Inhibitory Effect of 5-Aminoimidazole-4-Carbohydrazonamides Derivatives Against Candida spp. Biofilm on Nanohydroxyapatite Substrate.

Candida can adhere and form biofilm on biomaterials commonly used in medical devices which is a key attribute that enhances its ability to cause infections in humans. Furthermore, biomaterial-related infections represent a major therapeutic challenge since Candida biofilms are implicated in antifungal therapies failure. The goals of the present work were to investigate the effect of three 5-aminoimidazole-4-carbohydrazonamides, namely (Z)-5-amino-1-methyl-N'-aryl-1H-imidazole-4-carbohydrazonamides [aryl = phenyl (1a), 4-fluorophenyl (1b), 3-fluorophenyl (1c)], on Candida albicans and Candida krusei biofilm on nanohydroxyapatite substrate, a well-known bioactive ceramic material. To address these goals, both quantitative methods (by cultivable cell numbers) and qualitative evaluation (by scanning electron microscopy) were used. Compounds cytocompatibility towards osteoblast-like cells was also evaluated after 24 h of exposure, through resazurin assay. The three tested compounds displayed a strong inhibitory effect on biofilm development of both Candida species as potent in vitro activity against C. albicans sessile cells. Regarding cytocompatibility, a concentration-dependent effect was observed. Together, these findings indicated that the potent activity of imidazole derivatives on Candida spp. biofilms on nanohydroxyapatite substrate, in particular compound 1c, is worth further investigating.

Osteoclastogenic differentiation of human precursor cells over micro- and nanostructured hydroxyapatite topography.

BACKGROUND: Surface topography is a key parameter in bone cells-biomaterials interactions. This study analyzed the behavior of human osteoclast precursor cells cultured over three hydroxyapatite (HA) surfaces ranging from a micro- to nanoscale topography. METHODS: HA surfaces were prepared with microsized HA particles, at 1300°C (HA1), and with nanosized HA particles at 1000°C (HA2) and 830°C (HA3). Human osteoclast precursors were cultured in the absence or presence of M-SCF and RANKL. RESULTS: HA surfaces had similar chemical composition, however, HA1 and HA3 presented typical micro- and nanostructured topographies, respectively, and HA2 profile was between those of HA1 and HA3. The decrease on the average grain diameter to the nanoscale range (HA3) was accompanied by an increase in surface area, porosity and hydrophilicity and a decrease in roughness. Compared to HA1 surface, HA3 allowed a lower osteoclastic adhesion, differentiation and function. Differences in the cell response appeared to be associated with the modulation of relevant intracellular signaling pathways. CONCLUSIONS: The decrease in HA grain size to a biomimetic nanoscale range, appears less attractive to osteoclastic differentiation and function, compared to the HA microsized topography. GENERAL SIGNIFICANCE: This observation emphasizes the role of surface topography in designing advanced biomaterials for tailored bone cells response in regenerative strategies.

Antibacterial activity and biocompatibility of three-dimensional nanostructured porous granules of hydroxyapatite and zinc oxide nanoparticles--an in vitro and in vivo study.

Ceramic scaffolds are widely studied in the bone tissue engineering field due to their potential in regenerative medicine. However, adhesion of microorganisms on biomaterials with subsequent formation of antibiotic-resistant biofilms is a critical factor in implant-related infections. Therefore, new strategies are needed to address this problem. In the present study, three-dimensional and interconnected porous granules of nanostructured hydroxyapatite (nanoHA) incorporated with different amounts of zinc oxide (ZnO) nanoparticles were produced using a simple polymer sponge replication method. As in vitro experiments, granules were exposed to Staphylococcus aureus and Staphylococcus epidermidis and, after 24 h, the planktonic and sessile populations were assessed. Cytocompatibility towards osteoblast-like cells (MG63 cell line) was also evaluated for a period of 1 and 3 days, through resazurin assay and imaging flow cytometry analysis. As in vivo experiments, nanoHA porous granules with and without ZnO nanoparticles were implanted into the subcutaneous tissue in rats and their inflammatory response after 3, 7 and 30 days was examined, as well as their antibacterial activity after 1 and 3 days of S. aureus inoculation. The developed composites proved to be especially effective at reducing bacterial activity in vitro and in vivo for a weight percentage of 2% ZnO, with a low cell growth inhibition in vitro and no differences in the connective tissue growth and inflammatory response in vivo. Altogether, these results suggest that nanoHA-ZnO porous granules have a great potential to be used in orthopaedic and dental applications as a template for bone regeneration and, simultaneously, to restrain biomaterial-associated infections.

