Comparative analysis of the physical, chemical, and microbiological properties of Ti-6Al-4V disks produced by different methods and subjected to surface treatments.

STATEMENT OF PROBLEM: To improve the osseointegration of dental implants and reduce microbiological growth, different micro- and nanoscale surface topographies can be used. PURPOSE: The purpose of this in vitro study was to evaluate the influence of Ti-6Al-4V with 4 surfaces, machined (DU), machined+hydroxyapatite (DUHAp), machined+acid-alkali treatment (DUAA), and additive manufacturing (DMA), on the physical, chemical, and microbiological properties. MATERIAL AND METHODS: The topography of Ti-6Al-4V disks with the 4 surfaces was evaluated by scanning electron microscopy (SEM), the chemical composition by energy dispersive X-ray spectroscopy (EDS), and the crystalline structure by X-ray diffraction (XRD). Physical and chemical properties were analyzed by using wettability and surface free energy, roughness, and microbial adhesion against Staphylococcus aureus by colony forming units (CFU). One-way ANOVA analysis of variance and the Tukey multiple comparisons test were applied to evaluate the data, except CFU, which was submitted to the Kruskal-Wallis nonparametric test (α=.05). RESULTS: DU photomicrographs showed a topography characteristic of a polished machined surface, DUHAp and DUAA exhibited patterns corresponding to the surface modifications performed, and in DMA the presence of partially fused spherical particles was observed. The EDS identified chemical elements inherent in the Ti-6Al-4V, and the DUHAp and DUAA disks also had the ions from the treatments applied. XRD patterns revealed similarities between DU and DMA, as well as characteristic peaks of hydroxyapatite (HA) in the DUHAp disk and the DUAA. Compared with DU and DMA the DUHAp and DUAA groups showed hydrophilic behavior with smaller contact angles and higher surface free energy (P<.05). DMA showed a higher mean value of roughness, different from the others (P<.05), and a higher CFU for S. aureus (P=.006). CONCLUSIONS: DUHAp and DUAA showed similar behaviors regarding wettability, surface free energy, and bacterial adhesion. Among the untreated groups, DMA exhibited higher roughness, bacterial adhesion, and lower wettability and surface free energy.

Collagen/κ-Carrageenan-Based Scaffolds as Biomimetic Constructs for In Vitro Bone Mineralization Studies.

Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular matrix made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macromolecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro, a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate the anatomical and biological properties of native bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated polysaccharide κ-carrageenan was added to the scaffolds to fulfill the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/κ-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibrils with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of κ-carrageenan. The presence of κ-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), and osteopontin (Opn), as well as the ability to mineralize the extracellular matrix after 14 and 21 days of culture. Taken together, the results described in this study shed light on the potential use of collagen/κ-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.

Annexin A5 stabilizes matrix vesicle-biomimetic lipid membranes: unravelling a new role of annexins in calcification.

Matrix vesicles are a special class of extracellular vesicles thought to actively contribute to both physiologic and pathologic mineralization. Proteomic studies have shown that matrix vesicles possess high amounts of annexin A5, suggesting that the protein might have multiple roles at the sites of calcification. Currently, Annexin A5 is thought to promote the nucleation of apatitic minerals close to the inner leaflet of the matrix vesicles' membrane enriched in phosphatidylserine and Ca2+. Herein, we aimed at unravelling a possible additional role of annexin A5 by investigating the ability of annexin A5 to adsorb on matrix-vesicle biomimetic liposomes and Langmuir monolayers made of dipalmitoylphosphatidylserine (DPPS) and dipalmitoylphosphatidylcholine (DPPC) in the absence and in the presence of Ca2+. Differential scanning calorimetry and dynamic light scattering measurements showed that Ca2+ at concentrations in the 0.5-2.0 mM range induced the aggregation of liposomes probably due to the formation of DPPS-enriched domains. However, annexin A5 avoided the aggregation of liposomes at Ca2+ concentrations lower than 1.0 mM. Surface pressure versus surface area isotherms showed that the adsorption of annexin A5 on the monolayers made of a mixture of DPPC and DPPS led to a reduction in the area of excess compared to the theoretical values, which confirmed that the protein favored attractive interactions among the membrane lipids. The stabilization of the lipid membranes by annexin A5 was also validated by recording the changes with time of the surface pressure. Finally, fluorescence microscopy images of lipid monolayers revealed the formation of spherical lipid-condensed domains that became unshaped and larger in the presence of annexin A5. Our data support the model that annexin A5 in matrix vesicles is recruited at the membrane sites enriched in phosphatidylserine and Ca2+ not only to contribute to the intraluminal mineral formation but also to stabilize the vesicles' membrane and prevent its premature rupture.

