In Vitro Studies on 3D-Printed PLA/HA/GNP Structures for Bone Tissue Regeneration.

The successful regeneration of large-size bone defects remains one of the most critical challenges faced in orthopaedics. Recently, 3D printing technology has been widely used to fabricate reliable, reproducible and economically affordable scaffolds with specifically designed shapes and porosity, capable of providing sufficient biomimetic cues for a desired cellular behaviour. Natural or synthetic polymers reinforced with active bioceramics and/or graphene derivatives have demonstrated adequate mechanical properties and a proper cellular response, attracting the attention of researchers in the bone regeneration field. In the present work, 3D-printed graphene nanoplatelet (GNP)-reinforced polylactic acid (PLA)/hydroxyapatite (HA) composite scaffolds were fabricated using the fused deposition modelling (FDM) technique. The in vitro response of the MC3T3-E1 pre-osteoblasts and RAW 264.7 macrophages revealed that these newly designed scaffolds exhibited various survival rates and a sustained proliferation. Moreover, as expected, the addition of HA into the PLA matrix contributed to mimicking a bone extracellular matrix, leading to positive effects on the pre-osteoblast osteogenic differentiation. In addition, a limited inflammatory response was also observed. Overall, the results suggest the great potential of the newly developed 3D-printed composite materials as suitable candidates for bone tissue engineering applications.

In Vitro Evaluation of Ag- and Sr-Doped Hydroxyapatite Coatings for Medical Applications.

Osseointegration plays the most important role in the success of an implant. One of the applications of hydroxyapatite (HAp) is as a coating for metallic implants due to its bioactive nature, which improves osteoconduction. The purpose of this research was to assess the in vitro behavior of HAp undoped and doped with Ag and/or Sr obtained by galvanostatic pulsed electrochemical deposition. The coatings were investigated in terms of chemical bonds, contact angle and surface free energy, electrochemical behavior, in vitro biomineralization in acellular media (SBF and PBS), and biocompatibility with preosteoblasts cells (MC3T3-E1 cell line). The obtained results highlighted the beneficial impact of Ag and/or Sr on the HAp. The FTIR spectra confirmed the presence of hydroxyapatite within all coatings, while in terms of wettability, the contact angle and surface free energy investigations showed that all surfaces were hydrophilic. The in vitro behavior of MC3T3-E1 indicated that the presence of Sr in the HAp coatings as a unique doping agent or in combination with Ag elicited improved cytocompatibility in terms of cell proliferation and osteogenic differentiation. Therefore, the composite HAp-based coatings showed promising potential for bone regeneration applications.

Influence of Ceramic Particles Size and Ratio on Surface-Volume Features of the Naturally Derived HA-Reinforced Filaments for Biomedical Applications.

The intersection of the bone tissue reconstruction and additive manufacturing fields promoted the advancement to a prerequisite and new feedstock resource for high-performance bone-like-scaffolds manufacturing. In this paper, the proposed strategy was directed toward the use of bovine-bone-derived hydroxyapatite (HA) for surface properties enhancement and mechanical features reinforcement of the poly(lactic acid) matrix for composite filaments extrusion. The involvement of completely naturally derived materials in the technological process was based on factors such as sustainability, low cost, and a facile and green synthesis route. After the HA isolation and extraction from bovine bones by thermal processing, milling, and sorting, two dependent parameters­the HA particles size (<40 µm, <100 µm, and >125 µm) and ratio (0−50% with increments of 10%)­were simultaneously modulated for the first time during the incorporation into the polymeric matrix. The resulting melt mixtures were divided for cast pellets and extruded filaments development. Based on the obtained samples, the study was further designed to examine several key features by complementary surface−volume characterization techniques. Hence, the scanning electron microscopy and micro-CT results for all specimens revealed a uniform and homogenous dispersion of HA particles and an adequate adhesion at the ceramic/polymer interface, without outline pores, sustained by the shape and surface features of the synthesized ceramic particles. Moreover, an enhanced wettability (contact angle in the ~70−21° range) and gradual mechanical takeover were indicated once the HA ratio increased, independent of the particles size, which confirmed the benefits and feasibility of evenly blending the natural ceramic/polymeric components. The results correlation led to the selection of optimal technological parameters for the synthesis of adequate composite filaments destined for future additive manufacturing and biomedical applications.

TiO2 Nanotubes Functionalized with Icariin for an Attenuated In Vitro Immune Response and Improved In Vivo Osseointegration.