Poly(DL-lactic acid) scaffolds as a bone targeting platform for the co-delivery of antimicrobial agents against S. aureus-C.albicans mixed biofilms.

New strategies for the treatment of polymicrobial bone infections are required. In this study, the co-delivery of two antimicrobials by poly(D,L-lactic acid) (PDLLA) scaffolds was investigated in a polymicrobial biofilm model. PDLLA scaffolds were prepared by solvent casting/particulate leaching methodology, incorporating minocycline and voriconazole as clinically relevant antimicrobial agents. The scaffolds presented a sponge-like appearance, suitable to support cell proliferation and drug release. Single- and dual-species biofilm models of Staphylococcus aureus and Candida albicans were developed and characterized. S. aureus presented a higher ability to form single-species biofilms, compared to C. albicans. Minocycline and voriconazole-loaded PDLLA scaffolds showed activity against S. aureus and C. albicans single- and dual-biofilms. Ultimately, the cytocompatibility/functional activity of PDLLA scaffolds observed in human MG-63 osteosarcoma cells unveil their potential as a next-generation co-delivery system for antimicrobial therapy in bone infections.

Heparinized hydroxyapatite/collagen three-dimensional scaffolds for tissue engineering.

Currently, in bone tissue engineering research, the development of appropriate biomaterials for the regeneration of bony tissues is a major concern. Bone tissue is composed of a structural protein, collagen type I, on which calcium phosphate crystals are enclosed. For tissue engineering, one of the most applied strategies consists on the development and application of three dimensional porous scaffolds with similar composition to the bone. In this way, they can provide a physical support for cell attachment, proliferation, nutrient transport and new bone tissue infiltration. Hydroxyapatite is a calcium phosphate with a similar composition of bone and widely applied in several medical/dentistry fields. Therefore, in this study, hydroxyapatite three dimensional porous scaffolds were produced using the polymer replication method. Next, the porous scaffolds were homogeneously coated with a film of collagen type I by applying vacuum force. Yet, due to collagen degradability properties, it was necessary to perform an adequate crosslinking method. As a result, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) was employed as an efficient and non-toxic crosslinking method in this research. The composites were characterized by means of SEM, DSC and TNBS. Furthermore, heparin was incorporated in order to accomplish sustained delivery of a growth factor of interest namely, bone morphogenetic proteins (BMP-2). BMP-2 binding and release of non-heparinized and heparinized scaffolds was evaluated at specific time points. The incorporation of heparin leads to a reduced initial burst phase when compared to the non heparinized materials. The results show a beneficial effect with the incorporation of heparin and its potential as a localized drug delivery system for the sustained release of growth factors.

Synthesis and characterization of HAp nanorods from a cationic surfactant template method.

Hydroxyapatite (HAp) [Ca(10)(PO(4))(6)(OH)(2)] nanorods were synthesized using a surfactant templating method, with cetyltrimethylammonium bromide (CTAB) micelles acting as template for HAp growth. The effects of the sintering temperature on the morphological and crystallographic characteristics and on chemical composition of the "as-prepared" structures are discussed. The experimental results show that low heat-treatment temperatures are preferred in order to obtain high quality nanorods, with diameters ranging between 20 and 50 nm. High heat-treatment temperatures enhance the thermal decomposition of HAp into other calcium phosphate compounds, and the sintering of particles into micrometer ball-like structures. The stability of aqueous suspensions of HAp nanorods is also discussed.

Early spreading and propagation of human bone marrow stem cells on isotropic and anisotropic topographies of silica thin films produced via microstamping.