NPP1 and TNAP hydrolyze ATP synergistically during biomineralization.

Matrix vesicles (MVs) are a special class of extracellular vesicles released by mineralizing cells during bone and tooth mineralization that initiate the precipitation of apatitic minerals by regulating the extracellular ratio between inorganic phosphate (Pi), a calcification promoter, and pyrophosphate (PPi), a calcification inhibitor. The Pi/PPi ratio is thought to be controlled by two ecto-phosphatases present on the outer leaflet of the MVs' membrane: ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) that produces PPi as well as Pi from ATP and tissue-nonspecific alkaline phosphatase (TNAP) that hydrolyzes both ATP and PPi to generate Pi. However, if and how these enzymes act in concert in MVs are still unclear. Herein, we investigated the role of NPP1 and TNAP in ATP hydrolysis during MV-mediated biomineralization using proteoliposomes as a biomimetic model for MVs. Proteoliposomes composed by 1,2-dipalmitoylphosphatidylcholine (DPPC) and harboring NPP1 alone, TNAP alone, or both together at different molar ratios (1:1, 10:1, and 1:10) were fabricated. After 48 h of incubation with ATP, TNAP-containing proteoliposomes consumed more ATP than NPP1-containing vesicles (270 and 210 nmol, respectively). Both types of vesicles comparatively formed ADP (205 and 201 nmol, respectively), while NPP1-containing vesicles hydrolyzed AMP less efficiently than TNAP-containing proteoliposomes (10 and 25 nmol, respectively). In vitro mineralization assays showed that in the presence of ATP, TNAP-harboring proteoliposomes mineralized through a sigmoidal single-step process, while NPP1-harboring vesicles displayed a two-step mineralization process. ATR-FTIR analyses showed that the minerals produced by TNAP-harboring proteoliposomes were structurally more similar to hydroxyapatite than those produced by NPP1-harboring vesicles. Our results with proteoliposomes indicate that the pyrophosphohydrolase function of NPP1 and the phosphohydrolase activity of TNAP act synergistically to produce a Pi/PPi ratio conducive to mineralization and the synergism is maximal when the two enzymes are present at equimolar concentrations. The significance of these findings for hypophosphatasia is discussed.

Influence of Er:YAG laser irradiation on surface properties of Ti-6Al-4V machined and hydroxyapatite coated.

Surface treatment by laser irradiation can change the topography of titanium; however, little is known about the changes it causes when applied to other coatings. This study aimed to evaluate the influence of Er:YAG laser irradiation on the surface properties of titanium-aluminum-vanadium (Ti-6Al-4V) discs. Four Ti-6Al-4V surfaces were evaluated (n = 10): CON-control, machined without surface treatment; LT-machined + laser treatment; HA-hydroxyapatite coating; and LT-HA-hydroxyapatite coating + laser treatment. For the laser treatment, an Er:YAG laser with a wavelength of 2940 nm, a frequency of 10 Hz, and an energy density of 12.8 J/cm2 was used. The morphology of the coating was investigated by scanning electron microscopy and the surface composition by energy-dispersive X-ray spectroscopy. The influence of laser irradiation treatment on roughness and wettability was also evaluated. The Er:YAG laser promoted a significant reduction in the roughness Sa (p < 0.05) and in the contact angle (p = 0.002) of the LT surface compared to the CON surface. On the LT-HA surface, a significant decrease in roughness was observed only for the Rz parameter (p = 0.015) and an increase in the contact angle (p < 0.001) compared to the HA surface. The use of the Er:YAG laser with the evaluated parameters decreased the surface roughness and improved the wetting capacity of machined without surface treatment. In the group with hydroxyapatite coating, the laser influenced the surface roughness only for the parameter Rz and reduced their wetting capacity.

