nanoparticle HA activated surface implant (nano group), developed by the deposition of pure crystalline hydroxyapatite. Cell culture on the nano group surface resulted in higher cell adhesion and cultured osteoblast viability compared with the mach group. Scanning electron microscope (SEM) images revealed a stable interaction, indicated by the presence of focal cell adhesion formation. These results together with positive mineralization assays showed the nano group to be an excellent scaffold for bone-implant integration.

Study of Two Bovine Bone Blocks (Sintered and Non-Sintered) Used for Bone Grafts: Physico-Chemical Characterization and In Vitro Bioactivity and Cellular Analysis.

In this work, the physicochemical properties and in vitro bioactivity and cellular viability of two commercially available bovine bone blocks (allografts materials) with different fabrication processes (sintered and not) used for bone reconstruction were evaluated in order to study the effect of the microstructure in the in vitro behavior. Scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectrometry, mechanical resistance of blocks, mercury porosimetry analysis, in vitro bioactivity, and cell viability and proliferation were performed to compare the characteristics of both allograft materials against a synthetic calcium phosphate block used as a negative control. The herein presented results revealed a very dense structure of the low-porosity bovine bone blocks, which conferred the materials' high resistance. Moreover, relatively low gas, fluid intrusion, and cell adhesion were observed in both the tested materials. The structural characteristics and physicochemical properties of both ceramic blocks (sintered and not) were similar. Finally, the bioactivity, biodegradability, and also the viability and proliferation of the cells was directly related to the physicochemical properties of the scaffolds.

Analysis of Trauma Intensity during Surgical Bone Procedures Using NF-κB Expression Levels as a Stress Sensor: An Experimental Study in a Wistar Rat Model.

Aim and objectives: It is well known that the transcription factor NF-κB regulates multiple aspects of innate and adaptive immune functions and functions as a pivotal mediator of inflammatory responses. In the present study, we evaluated the trauma generated (inflammatory reaction) after osteotomy bone surgical procedures and placement of implants in the femoral cortical bone of Wistar rats. Surgical stress was evaluated measuring the release and activation of the NF-κB factor. Materials and Methods: Rats were divided into four groups (n = 10) and submitted to different surgical treatments: Control Group (G1 group), only bone perforation was performed without irrigation; Implant Group (G2 group), a titanium implant was inserted after bone perforation without irrigation; Irrigated Group (G3 group) perforations were performed with intense irrigation; and Vitaminic Compound Group (G4 group) surgical perforation was performed without irrigation and a vitaminic compound containing the principal ions present in the natural bone structure was used to fill the bone defect. All animals were euthanized six hours after the surgical procedure and NF-κB levels were determined through immunohistochemical stain followed by direct counting of labeled and unlabeled osteocytes. Results: Among different treated groups, the overall mean of the NF-κB positive cell count in all positions were higher for G1 group (33.4 ± 2.45 cells). NF-κB values were lower in the G2 group (28.9 ± 2.70 cells), whereas in the G3 group (24.3 ± 2.72 cells) as well as in G4 group still lesser NF-κB positive cells were counted (26.5 ± 2.60 cells). Conclusions: The results here presented suggest that maneuvers performed during osteotomy procedures can significantly affect inflammation levels. The NF-κB activation during the surgical procedures can be minimized and/or controlled thought the adequate irrigation or application of adequate substances.

Effect of Different Morphology of Titanium Surface on the Bone Healing in Defects Filled Only with Blood Clot: A New Animal Study Design.

Background: The objective of the present histologic animal study was to analyze whether roughness of the titanium surface can influence and/or stimulate the bone growth in defects filled with the blood using a rabbit tibia model. Materials and Methods: Forty sets (implant and abutment), dental implant (3.5 mm in diameter and 7 mm in length) plus healing abutment (2.5 mm in diameter), were inserted in the tibiae of 10 rabbits. Moreover, twenty titanium discs were prepared. The abutment and discs were treated by 4 different methods and divided into 4 groups: (group A) machined abutments (smooth); (group B) double acid etching treatment; (group C) treatment with blasting with particles of aluminum oxide blasted plus acid conditioning; (group D) treatment with thorough blasting with particles of titanium oxide plus acid conditioning. The discs were used to characterize the surfaces by a profilometer and scanning electronic microscopy. Results: After 8 weeks, the new bone formation around the sets of the samples was analyzed qualitatively and quantitatively in relation to bone height from the base of the implant and presence of osteocytes. Group C (1.50±0.20 mm) and group D (1.62±0.18 mm) showed bone growth on the abutment with higher values compared to group A (0.94±0.30 mm) and group B (1.19±0.23 mm), with significant difference between the groups (P < 0.05). In addition, osteocyte presence was higher in groups with surface treatment related to machined (P < 0.05). Conclusions: Within the limitations of the present study, it was possible to observe that there is a direct relationship between the roughness present on the titanium surface and the stimulus for bone formation, since the presence of larger amounts of osteocytes on SLA surfaces evidenced this fact. Furthermore, the increased formation of bone tissue in height demonstrates that there is an important difference between the physical and chemical methods used for surface treatment.

Effect of a new anti-T. cruzi metallic compound based on palladium.

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. It is estimated that 6 million people are infected in Latin America. Current treatment is not effective due to the severe side effects and the limited efficacy towards the chronic phase of the disease. Considering the growing need for specific anti-Trypanosoma cruzi drugs, organometallic Pt and Pd based compounds were previously synthesized. Although the Pt-based compound effects on T. cruzi death have been reported, no mechanism of action has been proposed for the Pd-based analogous compound. In this work, we determined excellent to very good values of IC50 and SI. To analyze the compound mode of action, we measured Pd uptake and its association to the macromolecules of the parasite by electrothermal atomic absorption spectrometry. We found a poor uptake, which reaches only 16% after 24 h of incubation using 10× IC50, being the scarce incorporated metal preferentially associated to DNA. However, this compound has a trypanocidal effect, leading to morphological changes such as shortening of the parasite cell body and inducing necrosis after 24 h of treatment. Furthermore, this compound impairs the parasite development in the host both at the trypomastigote infection process and the intracellular amastigotes replication. In conclusion, our findings support that Pd-dppf-mpo compound constitutes a promising anti-T. cruzi compound effective against the chronic phase of the disease.