While there has been rapid development of microfabrication techniques to produce high-resolution surface modifications on a variety of materials in the last decade, there is still a strong need to produce novel alternatives to induce guided tissue regeneration on dental implants. High-resolution microscopy provides qualitative and quantitative techniques to study cellular guidance in the first stages of cell-material interactions. The purposes of this work were (1) to produce and characterize the surface topography of isotropic and anisotropic microfabricated silica thin films obtained by sol-gel processing, and (2) to compare the in vitro biological behavior of human bone marrow stem cells on these surfaces at early stages of adhesion and propagation. The results confirmed that a microstamping technique can be used to produce isotropic and anisotropic micropatterned silica coatings. Atomic force microscopy analysis was an adequate methodology to study in the same specimen the sintering derived contraction of the microfabricated coatings, using images obtained before and after thermal cycle. Hard micropatterned coatings induced a modulation in the early and late adhesion stages of cell-material and cell-cell interactions in a geometry-dependent manner (i.e., isotropic versus anisotropic), as it was clearly determined, using scanning electron and fluorescence microscopies.

Alginate-nanohydroxyapatite hydrogel system: Optimizing the formulation for enhanced bone regeneration.

Ceramic/polymer-based biocomposites have emerged as potential biomaterials to fill, replace, repair or regenerate injured or diseased bone, due to their outstanding features in terms of biocompatibility, bioactivity, injectability, and biodegradability. However, these properties can be dependent on the amount of ceramic component present in the polymer-based composite. Therefore, in the present study, the influence of nanohydroxyapatite content (30 to 70 wt%) on alginate-based hydrogels was studied in order to evaluate the best formulation for maximizing bone tissue regeneration. The composite system was characterized in terms of physic-chemical properties and biological response, with in vitro cytocompatibility assessment with human osteoblastic cells and ex vivo functional evaluation in embryonic chick segmental bone defects. The main morphological characteristics of the alginate network were not affected by the addition of nanohydroxyapatite. However, physic-chemical features, like water-swelling rate, stability at extreme pH values, apatite formation, and Ca2+ release were nanoHA dose-dependent. Within in vitro cytocompatibility assays it was observed that hydrogels with nanoHA 30% content enhanced osteoblastic cells proliferation and expression of osteogenic transcription factors, while those with higher concentrations (50 and 70%) decreased the osteogenic cell response. Ex vivo data underlined the in vitro findings, revealing an enhanced collagenous deposition, trabecular bone formation and matrix mineralization with Alg-nanoHA30 composition, while compositions with higher nanoHA content induced a diminished bone tissue response. The outcomes of this study indicate that nanohydroxyapatite concentration plays a major role in physic-chemical properties and biological response of the composite system and the optimization of the components ratio must be met to maximize bone tissue regeneration.

Micropatterned Silica Films with Nanohydroxyapatite for Y-TZP Implants.

This investigation aimed at developing micropatterned silica thin films (MSTFs) containing nanohydroxyapatite (nano-HA) microaggregates that were not completely covered by silica so that they could directly interact with the surrounding cells. The objectives were 1) to evaluate the effect of the presence of 2 films (MSTF with or without nano-HA addition) on the characteristic strength (σ0) and Weibull modulus ( m) of a yttria-stabilized tetragonal zirconia polycrystal (Y-TZP) and 2) to evaluate the effect of these 2 films, as applied onto the Y-TZP surface, on the morphology, orientation, and proliferation of MG63 cells. Sol-gel process and soft lithography were used to apply the MSTF onto the Y-TZP specimens. Three experimental groups were produced: Y-TZP, Y-TZP + MSTF, and Y-TZP + MSTF + sprayed nano-HA. All surfaces were characterized by scanning electron microscopy and energy-dispersive X-ray spectroscopy and tested for 4-point flexural strength ( n = 30) in water at 37 °C. Weibull analysis was used to determine m and σ0 (maximum likelihood method). In vitro biological behavior was performed with human osteoblast-like cells (MG63). Y-TZP was successfully coated with MSFT and MSFT + nano-HA. Scanning electron microscopy micrographs indicated that the microaggregates of nano-HA were not entirely covered by the silica. There was no statistically significant difference among the experimental groups for σ0 and m. In the groups containing the films, the cells were elongated and aligned along the lines. The MSFT + nano-HA group showed significantly higher cell metabolic activity than that obtained for the Y-TZP group at day 7. This investigation was successful in producing an MSTF containing nano-HA microaggregates that remained exposed to the environment. The developed films did not jeopardize the structural reliability of a commercial Y-TZP, as confirmed by the Weibull statistics. The MG63 cells seeded over the films became elongated and aligned along the films' micropatterned lines. Y-TZP specimens coated with MSTF and nano-HA showed a higher cell metabolic activity and proliferation after 7 d of culture when compared with uncoated Y-TZP.