Synthesis of Antibacterial Hybrid Hydroxyapatite/Collagen/Polysaccharide Bioactive Membranes and Their Effect on Osteoblast Culture.

Inspired by the composition and confined environment provided by collagen fibrils during bone formation, this study aimed to compare two different strategies to synthesize bioactive hybrid membranes and to assess the role the organic matrix plays as physical confinement during mineral phase deposition. The hybrid membranes were prepared by (1) incorporating calcium phosphate in a biopolymeric membrane for in situ hydroxyapatite (HAp) precipitation in the interstices of the biopolymeric membrane as a confined environment (Methodology 1) or (2) adding synthetic HAp nanoparticles (SHAp) to the freshly prepared biopolymeric membrane (Methodology 2). The biopolymeric membranes were based on hydrolyzed collagen (HC) and chitosan (Cht) or κ-carrageenan (κ-carr). The hybrid membranes presented homogeneous and continuous dispersion of the mineral particles embedded in the biopolymeric membrane interstices and enhanced mechanical properties. The importance of the confined spaces in biomineralization was confirmed by controlled biomimetic HAp precipitation via Methodology 1. HAp precipitation after immersion in simulated body fluid attested that the hybrid membranes were bioactive. Hybrid membranes containing Cht were not toxic to the osteoblasts. Hybrid membranes added with silver nanoparticles (AgNPs) displayed antibacterial action against different clinically important pathogenic microorganisms. Overall, these results open simple and promising pathways to develop a new generation of bioactive hybrid membranes with controllable degradation rates and antimicrobial properties.

Shedding Light on the Role of Na,K-ATPase as a Phosphatase during Matrix-Vesicle-Mediated Mineralization.

Matrix vesicles (MVs) contain the whole machinery necessary to initiate apatite formation in their lumen. We suspected that, in addition to tissue-nonspecific alkaline phosphatase (TNAP), Na,K,-ATPase (NKA) could be involved in supplying phopshate (Pi) in the early stages of MV-mediated mineralization. MVs were extracted from the growth plate cartilage of chicken embryos. Their average mean diameters were determined by Dynamic Light Scattering (DLS) (212 ± 19 nm) and by Atomic Force Microcopy (AFM) (180 ± 85 nm). The MVs had a specific activity for TNAP of 9.2 ± 4.6 U·mg-1 confirming that the MVs were mineralization competent. The ability to hydrolyze ATP was assayed by a colorimetric method and by 31P NMR with and without Levamisole and SBI-425 (two TNAP inhibitors), ouabain (an NKA inhibitor), and ARL-67156 (an NTPDase1, NTPDase3 and Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) competitive inhibitor). The mineralization profile served to monitor the formation of precipitated calcium phosphate complexes, while IR spectroscopy allowed the identification of apatite. Proteoliposomes containing NKA with either dipalmitoylphosphatidylcholine (DPPC) or a mixture of 1:1 of DPPC and dipalmitoylphosphatidylethanolamine (DPPE) served to verify if the proteoliposomes were able to initiate mineral formation. Around 69-72% of the total ATP hydrolysis by MVs was inhibited by 5 mM Levamisole, which indicated that TNAP was the main enzyme hydrolyzing ATP. The addition of 0.1 mM of ARL-67156 inhibited 8-13.7% of the total ATP hydrolysis in MVs, suggesting that NTPDase1, NTPDase3, and/or NPP1 could also participate in ATP hydrolysis. Ouabain (3 mM) inhibited 3-8% of the total ATP hydrolysis by MVs, suggesting that NKA contributed only a small percentage of the total ATP hydrolysis. MVs induced mineralization via ATP hydrolysis that was significantly inhibited by Levamisole and also by cleaving TNAP from MVs, confirming that TNAP is the main enzyme hydrolyzing this substrate, while the addition of either ARL-6715 or ouabain had a lesser effect on mineralization. DPPC:DPPE (1:1)-NKA liposome in the presence of a nucleator (PS-CPLX) was more efficient in mineralizing compared with a DPPC-NKA liposome due to a better orientation of the NKA active site. Both types of proteoliposomes were able to induce apatite formation, as evidenced by the presence of the 1040 cm-1 band. Taken together, the findings indicated that the hydrolysis of ATP was dominated by TNAP and other phosphatases present in MVs, while only 3-8% of the total hydrolysis of ATP could be attributed to NKA. It was hypothesized that the loss of Na/K asymmetry in MVs could be caused by a complete depletion of ATP inside MVs, impairing the maintenance of symmetry by NKA. Our study carried out on NKA-liposomes confirmed that NKA could contribute to mineral formation inside MVs, which might complement the known action of PHOSPHO1 in the MV lumen.

