Anisoara Cimpean

Reduced inflammatory activity of RAW 264.7 macrophages on titania nanotube modified Ti surface.

Macrophages play a pivotal role in the hosts response to biomaterials being considered as an essential cell type during both optimal tissue-implant integration and pathologic process of implant failure. Hence, understanding of their cellular activity on biomaterials is important for improving evaluation and design of biomaterials for biomedical applications. In the present study, we have comparatively investigated the interactions of titania nanotubes (78 nm diameter) and commercial pure Ti with RAW 264.7 macrophages in both standard and pro-inflammatory (stimulation with lipopolysaccharide, LPS) culture conditions. In vitro tests showed that TiO2 nanotubes exhibited significantly decreased inflammatory activity of macrophages with respect to cytokine and chemokine gene expression/protein secretion, induction of foreign body giant cells (FBGCs) and nitric oxide (NO) release thereby mitigating the inflammatory response induced by LPS as compared to flat Ti surface. Therefore, our results suggest a novel role of TiO2 nanotubes in modulating macrophage response in biomaterial-associated bacterial infections. Overall, the current study provides new insight into how TiO2 nanotubes can be involved in macrophage activation and supports the great promise of such surface modifications for biomedical applications.

Comparative Effects of Boric Acid and Calcium Fructoborate on Breast Cancer Cells

Recent studies suggested that boron has a chemo-preventive role in prostate cancer. In the present report, we investigated the effects of calcium fructoborate (CF) and boric acid (BA) on activation of the apoptotic pathway in MDA-MB-231 human breast cancer cells. Exposure to BA and CF inhibited the proliferation of breast cancer cells in a dose-dependent manner. Treatment with CF but not BA resulted in a decrease in p53 and bcl-2 protein levels. Furthermore, after the treatment with CF, augmentation of pro-caspase-3 protein expression, cytosolic cytochrome c level, and caspase-3 activity were observed, indicating apoptotic cell death induction. This was also demonstrated by terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end-labeling assay. In conclusion, our data provide arguments to the fact that both BA and CF inhibited the growth of breast cancer cells, while only CF induced apoptosis. Additional studies will be needed to identify the underlying mechanism responsible for the observed cellular responses to these compounds and to determine if BA and CF may be further evaluated as chemotherapeutic agents for human cancer.

Layer-shaped alginate hydrogels enhance the biological performance of human adipose-derived stem cells

BackgroundThe reconstruction of adipose tissue defects is often challenged by the complications that may occur following plastic and reconstructive surgery, including donor-site morbidity, implant migration and foreign body reaction. To overcome these problems, adipose tissue engineering (ATE) using stem cell-based regeneration strategies has been widely explored in the last years. Mounting evidence has shown that adipose-derived stem cells (ADSCs) represent a promising cell source for ATE. In the context of a small number of reports concerning adipose tissue regeneration using three-dimensional (3-D) systems, the present study was designed to evaluate the biological performance of a novel alginate matrix that incorporates human ADSCs (hADSCs).ResultsCulture-expanded cells isolated from the stromal vascular fraction (SVF), corresponding to the third passage which showed the expression of mesenchymal stem cell (MSC) markers, were used in the 3-D culture systems. The latter represented a calcium alginate hydrogel, obtained by the diffusion of calcium gluconate (CGH matrix), and shaped as discoid-thin layer. For comparative purposes, a similar hADSC-laden alginate hydrogel cross-linked with calcium chloride was considered as reference hydrogel (RH matrix). Both hydrogels showed a porous structure under scanning electron microscopy (SEM) and the hADSCs embedded displayed normal spherical morphologies, some of them showing signs of mitosis. More than 85% of the entrapped cells survived throughout the incubation period of 7 days. The percentage of viable cells was significantly higher within CGH matrix at 2 days post-seeding, and approximately similar within both hydrogels after 7 days of culture. Moreover, both alginate-based hydrogels stimulated cell proliferation. The number of hADSC within hydrogels has increased during the incubation period of 7 days and was higher in the case of CGH matrix. Cells grown under adipogenic conditions for 21 days showed that both analyzed 3-D culture systems support adipogenic differentiation in terms of neutral lipid accumulation and perillipin expression. Furthermore, the cells encapsulated in CGH matrix displayed a more differentiated phenotype.ConclusionsThe results of this study suggest that both CGH and RH matrices successfully support the survival and adipogenesis of hADSC. An enhancement of biological performance was detected in the case of CGH matrix, suggesting its promising application in ATE.

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.

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-α (TNF-α), interleukins IL-1β 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-α 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-1β 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.