Supercritical CO2 assisted process for the production of high-purity and sterile nano-hydroxyapatite/chitosan hybrid scaffolds.

Hybrid scaffolds composed of hydroxyapatite (HAp), in particular in its nanometric form (n-HAp), and chitosan (CS) are promising materials for non-load-bearing bone graft applications. The main constraints of their production concern the successful implementation of the final purification/neutralization and sterilization steps. Often, the used purification strategies can compromise scaffold structural features, and conventional sterilization techniques can result in material's thermal degradation and/or contamination with toxic residues. In this context, this work presents a process to produce n-HAp/CS scaffolds mimicking bone composition and structure, where an innovative single step based on supercritical CO2 extraction was used for both purification and sterilization. A removal of 80% of the residual acetic acid was obtained (T = 75°C, p = 8.0 MPa, 2 extraction cycles of 2 h) giving rise to scaffolds exhibiting adequate interconnected porous structure, fast swelling and storage modulus compatible with non-load-bearing applications. Moreover, the obtained scaffolds showed cytocompatibility and osteoconductivity without further need of disinfection/sterilization procedures. Among the main advantages, the proposed process comprises only three steps (n-HAp/CS dispersion preparation; freeze-drying; and supercritical CO2 extraction), and the supercritical CO2 extraction show clear advantages over currently used procedures based on neutralization steps. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 965-975, 2018.

Laser surface treatment of hydroxyapatite for enhanced tissue integration: surface characterization and osteoblastic interaction studies.

Biocompatibility has long been associated with surface microtopography, microtexture, and microchemistry. The surface topography eventually affects the nature and the intensity of the interactions that occur at biomaterial-biological interface (cell adhesion, mobility, spreading, and proliferation). Therefore, it is necessary to produce and work with controlled microtopographical surfaces that present reproducible microdomains of a dimension similar to that of the biological elements of interest (in this case, osteoblasts). There are a number of substrates that already have been studied in terms of surface topography; however, few studies are related to hydroxyapatite (HA) substrates. As it is well established, HA is a well-known ceramic that is extremely used in medical applications, namely implants and coatings. In this work, the surface topography of dense HA substrates was altered by using KFr excimer laser. The surface was characterized by atomic force microscopy and contact angle measurements, while the cell distribution and morphology was assessed by scanning electron microscopy and confocal laser scanning microscopy. Results revealed that the surface is characterized by a homogeneous columnar structure with high specific area. Moreover, cells were able to attach and spread on the surface of the samples, and gradually grow into nearly confluent monolayers.

Nanohydroxyapatite microspheres as delivery system for antibiotics: release kinetics, antimicrobial activity, and interaction with osteoblasts.

Severe periodontitis treatment, where massive alveolar bone loss occurs, involves bone defect filling and intensive systemic log-term antibiotics administration. This study aims at developing novel injectable drug delivery systems (nanohydroxyapatite microspheres) with the drug releasing capability for periodontitis treatment and simultaneously initiating the osteointegration process. Materials were characterized by XRD, SEM, inverted stand optical microscope analysis, and mercury porosimetry method. Amoxicillin, amoxicillin + clavulanic acid, and erythromycin were the antibiotics used. Release properties during 28 days from the hydroxyapatite (HA) granules, and two types of nanoHA microspheres were investigated. Biocompatibility was assessed by cytotoxicity assays. HA granules were inadequate, releasing all antibiotic during the first hours. The concentration of antibiotics released in the first days from HA-2 was higher than from HA-1 microspheres, because of the increased porosity and surface area. The release profiles (fast initial release followed by long-term sustained release) of effective doses of antibiotics make these systems good alternatives for antibiotics delivery. Osteoblasts proliferated well on both types of microspheres, being cell growth enhanced in the presence of antibiotics. Erythromycin presented the most beneficial effect. Combining the sustained antibiotic release with the osteoconduction, resorbability, and potential use as injectable bone filling material of porous HA microspheres, these systems provided a forth fold beneficial effect.