Mineralization Profile of Annexin A6-Harbouring Proteoliposomes: Shedding Light on the Role of Annexin A6 on Matrix Vesicle-Mediated Mineralization.

The biochemical machinery involved in matrix vesicles-mediated bone mineralization involves a specific set of lipids, enzymes, and proteins. Annexins, among their many functions, have been described as responsible for the formation and stabilization of the matrix vesicles' nucleational core. However, the specific role of each member of the annexin family, especially in the presence of type-I collagen, remains to be clarified. To address this issue, in vitro mineralization was carried out using AnxA6 (in solution or associated to the proteoliposomes) in the presence or in the absence of type-I collagen, incubated with either amorphous calcium phosphate (ACP) or a phosphatidylserine-calcium phosphate complex (PS-CPLX) as nucleators. Proteoliposomes were composed of 1,2-dipalmitoylphosphatidylcholine (DPPC), 1,2-dipalmitoylphosphatidylcholine: 1,2-dipalmitoylphosphatidylserine (DPPC:DPPS), and DPPC:Cholesterol:DPPS to mimic the outer and the inner leaflet of the matrix vesicles membrane as well as to investigate the effect of the membrane fluidity. Kinetic parameters of mineralization were calculated from time-dependent turbidity curves of free Annexin A6 (AnxA6) and AnxA6-containing proteoliposomes dispersed in synthetic cartilage lymph. The chemical composition of the minerals formed was investigated by Fourier transform infrared spectroscopy (FTIR). Free AnxA6 and AnxA6-proteoliposomes in the presence of ACP were not able to propagate mineralization; however, poorly crystalline calcium phosphates were formed in the presence of PS-CPLX, supporting the role of annexin-calcium-phosphatidylserine complex in the formation and stabilization of the matrix vesicles' nucleational core. We found that AnxA6 lacks nucleation propagation capacity when incorporated into liposomes in the presence of PS-CPLX and type-I collagen. This suggests that AnxA6 may interact either with phospholipids, forming a nucleational core, or with type-I collagen, albeit less efficiently, to induce the nucleation process.

Surface Wettability of a Natural Rubber Composite under Stretching: A Model to Predict Cell Survival.

We report the stress-strain effect of a stretchable natural rubber (NR)-calcium phosphate composite on the surface wettability (SW) using an innovative approach coupling a uniaxial tensile micromachine, goniometer, and microscope. In situ contact angle measurements in real time were performed during mechanical tension. Our results show that SW is guided by the stress-strain relationship with two different characteristics, depending on the static or dynamic experiments. The results evidenced the limits of the classical theory of wetting. Furthermore, based on the mechanically tunable SW of the system associated with the cytocompatibility of the NR composite, we have modeled such a system for application as a cell support. From the experimental surface energy value, our proposed 3D modeling numerical simulation predicted a window of opportunities for cell-NR survival under mechanical stimuli. The presented data and the thermodynamics-based theoretical approach enable not only accurate correlation of SW with mechanical properties of the NR composite but also provide huge potential for future cell supportability in view of tissue engineering.

