Core-shell Fe@Fex_xOy_y nanoring system: A versatile platform for biomedical applications

Iron oxide (maghemite and magnetite) nanoparticles are the most commonly used magnetic materials in nanomedicine because of their high biocompatibility. However, their low saturation magnetization (60–90 emu/g) limits their applicability. Here, we report a new core–shell (Fe@Fe$_x$O$_y$) nanoring system, which combines the high magnetic saturation of a metallic iron core (220 emu/g) and the biocompatibility of an iron oxide shell. To produce these nanostructures, hematite (α-Fe$_2$O$_3$) nanorings were annealed in a H$_2$ gas atmosphere for different periods to optimize the amount of metallic iron percentage (δ) in the system. Thus, nanostructures with different magnetic saturation (97 to 178 emu/g) could be obtained; based on their metallic iron content, these particles are labeled as Vortex Iron oxide Particle δ (VIPδ). Micromagnetic simulations confirmed that the VIPδ nanorings exhibit a vortex configuration, guaranteeing low remanence and coercitivity. Moreover, the system shows good biocompatibility in various assays as determined through cell viability measurements performed using two different human cell lines, which were exposed to VIP78% for 24 h. Therefore, VIPδ nanorings combine a magnetic vortex state and biocompatibility with their high magnetic saturation and can thus serve as a platform that can be tuned during the synthesis based on desired biomedical application

Trojan-like internalization of anatase titanium dioxide nanoparticles by human osteoblast cells

Dentistry and orthopedics are undergoing a revolution in order to provide more reliable, comfortable and long-lasting implants to patients. Titanium (Ti) and titanium alloys have been used in dental implants and total hip arthroplasty due to their excellent biocompatibility. However, Ti-based implants in human body suffer surface degradation (corrosion and wear) resulting in the release of metallic ions and solid wear debris (mainly titanium dioxide) leading to peri-implant inflammatory reactions. Unfortunately, our current understanding of the biological interactions with titanium dioxide nanoparticles is still very limited. Taking this into consideration, this study focuses on the internalization of titanium dioxide nanoparticles on primary bone cells, exploring the events occurring at the nano-bio interface. For the first time, we report the selective binding of calcium (Ca), phosphorous (P) and proteins from cell culture medium to anatase nanoparticles that are extremely important for nanoparticle internalization and bone cells survival. In the intricate biological environment, anatase nanoparticles form bio-complexes (mixture of proteins and ions) which act as a kind of ‘Trojan-horse’ internalization by cells. Furthermore, anatase nanoparticles-induced modifications on cell behavior (viability and internalization) could be understand in detail. The results presented in this report can inspire new strategies for the use of titanium dioxide nanoparticles in several regeneration therapies

TiO2 bioactive implant surfaces doped with specific amount of Sr modulate mineralization

One of the main problems that remain in the implant industry is poor osseointegration due to bioinertness of implants. In order to promote bioactivity, calcium (Ca), phosphorus (P) and strontium (Sr) were incorporated into a TiO2 porous layer produced by micro-arc oxidation. Ca and P as bioactive elements are already well reported in the literature, however, the knowledge of the effect of Sr is still limited. In the present work, the effect of various amounts of Sr was evaluated and the morphology, chemical composition and crystal structure of the oxide layer were investigated. Furthermore, in vitro studies were carried out using human osteoblast-like cells. The oxide layer formed showed a triplex structure, where higher incorporation of Sr increased Ca/P ratio, amount of rutile and promoted the formation of SrTiO3 compound. Biological tests revealed that lower concentrations of Sr did not compromise initial cell adhesion neither viability and interestingly improved mineralization. However, higher concentration of Sr (and consequent higher amount of rutile) showed to induce collagen secretion but with compromised mineralization, possibly due to a delayed mineralization process or induced precipitation of deficient hydroxyapatite. Ca-P-TiO2 porous layer with less concentration of Sr seems to be an ideal candidate for bone implants.This work was supported by FCT with the reference projects UID/EEA/04436/2019, M-ERA-NET/0001/2015 and FCT/CAPES Procs.. 4.4.1.00. Also the support of FAPESP is acknowledged (Proc. 2017/24300-4). A.I. Costa is very grateful for the PhD grant through NORTE-08-5369-FSE-000051 project. The authors would like to thank the LABNANO/CBPF and Centro Nacional de Biologia Estrutural e Bioimagem (Cenabio) in Rio de Janeiro for technical support during electron microscopy work

Kinome Profiling Of Osteoblasts On Hydroxyapatite Opens New Avenues On Biomaterial Cell Signaling