Due to their superior mechanical and chemical properties, titanium (Ti) and its alloys have been widely used as orthopedic implantable devices. However, their bioinertness represents a limitation, which can be overcome by employing various surface modifications, such as TiO2 nanotube (TNT) fabrication via electrochemical anodization. Anodic TNTs present tunable dimensions and unique structures, turning them into feasible drug delivery platforms. In the present work, TNTs were loaded with icariin (Ica) through an adhesive intermediate layer of polydopamine (DP), and their in vitro and in vivo biological performance was evaluated. The successful fabrication of the modified surfaces was verified by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and contact angle measurements (CA), while the in vitro release of Ica was evaluated via UV-VIS spectrophotometry. In terms of in vitro behaviour, comparative studies on RAW 264.7 macrophages demonstrated that the TNT substrates, especially TNT-DP-Ica, elicited a lower inflammatory response compared to the Ti support. Moreover, the in vivo implantation studies evinced generation of a reduced fibrotic capsule around this implant and increased thickness of the newly formed bone tissue at 1 month and 3 months post-implantation, respectively. Overall, our results indicate that the controlled release of Ica from TNT surfaces could result in an improved osseointegration process.

Macrophage-like Cells Are Responsive to Titania Nanotube Intertube Spacing-An In Vitro Study.

With the introduction of a new interdisciplinary field, osteoimmunology, today, it is well acknowledged that biomaterial-induced inflammation is modulated by immune cells, primarily macrophages, and can be controlled by nanotopographical cues. Recent studies have investigated the effect of surface properties in modulating the immune reaction, and literature data indicate that various surface cues can dictate both the immune response and bone tissue repair. In this context, the purpose of the present study was to investigate the effects of titanium dioxide nanotube (TNT) interspacing on the response of the macrophage-like cell line RAW 264.7. The cells were maintained in contact with the surfaces of flat titanium (Ti) and anodic TNTs with an intertube spacing of 20 nm (TNT20) and 80 nm (TNT80), under standard or pro-inflammatory conditions. The results revealed that nanotube interspacing can influence macrophage response in terms of cell survival and proliferation, cellular morphology and polarization, cytokine/chemokine expression, and foreign body reaction. While the nanostructured topography did not tune the macrophages' differentiation into osteoclasts, this behavior was significantly reduced as compared to flat Ti surface. Overall, this study provides a new insight into how nanotubes' morphological features, particularly intertube spacing, could affect macrophage behavior.

Bioactive Ibuprofen-Loaded PLGA Coatings for Multifunctional Surface Modification of Medical Devices.

To modulate the biofunctionality of implantable medical devices commonly used in clinical practice, their surface modification with bioactive polymeric coatings is an attractive and successful emerging strategy. Biodegradable coatings based on poly(lactic acid-co-glycolic acid), PLGA, represent versatile and safe candidates for surface modification of implantable biomaterials and devices, providing additional tunable ability for topical delivery of desired therapeutic agents. In the present study, Ibuprofen-loaded PLGA coatings (PLGA/IBUP) were obtained by using the dip-coating and drop-casting combined protocol. The composite materials demonstrated long-term drug release under biologically simulated dynamic conditions. Reversible swelling phenomena of polymeric coatings occurred in the first two weeks of testing, accompanied by the gradual matrix degradation and slow release of the therapeutic agent. Irreversible degradation of PLGA coatings occurred after one month, due to copolymer's hydrolysis (evidenced by chemical and structural modifications). After 30 days of dynamic testing, the cumulative release of IBUP was ~250 µg/mL. Excellent cytocompatibility was revealed on human-derived macrophages, fibroblasts and keratinocytes. The results herein evidence the promising potential of PLGA/IBUP coatings to be used for surface modification of medical devices, such as metallic implants and wound dressings.

Evaluation of cytotoxic activity and anticancer potential of indigenous Rosemary (Rosmarinus officinalis L.) and Oregano (Origanum vulgare L.) dry extracts on MG-63 bone osteosarcoma human cell line.