Effect of phosphorylated chitosan and carbodiimide biomodification on the chemical composition of eroded dentin.

PURPOSE: To evaluate the chemical composition and morphological properties of eroded dentin after biomodification with phosphorylated chitosan (P-Chi) and carbodiimide (EDC). METHODS: 42 bovine dentin specimens were used; 21 of these specimens were subjected to erosive challenge with 0.3% citric acid (pH = 3.2) for 2 hours. The specimens were randomly divided into six groups according to dentin substrate (sound or eroded) and biomodification [with 2.5% P-Chi, with 0.5 mol/L EDC, or no biomodification (control)]. The specimens were analyzed by Fourier-transform infrared spectroscopy (FTIR, n= 5, in triplicate) and atomic force microscopy (AFM, n= 2) to verify the phosphate, carbonate, and organic matrix absorption peaks and to investigate surface morphology, respectively. The data were analyzed with Origin 6.0. RESULTS: Dentin erosion reduced the intensity of the phosphate (1,100 cm⁻¹) and carbonate (872 cm⁻¹) related bands, which evidenced demineralization. Eroded dentin consisted of a more irregular surface containing slightly more open tubules. Modification with P-Chi removed intertubular dentin, which was compatible with surface demineralization; however, this modification obliterated dentin tubules. EDC did not promote demineralization. Biomodified dentin had a more irregular surface, irrespective of substrate type. CLINICAL SIGNIFICANCE: Eroded dentin demineralization promoted by biomodification with 2.5% phosphorylated chitosan (P-Chi) is a promising indicator for further studies and highlights the dentin intrinsic characteristics. From the point of view of dentin surface chemical analysis, more studies with P-Chi should be conducted to achieve greater interactions with surfaces and to improve the adhesive interface.

Storm-Drain and Manhole Detection Using the RetinaNet Method.

As key-components of the urban-drainage system, storm-drains and manholes are essential to the hydrological modeling of urban basins. Accurately mapping of these objects can help to improve the storm-drain systems for the prevention and mitigation of urban floods. Novel Deep Learning (DL) methods have been proposed to aid the mapping of these urban features. The main aim of this paper is to evaluate the state-of-the-art object detection method RetinaNet to identify storm-drain and manhole in urban areas in street-level RGB images. The experimental assessment was performed using 297 mobile mapping images captured in 2019 in the streets in six regions in Campo Grande city, located in Mato Grosso do Sul state, Brazil. Two configurations of training, validation, and test images were considered. ResNet-50 and ResNet-101 were adopted in the experimental assessment as the two distinct feature extractor networks (i.e., backbones) for the RetinaNet method. The results were compared with the Faster R-CNN method. The results showed a higher detection accuracy when using RetinaNet with ResNet-50. In conclusion, the assessed DL method is adequate to detect storm-drain and manhole from mobile mapping RGB images, outperforming the Faster R-CNN method. The labeled dataset used in this study is available for future research.

Localization of Annexin A6 in Matrix Vesicles During Physiological Mineralization.

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca2+, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-ß-cyclodextrin (MßCD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in πexc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca2+ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca2+ as observed by the larger change in Δt1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.

Lipid microenvironment affects the ability of proteoliposomes harboring TNAP to induce mineralization without nucleators.

Tissue-nonspecific alkaline phosphatase (TNAP), a glycosylphosphatidylinositol-anchored ectoenzyme present on the membrane of matrix vesicles (MVs), hydrolyzes the mineralization inhibitor inorganic pyrophosphate as well as ATP to generate the inorganic phosphate needed for apatite formation. Herein, we used proteoliposomes harboring TNAP as MV biomimetics with or without nucleators of mineral formation (amorphous calcium phosphate and complexes with phosphatidylserine) to assess the role of the MVs' membrane lipid composition on TNAP activity by means of turbidity assay and FTIR analysis. We found that TNAP-proteoliposomes have the ability to induce mineralization even in the absence of mineral nucleators. We also found that the addition of cholesterol or sphingomyelin to TNAP-proteoliposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine reduced the ability of TNAP to induce biomineralization. Our results suggest that the lipid microenvironment is essential for the induction and propagation of minerals mediated by TNAP.