In degenerative diseases or lesions, bone tissue replacement and regeneration are important clinical goals. The most used bone substitutes today are hydroxyapatite (HA) scaffolds. These scaffolds, developed over the last few decades, present high porosity and good osteointegration, but haven't completely solved issues related to bone defects. Moreover, the exact intracellular mechanisms involved in the response to HA have yet to be addressed. This prompted us to investigate the protein networks responsible for signal transduction during early osteoblast adhesion on synthetic HA scaffolds. By performing a global kinase activity assay, we showed that there is a specific molecular machinery responding to HA contact, immediately triggering pathways leading to cytoskeleton rearrangement due to activation of Adducin 1 (ADD1), protein kinase A (PKA), protein kinase C (PKC), and vascular endothelial growth factor (VEGF). Moreover, we found a significantly increased phosphorylation of the activating site Ser-421 in histone deacetylase 1 (HDAC1), a substrate of Cyclin-Dependent Kinase 5 (CDK5). These phosphorylation events are hallmarks of osteoblast differentiation, pointing to HA surfaces ability to promote differentiation. We also found that AKT was kept active, suggesting the maintenance of survival pathways. Interestingly, though, the substrate sequence of CDK5 also presented higher phosphorylation levels when compared to control conditions. To our knowledge, this kinase has never before been related to osteoblast biology, opening a new avenue of investigation for novel pathways involved in this matter. These results suggest that HA triggers a specific intracellular signal transduction cascade during early osteoblast adhesion, activating proteins involved with cytoskeleton rearrangement, and induction of osteoblast differentiation. Biotechnol. Bioeng. 2014;111: 1900-1905. © 2014 Wiley Periodicals, Inc.111919001905Alves, N.M., Pashkuleva, I., Reis, R.L., Mano, J.F., Controlling cell behavior through the design of polymer surfaces (2010) Small, 6, pp. 2208-2220Anselme, K., Osteoblast adhesion on biomaterials (2000) Biomaterials, 21, pp. 667-681Barkalow, K.L., Italiano Jr., J.E., Chou, D.E., Matsuoka, Y., Bennett, V., Hartwig, J.H., Alpha-adducin dissociates from F-actin and spectrin during platelet activation (2003) J Cell Biol, 161, pp. 557-570Bertazzo, S., Bertran, C.A., Effect of hydrazine deproteination on bone mineral phase: A critical view (2008) J Inorg Biochem, 102, pp. 137-145Bertazzo, S., Zambuzzi, W.F., Campos, D.D., Ferreira, C.V., Bertran, C.A., A simple method for enhancing cell adhesion to hydroxyapatite surface (2010) Clin Oral Implants Res, 21, pp. 1411-1413Bertazzo, S., Zambuzzi, W.F., Campos, D.D., Ogeda, T.L., Ferreira, C.V., Bertran, C.A., Hydroxyapatite surface solubility and effect on cell adhesion (2010) Colloids Surf B Biointerfaces, 78, pp. 177-184Bertazzo, S., Zambuzzi, W.F., da Silva, H.A., Ferreira, C.V., Bertran, C.A., Bioactivation of alumina by surface modification: A possibility for improving the applicability of alumina in bone and oral repair (2009) Clin Oral Implants Res, 20, pp. 288-293Bose, S., Tarafder, S., Calcium phosphate ceramic systems in growth factor and drug delivery for bone tissue engineering: A review (2012) Acta Biomater, 8, pp. 1401-1421Caplan, M.R., Shah, M.M., Translating biomaterial properties to intracellular signaling (2009) Cell Biochem Biophys, 54, pp. 1-10Coelho, P.G., Granjeiro, J.M., Romanos, G.E., Suzuki, M., Silva, N.R., Cardaropoli, G., Thompson, V.P., Lemons, J.E., Basic research methods and current trends of dental implant surfaces (2009) J Biomed Mater Res B Appl Biomater, 88, pp. 579-596Cooper, C.D., Lampe, P.D., Casein kinase 1 regulates connexin-43 gap junction assembly (2002) J Biol Chem, 277, pp. 44962-44968Donahue, H.J., Li, Z., Zhou, Z., Yellowley, C.E., Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication (2000) Am J Physiol Cell Physiol, 278, pp. C315-C322Dorozhkin, S., Calcium