We aimed to investigate the cytotoxic activity of indigenous Rosemary and Oregano freeze-dried extracts upon MG-63 osteosarcoma human cell line. We have determined the influence of analyzed dry extracts on cell morphology, cell survival and cell proliferation. The evaluation of dry extracts effect upon cell proliferation and viability was assessed by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) method. For cytotoxicity evaluation, Live & Dead and lactate dehydrogenase assays have been used. To further investigate the potential anticancer effect, we have studied the influence of dry extracts upon cells, by means of caspase-3∕7 assay and proliferation cell nuclear antigen (PCNA) expression. Cells were incubated with extracts in the following concentration range (100-700 µg∕mL) for 24 hours. According to our results, both dry extracts have shown cytotoxic effects by means of all used methods. Bone osteosarcoma cells viability significantly decreased with increasing concentration of analyzed extracts (beyond 300 µg∕mL for Rosemary dry extract and only at 700 µg∕mL for Oregano dry extract). According to our results, apoptosis is one of the main mechanisms involved in the cytotoxic properties of analyzed extracts. Moreover, Rosemary extract has also shown decreased expression of PCNA, when compared to control (untreated cells). Both extracts were standardized in phenolic compounds (being a rich source of flavones and phenolcarboxylic acids), so we assume that these are the main constituents involved in the cytotoxic effect. Still, further preclinical studies are needed to confirm the antitumor properties and to go deeply in the molecular mechanisms involved.

The Influence of Severe Plastic Deformation on Microstructure and In Vitro Biocompatibility of the New Ti-Nb-Zr-Ta-Fe-O Alloy Composition.

In this work, severe plastic deformation (SPD) of the newly designed Ti-Nb-Zr-Ta-Fe-O GUM metal was successfully conducted at room temperature using high speed high pressure torsion (HSHPT) followed by cold rolling (CR) to exploit the suitability of the processed alloy for bone staples. The Ti-31.5Nb-3.1Zr-3.1Ta-0.9Fe-0.16O GUM alloy was fabricated in a levitation melting furnace using a cold crucible and argon protective atmosphere. The as-cast specimens were subjected to SPD, specifically HSHPT, and then processed by the CR method to take the advantages of both grain refinement and larger dimensions. This approach creates the opportunity to obtain temporary orthopedic implants nanostructured by SPD. The changes induced by HSHPT technology from the coarse dendrite directly into the ultrafine grained structure were examined by optical microscopy, scanning electron microscopy and X-ray diffraction. The structural investigations showed that by increasing the deformation, a high density of grain boundaries is accumulated, leading gradually to fine grain size. In addition, the in vitro biocompatibility studies were conducted in parallel on the GUM alloy specimens in the as-cast state, and after HSHPT- and HSHPT+CR- processing. For comparative purposes, in vitro behavior of the bone-derived MC3T3-E1 cells on the commercially pure titanium has also been investigated regarding the viability and proliferation, morphology and osteogenic differentiation. The results obtained support the appropriateness of the HSHPT technology for developing compression staples able to ensure a better fixation of bone fragments.

Drug Delivery Systems Based on Titania Nanotubes and Active Agents for Enhanced Osseointegration of Bone Implants.

TiO2 nanotubes (TNTs) are attractive nanostructures for localized drug delivery. Owing to their excellent biocompatibility and physicochemical properties, numerous functionalizations of TNTs have been attempted for their use as therapeutic agent delivery platforms. In this review, we discuss the current advances in the applications of TNT-based delivery systems with an emphasis on the various functionalizations of TNTs for enhancing osteogenesis at the bone-implant interface and for preventing implant-related infection. Innovation of therapies for enhancing osteogenesis still represents a critical challenge in regeneration of bone defects. The overall concept focuses on the use of osteoconductive materials in combination with the use of osteoinductive or osteopromotive factors. In this context, we highlight the strategies for improving the functionality of TNTs, using five classes of bioactive agents: growth factors (GFs), statins, plant derived molecules, inorganic therapeutic ions/nanoparticles (NPs) and antimicrobial compounds.

Nitrodopamine vs dopamine as an intermediate layer for bone regeneration applications.

The aim of this paper was to present a parallel investigation of the poly(dopamine) (DP) and nitrodopamine (NDP) structures deposited on titanium surface (Ti) and titanium oxide nanotubes (NT-TiO2/Ti) and to highlight their advantages and drawbacks to serve as an intermediary layer for bone regeneration applications. This study outlines some hypotheses regarding the manner in which these compounds are able to form a stable film that could serve as bioadhesive. The paper is also a study of structuring and evolution of film architecture for two coatings, polydopamine and nitrodopamine in terms of surface structure, stability, wettability, morphology, adhesion and ability to protect the titanium surface. All investigations are based on the data provided by surface characterization techniques: SEM, RAMAN, XRD, XPS, wettability and flexural strength. The impact of polydopamine and nitrodopamine coatings on the biocompatibility of titanium nanotubes was investigated in vitro. Cell morphology, viability, proliferation and pre-osteoblast differentiation were examined in detail. It was highlighted that both DP and NDP functionalized TiO2 nanotubes display good cell response in terms of cell spreading, formation of focal adhesions, cell viability and proliferation, suggesting their suitability for applications in bone regeneration field. However, NDP coated TiO2 nanotubes demonstrated an enhanced osteogenic potential compared to DP coated substrates.