Interaction of Artepillin C with model membranes.

Green propolis, a mixture of beeswax and resinous compounds processed by Apis mellifera, displays several pharmacological properties. Artepillin C, the major compound in green propolis, consists of two prenylated groups bound to a phenyl group. Several studies have focused on the therapeutic effects of Artepillin C, but there is no evidence that it interacts with amphiphilic aggregates to mimic cell membranes. We have experimentally and computationally examined the interaction between Artepillin C and model membranes composed of dimyristoylphosphatidylcholine (DMPC) because phosphatidylcholine (PC) is one of the most abundant phospholipids in eukaryotic cell membranes. PC is located in both outer and inner leaflets and has been used as a simplified membrane model and a non-specific target to study the action of amphiphilic molecules with therapeutic effects. Experimental results indicated that Artepillin C adsorbed onto the DMPC monolayers. Its presence in the lipid suspension pointed to an increased tendency toward unilamellar vesicles and to decreased bilayer thickness. Artepillin C caused point defects in the lipid structure, which eliminated the ripple phase and the pre-transition in thermotropic chain melting. According to molecular dynamics (MD) simulations, (1) Artepillin C aggregated in the aqueous phase before it entered the bilayer; (2) Artepillin C was oriented along the direction normal to the surface; (3) the negatively charged group on Artepillin C was accommodated in the polar region of the membrane; and (4) thinner regions emerged around the Artepillin C molecules. These results help an understanding of the molecular mechanisms underlying the biological action of propolis.

Enhancing and quenching luminescence with gold nanoparticle films: the influence of substrate on the luminescent properties.

Gold nanoparticle (AuNP) films were sputtered over glass and aluminum substrates to enhance optically stimulated luminescence (OSL), a luminescent technique employed for radiation detection, from x-ray irradiated NaCl nanocrystals. The AuNP films deposited over glass led to enhanced-OSL emission, whereas the AuNP films deposited on aluminum substrates quenched the OSL emission. The enhanced-OSL intensity is proportional to the optical density of the film's plasmon resonance band at the stimulation wavelength. For the case of the AuNP/aluminum films, the luminescence quenching diminishes, and OSL intensity partially recovers upon increasing the distance between the AuNPs and the aluminum substrates, and between the luminescent nanocrystals and the AuNP films. These results suggest that plasmonic interactions between the emitter nanocrystals, the localized surface plasmons (LSP) of the AuNPs, and the substrate are responsible for the OSL enhancement and quenching. In this sense, the substrate dictates whether LSP relaxation occurs by radiative or non-radiative transisitions, leading to enhanced or quenched OSL, respectively. Therefore, besides showing that AuNP films can enhance and/or tune the sensitivity of luminescent radiation detectors, and demonstrating OSL as a new technique to investigate mechanisms of plasmon-enhanced luminescence, these results bring insights on how substrates strongly modify the optical properties of AuNP films.

Multi and single walled carbon nanotubes: effects on cell responses and biomineralization of osteoblasts cultures.