orthophosphates and human beings: A historical perspective from the 1770s until 1940 (2012) Biomaterials, 2, pp. 53-70Kokubo, T., Bioactive glass ceramics: Properties and applications (1991) Biomaterials, 12, pp. 155-163Krüger, M., Kratchmarova, I., Blagoev, B., Tseng, Y.H., Kahn, C.R., Mann, M., Dissection of the insulin signaling pathway via quantitative phosphoproteomics (2008) Proc Natl Acad Sci USA, 105, pp. 2451-2456Kuhlman, P.A., Hughes, C.A., Bennett, V., Fowler, V.M., A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments (1996) J Biol Chem, 27, pp. 7986-7991Lee, H.W., Suh, J.H., Kim, A.Y., Lee, Y.S., Park, S.Y., Kim, J.B., Histone deacetylase 1-mediated histone modification regulates osteoblast differentiation (2006) Mol Endocrinol, 20, pp. 2432-2443Liu, Y., Berendsen, A.D., Jia, S., Lotinun, S., Baron, R., Ferrara, N., Olsen, B.R., Intracellular VEGF regulates the balance between osteoblast and adipocyte differentiation (2012) J Clin Invest, 122, pp. 3101-3136Lopez-Macipe, A., Rodrigues-Clemente, R., Hidalgo-López, A., Arita, I., García-Gaduño, M.V., Rivera, E., Castaño, V.M., Wet chemical synthesis of hydroxyapatite particles from nonstoichiometric solutions (1998) J Mater Synth Process, 6, pp. 21-26Matassi, F., Nistri, L., Chicon Paez, D., Innocenti, M., New biomaterials for bone regeneration (2011) Clin Cases Miner Metab, 8 (1), pp. 21-24Matsuoka, Y., Li, X., Bennett, V., Adducin: Structure, function and regulation (2000) Cell Mol Life Sci, 57, pp. 884-895Milani, R., Ferreira, C.V., Granjeiro, J.M., Paredes-Gamero, E.J., Silva, R.A., Justo, G.Z., Nader, H.B., Zambuzzi, W.F., Phosphoproteome reveals an atlas of protein signaling networks during osteoblast adhesion (2010) J Cell Biochem, 109, pp. 957-966Paglini, G., Cáceres, A., The role of the Cdk5-p35 kinase in neuronal development (2001) Eur J Biochem, 268, pp. 1528-1533Pflum, M.K., Tong, J.K., Lane, W.S., Schreiber, S.L., Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation (2001) J Biol Chem, 276, pp. 47733-47741Rigbolt, K.T., Prokhorova, T.A., Akimov, V., Henningsen, J., Johansen, P.T., Kratchmarova, I., Kassem, M., Blagoev, B., System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation (2011) Sci Signal, 4, pp. rs3Ruggiu, A., Ulivi, V., Sanguineti, F., Cancedda, R., Descalzi, F., The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration (2013) Biomaterials, 34, pp. 9318-9330Sasaki, Y., Cheng, C., Uchida, Y., Nakajima, O., Ohshima, T., Yagi, T., Taniguchi, M., Goshima, Y., Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex (2002) Neuron, 35, pp. 907-920Wang, Y., Wan, C., Deng, L., Liu, X., Cao, X., Gilbert, S.R., Bouxsein, M.L., Clemens, T.L., The hypoxia-inducible factor alpha pathway couples angiogenesis to osteogenesis during skeletal development (2007) J Clin Invest, 117, pp. 1616-1626Yang, W., Hong, Y.H., Shen, X.Q., Frankowski, C., Camp, H.S., Leff, T., Regulation of transcription by AMP-activated protein kinase: Phosphorylation of p300 blocks its interaction with nuclear receptors (2001) J Biol Chem, 276, pp. 38341-38344Zambuzzi, W.F., Bruni-Cardoso, A., Granjeiro, J.M., Peppelenbosch, M.P., de Carvalho, H.F., Aoyama, H., Ferreira, C.V., On the road to understanding of the osteoblast adhesion: Cytoskeleton organization is rearranged by distinct signaling pathways (2009) J Cell Biochem, 108, pp. 134-144Zambuzzi, W.F., Coelho, P.G., Alves, G.G., Granjeiro, J.M., Intracellular signal transduction as a factor in the development of "smart" biomaterials for bone tissue engineering (2011) Biotechnol Bioeng, 108, pp. 1246-1250Zambuzzi, W.F., Fernandes, G.V., Iano, F.G., Fernandes, M.S., Granjeiro, J.M., Oliveira, R.C., Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: A study in rat critical-size calvarial defects (2012) Braz Dent J, 23, pp. 315-321Zambuzzi, W.F., Milani, R., Teti, A., Expanding the role of Src and protein-tyrosine phosphatases balance in modulating osteoblast metabolism: Lessons from mice (2010) Biochimie, 92, pp. 327-33