Naturally-Derived Biphasic Calcium Phosphates through Increased Phosphorus-Based Reagent Amounts for Biomedical Applications.

Calcium carbonate from marble and seashells is an eco-friendly, sustainable, and largely available bioresource for producing natural bone-like calcium phosphates (CaPs). Based on three main objectives, this research targeted the: (i) adaptation of an indirect synthesis route by modulating the amount of phosphorus used in the chemical reaction, (ii) comprehensive structural, morphological, and surface characterization, and (iii) biocompatibility assessment of the synthesized powdered samples. The morphological characterization was performed on digitally processed scanning electron microscopy (SEM) images. The complementary 3D image augmentation of SEM results also allowed the quantification of roughness parameters. The results revealed that both morphology and roughness were modulated through the induced variation of the synthesis parameters. Structural investigation of the samples was performed by Fourier transform infrared spectroscopy and X-ray diffraction. Depending on the phosphorus amount from the chemical reaction, the structural studies revealed the formation of biphasic CaPs based on hydroxyapatite/brushite or brushite/monetite. The in vitro assessment of the powdered samples demonstrated their capacity to support MC3T3-E1 pre-osteoblast viability and proliferation at comparable levels to the negative cytotoxicity control and the reference material (commercial hydroxyapatite). Therefore, these samples hold great promise for biomedical applications.

Osteoblast Cell Response to Naturally Derived Calcium Phosphate-Based Materials.

The demand of calcium phosphate bioceramics for biomedical applications is constantly increasing. Efficient and cost-effective production can be achieved using naturally derived materials. In this work, calcium phosphate powders, obtained from dolomitic marble and Mytilus galloprovincialis seashells by a previously reported and improved Rathje method were used to fabricate microporous pellets through cold isostatic pressing followed by sintering at 1200 °C. The interaction of the developed materials with MC3T3-E1 pre-osteoblasts was explored in terms of cell adhesion, morphology, viability, proliferation, and differentiation to evaluate their potential for bone regeneration. Results showed appropriate cell adhesion and high viability without distinguishable differences in the morphological features. Likewise, the pre-osteoblast proliferation overtime on both naturally derived calcium phosphate materials showed a statistically significant increase comparable to that of commercial hydroxyapatite, used as reference material. Furthermore, evaluation of the intracellular alkaline phosphatase activity and collagen synthesis and deposition, used as markers of the osteogenic ability of these bioceramics, revealed that all samples promoted pre-osteoblast differentiation. However, a seashell-derived ceramic demonstrated a higher efficacy in inducing cell differentiation, almost equivalent to that of the commercial hydroxyapatite. Therefore, data obtained demonstrate that this naturally sourced calcium-phosphate material holds promise for applications in bone tissue regeneration.

Graphene nanoplatelets-sericin surface-modified Gum alloy for improved biological response.

In this study a "Gum Metal" titanium-based alloy, Ti-31.7Nb-6.21Zr-1.4Fe-0.16O, was synthesized by melting and characterized in order to evaluate its potential for biomedical applications. The results showed that the newly developed alloy presents a very high strength, high plasticity and a low Young's modulus relative to titanium alloys currently used in medicine. For further bone implant applications, the newly synthesized alloy was surface modified with graphene nanoplatelets (GNP), sericin (SS) and graphene nanoplatelets/sericine (GNP-SS) composite films via Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique. The characterization of each specimen was monitored by scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (CA) measurements, and Fourier Transform Infrared Spectroscopy (FTIR). The materials' surface analyses suggested the successful coating of GNP, SS and GNP-SS onto the alloy surface. Additionally, the activities of pre-osteoblasts such as cell adhesion, cytoskeleton organization, cell proliferation and differentiation potentials exhibited on these substrates were investigated. Results showed that the GNP-SS-coated substrate significantly enhanced the growth and osteogenic differentiation of MC3T3-E1 cells when compared to bare and GNP-coated alloy. Collectively, the results show that GNP-SS surface-modified Gum alloy can modulate the bioactivity of the pre-osteoblasts holding promise for improved biological response in vivo.

Biological Behaviour and Enhanced Anticorrosive Performance of the Nitrided Superelastic Ti-23Nb-0.7Ta-2Zr-0.5N Alloy.