The use of carbon nanotubes (CNTs) on the development of biomaterials has been motivated by their excellent mechanical properties that could improve synthetic bone materials. However, the toxicity of CNTs on the tissue/implant interface and their influence on the biomineralization process have some contradictions. We investigated the influence of CNTs on osteoblasts plated on titanium (Ti) discs or plastic surfaces. We evaluated osteoblasts viability, alkaline phosphatase (ALP) activity, and mineralized matrix formation in the different phases of osteoblasts growth in the presence of single-walled CNTs (SWCNTs) and multi-walled CNTs (MWCNTs). An increase in osteoblasts viability was observed at the 21st day for both CNTs on plastic surface, while viability increased for MWCNTs at the 7th and 14th days and at the 7th day for SWCNTs on Ti discs compared to control. ALP activity increased at the 14th and 21st days for MWCNTs on plastic surfaces. For cells incubated with SWCNTs, an increase in ALP activity at the 7th day for plastic surface and at the 14th day for both materials (plastic and Ti) was observed. The mineralized matrix formation increased at the 21st day on plastic surface with SWCNTs, and at the 14th and 21st days for both CNTs on Ti discs. In conclusion, both SWCNTs and MWCNTs are not toxic to osteoblasts at concentrations up to 5 × 10(-5) and 1.3 × 10(-2) mg/mL, respectively, either in Ti discs or plastic surfaces. In the long term, the cells grown in contact with both CNTs and Ti presented better results regarding bone-like nodules formation.

Graphene oxide and titanium: synergistic effects on the biomineralization ability of osteoblast cultures.

Graphene oxide (GO) has attracted remarkable attention in recent years due to properties such as extremely large surface area, biocompatibility, biostability, and easy chemical functionalization. Osteoblasts underlie the deposition of hydroxyapatite crystals in the bone protein matrix during biomineralization; hydroxyapatite deposition involves extracellular matrix vesicles that are rich in alkaline phosphatase (ALP). Here, we have investigated how GO affects osteoblast viability, ALP activity, and mineralized matrix formation in osteoblast cultures in three different phases of cell growth, in the presence and in the absence of titanium (Ti). Scanning electron microscopy (SEM), Raman spectra, and energy dispersive spectroscopy aided GO characterization. The presence of GO increased the viability of osteoblast cells grown on a plastic surface. However, osteoblast viability on Ti discs was lower in the presence than in the absence of GO. ALP activity emerged at 14 days for the cell culture incubated with GO. The total protein concentration also increased at 21 days on both the Ti discs and plastic surface. Osteoblasts grown on Ti discs had increased mineralized matrix formation in the presence of GO as compared to the cells grown in the absence of GO. SEM images of the cell cultures on plastic surfaces in the presence of GO suggested delayed mineralized matrix formation. In conclusion, applications requiring the presence of Ti, such as prostheses and implants, should benefit from the use of GO, which may increase mineralized nodule formation, stimulate biomineralization, and accelerate bone regeneration.

Evaluation of surface roughness, wettability and adhesion of multispecies biofilm on 3D-printed resins for the base and teeth of complete dentures.

OBJECTIVE: This study evaluated the surface roughness, wettability and adhesion of multispecies biofilms (Candida albicans, Staphylococcus aureus and Streptococcus mutans) on 3D-printed resins for complete denture bases and teeth compared to conventional resins (heat-polymerized acrylic resin; artificial pre-fabricated teeth). METHODOLOGY: Circular specimens (n=39; 6.0 mm Ø × 2.0 mm) of each group were subjected to roughness (n=30), wettability (n=30) and biofilm adhesion (n=9) tests. Three roughness measurements were taken by laser confocal microscopy and a mean value was calculated. Wettability was evaluated by the contact angle of sessile drop method, considering the mean of the three evaluations per specimen. In parallel, microorganism adhesion to resin surfaces was evaluated using a multispecies biofilm model. Microbial load was evaluated by determining the number of Colony Forming Units (CFU/mL) and by scanning electron microscopy (SEM). Data were subjected to the Wald test in a generalized linear model with multiple comparisons and Bonferroni adjustment, as well as two-way ANOVA (α=5%). RESULTS: The roughness of the conventional base resin (0.01±0.04) was lower than that of the conventional tooth (0.14±0.04) (p=0.023) and 3D-printed base (0.18±0.08) (p<0.001). For wettability, conventional resin (84.20±5.57) showed a higher contact angle than the 3D-printed resin (60.58±6.18) (p<0.001). Higher microbial loads of S. mutans (p=0.023) and S. aureus (p=0.010) were observed on the surface of the conventional resin (S. mutans: 5.48±1.55; S. aureus: 7.01±0.57) compared to the 3D-printed resin (S. mutans: 4.11±1.96; S. aureus: 6.42±0.78). The adhesion of C. albicans was not affected by surface characteristics. The conventional base resin showed less roughness than the conventional dental resin and the printed base resin. CONCLUSION: The 3D-printed resins for base and tooth showed less hydrophobicity and less adhesion of S. mutans and S. aureus than conventional resins.