Cellular behavior as a dynamic field for exploring bone bioengineering: A closer look at cell-biomaterial interface

Bone is a highly dynamic and specialized tissue, capable of regenerating itself spontaneously when afflicted by minor injuries. Nevertheless, when major lesions occur, it becomes necessary to use biomaterials, which are not only able to endure the cellular proliferation and migration, but also to substitute the original tissue or integrate itself to it. With the life expectancy growth, regenerative medicine has been gaining constant attention in the reconstructive field of dentistry and orthopedy. Focusing on broadening the therapeutic possibilities for the regeneration of injured organs, the development of biomaterials allied with the applicability of gene therapy and bone bioengineering has been receiving vast attention over the recent years. The progress of cellular and molecular biology techniques gave way to new-guided therapy possibilities. Supported by multidisciplinary activities, tissue engineering combines the interaction of physicists, chemists, biologists, engineers, biotechnologist, dentists and physicians with common goals: the search for materials that could promote and lead cell activity. A well-oriented combining of scaffolds, promoting factors, cells, together with gene therapy advances may open new avenues to bone healing in the near future. In this review, our target was to write a report bringing overall concepts on tissue bioengineering, with a special attention to decisive biological parameters for the development of biomaterials, as well as to discuss known intracellular signal transduction as a new manner to be explored within this field, aiming to predict in vitro the quality of the host cell/material and thus contributing with the development of regenerative medicine. (C) 2014 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Biocompatible Au@Carbynoid/Pluronic-F127 nanocomposites synthesized by pulsed laser ablation assisted CO2 recycling

Ligand-free carbynoid-encapsulated gold nanocomposites (Au@Carbynoid NCs) with blue-shifted localized surface plasmon resonance (LSPR) have been synthesized by CO2 recycling induced by pulsed laser ablation (PLA) of a solid gold target in aqueous solution with NaOH at pH 7.0. High Resolution Transmission Electron Microscopy (HRTEM) images at not destructive acceleration voltage of 80 kV revealed carbynoid nanocrystals around the gold core, associated to the intense bond length alternation (BLA) Raman mode of the carbon atomic wires (CAWs), centered at 2124 cm 1, observed in the Surface Enhanced Raman Scattering (SERS) spectra. It was verified that interlinking process with sp to sp(2) conversion of the CAWs is induced both by high acceleration voltage in HRTEM and high irradiance of the excitation beam used in SERS measurements. Post synthesis mixing of Pluronic-F127 copolymer with pre-synthesized Au@Carbynoid NCs allows the formation of a fully biocompatible colloidal solution of Au@Carbynoid/Copolymer NCs. SERS investigation highlights that the Raman band of the BLA mode can be used as efficient Raman tag to monitor the functionalization of the NCs with the copolymer. The biocompatibility of the NCs was demonstrated performing a study of cytotoxicity using human skin fibroblasts. As proof of principle, it was demonstrated that the photodynamic activity of the bifunctional Au@Carbynoid/PF127 NCs in the presence of chlorin e6 (Ce6) drug can be enhanced inducing the aggregation state of the colloidal suspension. The stability of the colloidal dispersions of Au@Carbynoid NCs functionalized with Pluronic-F127 is verified after centrifugation in PBS (0.15 mol L-1 NaCl) solutions, confirming the possibility to use the green carbynoid based NCs as drug-carrier in biological applications. (C) 2018 Elsevier B.V. All rights reserved

Biocompatible Au@Carbynoid/Pluronic-F127 nanocomposites synthesized by pulsed laser ablation assisted CO2 recycling

Ligand-free carbynoid-encapsulated gold nanocomposites (Au@Carbynoid NCs) with blue-shifted localized surface plasmon resonance (LSPR) have been synthesized by CO2 recycling induced by pulsed laser ablation (PLA) of a solid gold target in aqueous solution with NaOH at pH 7.0. High Resolution Transmission Electron Microscopy (HRTEM) images at not destructive acceleration voltage of 80 kV revealed carbynoid nanocrystals around the gold core, associated to the intense bond length alternation (BLA) Raman mode of the carbon atomic wires (CAWs), centered at 2124 cm 1, observed in the Surface Enhanced Raman Scattering (SERS) spectra. It was verified that interlinking process with sp to sp(2) conversion of the CAWs is induced both by high acceleration voltage in HRTEM and high irradiance of the excitation beam used in SERS measurements. Post synthesis mixing of Pluronic-F127 copolymer with pre-synthesized Au@Carbynoid NCs allows the formation of a fully biocompatible colloidal solution of Au@Carbynoid/Copolymer NCs. SERS investigation highlights that the Raman band of the BLA mode can be used as efficient Raman tag to monitor the functionalization of the NCs with the copolymer. The biocompatibility of the NCs was demonstrated performing a study of cytotoxicity using human skin fibroblasts. As proof of principle, it was demonstrated that the photodynamic activity of the bifunctional Au@Carbynoid/PF127 NCs in the presence of chlorin e6 (Ce6) drug can be enhanced inducing the aggregation state of the colloidal suspension. The stability of the colloidal dispersions of Au@Carbynoid NCs functionalized with Pluronic-F127 is verified after centrifugation in PBS (0.15 mol L-1 NaCl) solutions, confirming the possibility to use the green carbynoid based NCs as drug-carrier in biological applications