The influence of gas nitriding surface treatment on the superelastic Ti-23Nb-0.7Ta-2Zr-0.5N alloy was evaluated. A thorough characterization of bare and nitrided Ti-based alloy and pure Ti was performed in terms of surface film composition and morphology, electrochemical behaviour, and short term osteoblast response. XPS analysis showed that the nitriding treatment strongly influenced the composition (nitrides and oxynitrides) and surface properties both of the substrate and of the bulk alloy. SEM images revealed that the nitrided surface appears as a similar dotted pattern caused by the formation of N-rich domains coexisting with less nitrided domains, while before treatment only topographical features could be observed. All the electrochemical results confirmed the high chemical stability of the nitride and oxynitride coating and the superiority of the applied treatment. The values of the corrosion parameters ascertained the excellent corrosion resistance of the coated alloy in the real functional conditions from the human body. Cell culture experiments with MG63 osteoblasts demonstrated that the studied biomaterials do not elicit any toxic effects and support cell adhesion and enhanced cell proliferation. Altogether, these data indicate that the nitrided Ti-23Nb-0.7Ta-2Zr-0.5N alloy is the most suitable substrate for application in bone implantology.

In vitro performance assessment of new beta Ti-Mo-Nb alloy compositions.

New ß-titanium based alloys with low Young's modulus are currently required for the next generation of metallic implant materials to ensure good mechanical compatibility with bone. Several of these are representatives of the ternary Ti-Mo-Nb system. The aim of this paper is to assess the in vitro biological performance of five new low modulus alloy compositions, namely Ti12Mo, Ti4Mo32Nb, Ti6Mo24Nb, Ti8Mo16Nb and Ti10Mo8Nb. Commercially pure titanium (cpTi) was used as a reference material. Comparative studies of cell activity exhibited by MC3T3-E1 pre-osteoblasts over short- and long-term culture periods demonstrated that these newly-developed metallic substrates exhibited an increased biocompatibility in terms of osteoblast proliferation, collagen production and extracellular matrix mineralization. Furthermore, all analyzed biomaterials elicited an almost identical cell response. Considering that macrophages play a pivotal role in bone remodeling, the behavior of a monocyte-macrophage cell line, RAW 264.7, was also investigated showing a slightly lower inflammatory response to Ti-Mo-Nb biomaterials as compared with cpTi. Thus, the biological performances together with the superior mechanical properties recommend these alloys for bone implant applications.

Enhancement of the electrochemical behaviour and biological performance of Ti-25Ta-5Zr alloy by thermo-mechanical processing.

A new Ti-25Ta-5Zr alloy based only on non-toxic and non-allergic elements was elaborated in as-cast and thermo-mechanical processed, recrystallized states (XRD and SEM) in order to be used as candidate material for implant applications. Its long-term interactions with Ringer-Brown and Ringer solutions of different pH values and its cytocompatibility were determined. The thermo-mechanically processed alloy has nobler electrochemical behaviour than as-cast alloy due to finer microstructure obtained after the applied treatment. Corrosion and ion release rates presented the lowest values for the treated alloy. Nyquist and Bode plots displayed higher impedance values and phase angles for the processed alloy, denoting a more protective passive film. SEM micrographs revealed depositions from solutions that contain calcium, phosphorous and oxygen ions (EDX analysis), namely calcium phosphate. An electric equivalent circuit with two time constants was modelled. Cell culture experiments with MC3T3-E1 pre-osteoblasts demonstrated that thermo-mechanically processed Ti-25Ta-5Zr alloy supports a better cell adhesion and spreading, and enhanced cell proliferation. Altogether, these data indicate that thermo-mechanical treatment endows the alloy with improved anticorrosion and biological performances.

In vitro bio-functional performances of the novel superelastic beta-type Ti-23Nb-0.7Ta-2Zr-0.5N alloy.