Characterization of gelatin and chitosan scaffolds cross-linked by addition of dialdehyde starch.

In this study the influence of the addition of dialdehyde starch on the properties of scaffolds based on gelatin and chitosan obtained by the freeze-drying method was investigated. In addition, the adhesion and proliferation of human osteosarcoma SaOS-2 cells on the obtained scaffolds was examined. Chitosan and gelatin were mixed in different weight ratios (75/25, 50/50, 25/75) with 1, 2 and 5 wt% addition of dialdehyde starch. The obtained scaffolds were subjected to mechanical testing, infrared spectroscopy, swelling measurements, low-pressure porosimetry and zeta potential measurement. Internal material structures were observed by scanning electron microscopy. The results showed that the cross-linking process occurred after the addition of dialdehyde starch and resulted in increased mechanical strength, swelling properties, zeta potential and porosity of studied materials. The attachment of SaOS-2 cells to all modified materials was better compared to an unmodified control and the proliferation of these cells was markedly increased on modified scaffolds.

Anti-sessile bacterial and cytocompatibility properties of CHX-loaded nanohydroxyapatite.

Nanohydroxyapatite possesses exceptional biocompatibility and bioactivity regarding bone cells and tissues, justifying its use as a coating material or as a bone substitute. Unfortunately, this feature may also encourage bacterial adhesion and biofilm formation. Surface functionalization with antimicrobials is a promising strategy to reduce the likelihood of bacterial infestation and colonization on medical devices. Chlorhexidine digluconate is a common and effective antimicrobial agent used for a wide range of medical applications. The purpose of this work was the development of a nanoHA biomaterial loaded with CHX to prevent surface bacterial accumulation and, simultaneously, with good cytocompatibility, for application in the medical field. CHX (5-1500 mg/L) was loaded onto nanoHA discs and the materials were evaluated for CHX adsorption and release profile, physic-chemical features, antibacterial activity against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, and cytocompatibility toward L929 fibroblasts. Results showed that the adsorption of CHX on nanoHA surface occurred by electrostatic interactions between the cationic group of CHX and the phosphate group of nanoHA. The release of CHX from CHX-loaded nanoHA showed a fast initial rate followed by a slower kinetics release, due to constraints caused by dilution and diffusion-limiting processes. NanoHA.50 to nanoHA.1500 showed strong anti-sessile activity, inhibiting bacterial adhesion and the biofilm formation. CHX-nanoHA caused a dose- and time-dependent inhibitory effect on the proliferation of fibroblasts for nanoHA.100 to nanoHA.1500. Cellular behavior on nanoHA.5 and nanoHA.50 was similar to control. Therefore, CHX-loaded nanoHA surfaces appear as a promising alternative to prevention of devices-related infections.

In vitro antimicrobial activity and biocompatibility of propolis containing nanohydroxyapatite.

The high number of biomaterial associated infections demands new strategies to prevent this problem. In this study the suitability of nanohydroxyapatite (nanoHA)-based surfaces containing two Brazilian extracts of propolis (green and red ones) to prevent bacterial growth and biofilm formation, as well as its non-cytotoxic nature, was investigated. Optical density, colony forming units and MTT reduction assay were used to assess the materials' antibacterial activity against planktonic and sessile growth of Staphylococcus aureus. NanoHA matrix was able to absorb both types of propolis and the obtained results revealed the antibacterial effectiveness of the novel materials expressed as the reduction of bacterial growth and biofilm formation ability. Additionally, cell culture tests showed the growth of fibroblasts with high metabolic activity and without membrane damage. Therefore, these nanoHA-based surfaces containing natural products deriving from bees may be a promising bioactive biomaterial to be further studied with the aim of application to orthopaedic or dental devices.

Direct and indirect effects of P2O5 glass reinforced-hydroxyapatite composites on the growth and function of osteoblast-like cells.