Sericin Enhances the Bioperformance of Collagen-Based Matrices Preseeded with Human-Adipose Derived Stem Cells (hADSCs)

Current clinical strategies for adipose tissue engineering (ATE), including autologous fat implants or the use of synthetic surrogates, not only are failing in the long term, but also can’t face the latest requirements regarding the aesthetic restoration of the resulted imperfections. In this context, modern strategies in current ATE applications are based on the implantation of 3D cell-scaffold bioconstructs, designed for prospective achievement of in situ functional de novo tissue. Thus, in this paper, we reported for the first time the evaluation of a spongious 60% collagen and 40% sericin scaffold preseeded with human adipose-derived stem cells (hADSCs) in terms of biocompatibility and adipogenic potential in vitro. We showed that the addition of the sticky protein sericin in the composition of a classical collagen sponge enhanced the adhesion and also the proliferation rate of the seeded cells, thus improving the biocompatibility of the novel scaffold. In addition, sericin stimulated PPARγ2 overexpression, triggering a subsequent upregulated expression profile of FAS, aP2 and perilipin adipogenic markers. These features, together with the already known sericin stimulatory potential on cellular collagen production, promote collagen-sericin biomatrix as a good candidate for soft tissue reconstruction and wound healing applications.

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 (β)-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 β-type Ti-23Nb-0.7Ta-2Zr-0.5N alloy with a Youngs 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.

Design of a nitrogen-implanted titanium-based superelastic alloy with optimized properties for biomedical applications.

In this study, a superelastic Ni-free Ti-based biomedical alloy was treated in surface by the implantation of nitrogen ions for the first time. The N-implanted surface was characterized by X-ray diffraction, X-ray photoelectron spectroscopy, and secondary ion mass spectroscopy, and the superficial mechanical properties were evaluated by nano-indentation and by ball-on-disk tribological tests. To investigate the biocompatibility, the corrosion resistance of the N-implanted Ti alloy was evaluated in simulated body fluids (SBF) complemented by in-vitro cytocompatibility tests on human fetal osteoblasts. After implantation, surface analysis methods revealed the formation of a titanium-based nitride on the substrate surface. Consequently, an increase in superficial hardness and a significant reduction of friction coefficient were observed compared to the non-implanted sample. Also, a better corrosion resistance and a significant decrease in ion release rates have been obtained. Cell culture experiments indicated that the cytocompatibility of the N-implanted Ti alloy was superior to that of the corresponding non-treated sample. Thus, this new functional N-implanted titanium-based superelastic alloy presents the optimized properties that are required for various medical devices: superelasticity, high superficial mechanical properties, high corrosion resistance and excellent cytocompatibility.

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

Among metallic materials used as bone substitutes, β 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 β-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+β 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 Youngs modulus and high corrosion resistance, recommend Ti-25Ta-25Nb as good candidate for applications in bone implantology.

Attenuation of the macrophage inflammatory activity by TiO2 nanotubes via inhibition of MAPK and NF-κB pathways

Biomaterial implantation in a living tissue triggers the activation of macrophages in inflammatory events, promoting the transcription of pro-inflammatory mediator genes. The initiation of macrophage inflammatory processes is mainly regulated by signaling proteins of mitogen-activated protein kinase (MAPK) and by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. We have previously shown that titania nanotubes modified Ti surfaces (Ti/TiO2) mitigate the immune response, compared with flat Ti surfaces; however, little is known regarding the underlying mechanism. Therefore, the aim of this study is to investigate the mechanism(s) by which this nanotopography attenuates the inflammatory activity of macrophages. Thus, we analyzed the effects of TiO2 nanotubes on the activation of MAPK and NF-κB signaling pathways in standard and lipopolysaccharide-evoked conditions. Results showed that the Ti/TiO2 significantly reduce the expression levels of the phosphorylated forms of p38, ERK1/2, c-Jun NH2-terminal kinase (JNK), IKKβ, and IkB-α. Furthermore, a significant reduction in the p65 nuclear accumulation on the nanotubular surface was remarked. Following, by using specific MAPK inhibitors, we observed that lipopolysaccharide-induced production of monocyte chemotactic protein-1 and nitric oxide was significantly inhibited on the Ti/TiO2 surface via p38 and ERK1/2, but not via JNK. However, the selective inhibitor for JNK signaling pathway (SP600125) was effective in reducing tumor necrosis factor alpha release as well as monocyte chemotactic protein-1 and nitric oxide production. Altogether, these data suggest that titania nanotubes can attenuate the macrophage inflammatory response via suppression of MAPK and NF-κB pathways providing a potential mechanism for their anti-inflammatory activity.