The materials used for internal fracture fixations and joint replacements are mainly made of metals which still face problems ranging from higher rigidity than that of natural bone to leaching cytotoxic metallic ions. Beta (ß)-type titanium alloys with low elastic modulus made from non-toxic and non-allergenic elements are desirable to reduce stress shielding effect and enhance bone remodeling. In this work, a new ß-type Ti-23Nb-0.7Ta-2Zr-0.5N alloy with a Young's modulus of approximately 50 GPa was designed and characterized. The behavior of MC3T3-E1 pre-osteoblasts on the new alloy, including adhesion, proliferation and differentiation, was evaluated by examining the cytoskeleton, focal adhesion formation, metabolic activity and extracellular matrix mineralization. Results indicated that the pre-osteoblast cells exhibited a similar degree of attachment and growth on Ti-23Nb-0.7Ta-2Zr-0.5N and Ti-6Al-4V. However, the novel alloy proved to be significantly more efficient in sustaining mineralized matrix deposition upon osteogenic induction of the cells than Ti-6Al-4V control. Further, the analysis of RAW 264.7 macrophages cytokine gene and protein expression indicated no significant inflammatory response. Collectively, these findings suggest that the Ti-23Nb-0.7Ta-2Zr-0.5N alloy, which has an increased mechanical biocompatibility with bone, allows a better osteogenic differentiation of osteoblast precursor cells than Ti-6Al-4V and holds great potential for future clinical prosthetic applications.

Collagen-hydroxyapatite/cisplatin drug delivery systems for locoregional treatment of bone cancer.

In this paper, the synthesis and characterization of novel cisplatin-loaded collagen (COLL)/hydroxyapatite (HA) composite materials are presented. The composite materials were designed to obtain a COLL: HA weight ratio close to the bone composition. The content of embedded cisplatin was chosen to assure a concentration of cisplatin of 6 and 10 µM, respectively, into the culture media used in cell culture experiments. These cisplatin delivery systems were characterized by determining the physico-chemical properties of the composite material, the drug release process as well as their biological activity. Based on the in vitro data that showed the cytotoxic, anti-proliferative and anti-invasive activities of these multifunctional systems on G292 osteosarcoma cells in dependence on the cisplatin concentration released in culture medium, we conclude that the newly developed COLL/HA-cisplatin drug delivery system could be a feasible approach for locoregional chemotherapy of bone cancer.

Osteoblast cell behavior on the new beta-type Ti-25Ta-25Nb alloy.

Among metallic materials used as bone substitutes, ß titanium alloys gain an increasing importance because of their low modulus, high corrosion resistance and good biocompatibility. In this work, an investigation of the in vitro cytocompatibility of a recently new developed ß-type Ti-25Ta-25Nb alloy was carried out by evaluating the behavior of human osteoblasts. The metallic Ti-6Al-4V biomaterial, which is one of representative α+ß type titanium alloys for biomedical applications, and Tissue Culture Polystyrene (TCPS), were also investigated as reference Ti-based material and control substrate, respectively. Both metallic surfaces were analyzed by X-ray diffraction, atomic force microscopy and X-ray photoelectron spectroscopy. The cellular response was quantified by assessments of viability, cell attachment and spreading, cell morphology, production and extracellular organization of fibronectin and cell proliferation. Polished surfaces from both materials having an equiaxed grain microstructure and nanometre scale surface roughness elicited an essentially identical osteoblast response in terms of all analyzed cellular parameters. Thus, on both surfaces the cells displayed high survival rates, good cell adhesion and spreading, a dense and randomly dispersed fibronectin matrix and increasing cell proliferation rates over the incubation time. Furhermore, the enhanced biological performance of Ti-25Ta-25Nb was highly supported by the results obtained in comparison with TCPS. These findings, together with previously shown superelastic behavior, low Young's modulus and high corrosion resistance, recommend Ti-25Ta-25Nb as good candidate for applications in bone implantology.

In vitro effects of calcium fructoborate upon production of inflammatory mediators by LPS-stimulated RAW 264.7 macrophages.

The present study is supported by our previous findings suggesting that calcium fructoborate (CF) has anti-inflammatory and antioxidant properties. Thus, we investigated the effects of CF on a model for studying inflammatory disorders in vitro represented by lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. This investigation was performed by analyzing the levels of some mediators released during the inflammatory process: cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukins IL-1beta and IL-6 as well as cyclooxygenase-2 (COX-2), the main enzyme responsible for endotoxin/LPS-induced prostaglandin synthesis by macrophages. We also measured production of nitric oxide (NO) that plays an important role in the cytotoxicity activity of macrophages towards microbial pathogens. After CF treatment of LPS-stimulated macrophages we found an up-regulation of TNF-alpha protein level in culture medium, no significant changes in the level of COX-2 protein expression and a decrease in NO production as well as in IL-1beta and IL-6 release. Collectively, this series of experiments indicate that CF affect macrophage production of inflammatory mediators. However, further research is required in order to establish whether CF treatment can be beneficial in suppression of pro-inflammatory cytokine production and against progression of endotoxin-related diseases.