Human osteoblast-like cells were plated on hydroxyapatite and P2O5-glass reinforced hydroxyapatite composite discs. They were also cultured in the presence of media obtained by incubating the discs in the absence of cells. The effects of these direct and indirect interactions were examined by measuring cell proliferation and the expression of certain key extracellular matrix antigens. One composite was found to initially delay cell growth, while the extract of a different composite appeared to down-regulate DNA synthesis. Flow cytometry analysis showed that growth directly on the discs had little effect on collagen type I, but reduced fibronectin and osteocalcin levels. The extracts of the materials generally had less effect, although one extract obtained from the glass-reinforced hydroxyapatite significantly down-regulated fibronectin. These in vitro studies thus suggest that there were only few differences overall in the growth of the cells directly on the glass-reinforced compared with the hydroxyapatite discs and also only relatively small effects of the extracts on the cells. However, the flow cytometry results suggest that both the materials and the extracts may have a potentially important influence on connective tissue production, and that these effects are both material- and antigen-specific.

Glass-reinforced hydroxyapatite composites: fracture toughness and hardness dependence on microstructural characteristics.

Fracture toughness and hardness properties of CaO-P2O5 glass-reinforced hydroxyapatite composites have been assessed using indentation techniques and results calculated according to Laugier and Evans' equations. Both properties showed to be dependent upon several microstructural characteristics, namely residual porosity and the percentage of secondary beta and alpha tricalcium phosphate phases in the structure of the composites. Composites presented a Palmqvist-type indentation crack system, which is the specific crack system addressed by Laugier's approach. Fracture toughness determinations according to Evan's equation, which is a universal one and adapted to both median and Palmqvist crack systems, did not correlate well with Laugier determinations.

Surface modifications of glass-reinforced hydroxyapatite composites.

Surface modifications of glass-reinforced hydroxyapatite composites immersed in a simulated physiological solution were studied using X-ray photoelectron spectroscopy, scanning electron microscopy and energy dispersive X-ray spectroscopy. In the first stages of the apatite formation process, calcium and silicon ions were leached out from the surface of the composites. After 12 days of immersion, apatite crystals were detected on the surface indicating bioactive behaviour. Sodium ions attributed to sodium sulphate were also found in the apatite layer. Bound water on the surface also increased with immersion time.

Microstructural dependence of Young's and shear moduli of P2O5 glass reinforced hydroxyapatite for biomedical applications.

P2O5 glass reinforced hydroxyapatite composite materials were prepared through a liquid-phase sintering process. Secondary phases, beta- and alpha-tricalcium phosphates (beta-TCP and alpha-TCP), were formed in the microstructure of the composites, due to the reaction between the liquid glassy phase and the hydroxyapatite matrix. The dynamic Young's modulus (E) and shear modulus (G) of these composites were determined using an impulse excitation method. By applying the Duckworth-Knudsen equation, the elastic property results were correlated with the relative proportion of beta-TCP and alpha-TCP phases and with the porosity percentage present in the microstructure. Glass reinforced hydroxyapatite composites showed lower Young's and shear moduli than unmodified hydroxyapatite, mainly due to the presence of beta-TCP phase. The Duckworth-Knudsen model demonstrated an exponential dependence of E and G modulus with porosity and mathematical equations were derived for composite materials with porosity correction factors (b) of 4.04 and 4.11, respectively, indicating that porosity largely decreased both E and G moduli.

Flow cytometry analysis of the effects of pre-immersion on the biocompatibility of glass-reinforced hydroxyapatite plasma-sprayed coatings.

Multilayered coatings composed of mixtures of HA and P2O5-based bioactive glasses are of potential clinical benefit in orthopaedic and dental surgery. Pre-immersion of these materials has been reported to further enhance their efficacy in vivo, although the precise biological effects of this treatment are not yet known. In this study we have therefore prepared double-layer plasma-sprayed coatings and evaluated the effects of pre-immersion on the growth and function of human osteosarcoma cells in vitro, using the MTT assay and flow cytometry analysis, respectively. The results showed that the increase in numbers of viable cells was the same or elevated following incubation on the pre-immersed HA and glass-reinforced HA coatings compared with the non-immersed materials. In addition, the expression of bone sialoprotein and fibronectin, two key connective tissue antigens, was up-regulated in cultures grown on the pre-immersed surfaces compared with the non-treated materials. Moreover, cell numbers and antigen expression both improved as the proportion of glass increased, particularly in the pre-immersed samples. Our findings thus suggest that the immersion treatment of these materials appeared to improve the response of these bone-like cells.