A nontoxic strontium nanoparticle that holds the potential to act upon osteocompetent cells: An in vitro and in vivo characterization.

Estrogen deficiency, long-term immobilization, and/or aging are commonly related to bone mass loss, thus increasing the risk of fractures. One option for bone replacement in injuries caused by either traumas or pathologies is the use of orthopedic cement based on polymethylmethacrylate (PMMA). Nevertheless, its reduced bioactivity may induce long-term detachment from the host tissue, resulting in the failure of the implant. In view of this problem, we developed an alternative PMMA-based porous cement (pPMMA) that favors cell invasion and improves osteointegration with better biocompatibility. The cement composition was changed by adding bioactive strontium-nanoparticles that mimic the structure of bone apatite. The nanoparticles were characterized regarding their physical-chemical properties, and their effects on osteoblasts and osteoclast cultures were assessed. Initial in vivo tests were also performed using 16 New Zealand rabbits as animal models, in which the pPMMA-cement containing the strontium nanoparticles were implanted. We showed that the apatite nanoparticles in which 90% of Ca2+ ions were substituted by Sr2+ (NanoSr 90%) upregulated TNAP activity and increased matrix mineralization. Moreover, at the molecular level, NanoSr 90% upregulated the mRNA expression levels of, Sp7, and OCN. Runx2 was increased at both mRNA and protein levels. In parallel, in vivo tests revealed that pPMMA-cement containing NanoSr 90%, upregulated two markers of bone maturation, OCN and BMP2, as well as the formation of apatite minerals after implantation in the femur of rabbits. The overall data support that strontium nanoparticles hold the potential to up-regulate mineralization in osteoblasts when associated with synthetic biomaterials.

Electrostimulable polymeric films with hyaluronic acid and lipid nanoparticles for simultaneous topical delivery of macromolecules and lipophilic drugs.

This study focused on developing electrically stimulable hyaluronic acid (HA) films incorporating lipid nanoparticles (NPs) designed for the topical administration of lipophilic drugs and macromolecules. Based on beeswax and medium-chain triglycerides, NPs were successfully integrated into silk fibroin/chitosan films containing HA (NP-HA films) at a density of approximately 1011 NP/cm2, ensuring a uniform distribution. This integration resulted in a 40% increase in film roughness, a twofold decrease in Young's modulus, and enhanced film flexibility and bioadhesion work. The NP-HA films, featuring Ag/AgCl electrodes, demonstrated the capability to conduct a constant electrical current of 0.2 mA/cm2 without inducing toxicity in keratinocytes and fibroblasts during a 15-min application. Moreover, the NPs facilitated the homogeneous distribution of lipophilic drugs within the film, effectively transporting them to the skin and uniformly distributing them in the stratum corneum upon film administration. The sustained release of HA from the films, following Higuchi kinetics, did not alter the macroscopic characteristics of the film. Although anodic iontophoresis did not noticeably affect the release of HA, it did enhance its penetration into the skin. This enhancement facilitated the permeation of HA with a molecular weight (MW) of up to 2 × 105 through intercellular and transcellular routes. Confocal Raman spectroscopy provided evidence of an approximate 100% increase in the presence of HA with a MW in the range of 1.5-1.8 × 106 in the viable epidermis of human skin after only 15 min of iontophoresis applied to the films. Combining iontophoresis with NP-HA films exhibits substantial potential for noninvasive treatments focused on skin rejuvenation and wound healing.