Photobiomodulation of Na,K-ATPase in native membrane fraction and reconstituted in DPPC:DPPE-liposome.

Studies focusing on how photobiomodulation (PBM) can affect the structure and function of proteins are scarce in the literature. Few previous studies have shown that the enzymatic activity of Na,K-ATPAse (NKA) can be photo-modulated. However, the variability of sample preparation and light irradiation wavelengths have not allowed for an unequivocal conclusion about the PBM of NKA. Here, we investigate minimal membrane models containing NKA, namely, native membrane fraction and DPPC:DPPE proteoliposome upon laser irradiation at wavelengths 532, 650, and 780 nm. Interestingly, we show that the PBM on the NKA enzymatic activity has a bell-shaped profile with a stimulation peak (~15% increase) at around 20 J.cm-2 and 6 J.cm-2 for the membrane-bound and the proteoliposome samples, respectively, and are practically wavelength independent. Further, by normalizing the enzymatic activity by the NKA enzyme concentration, we show that the PBM response is related to the protein amount with small influence due to protein's environment. The stimulation decays over time reaching the basal level around 6 h after the irradiation for the three lasers and both NKA samples. Our results demonstrate the potential of using low-level laser therapy to modulate NKA activity, which may have therapeutic implications and benefits.

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.

Differential effects of the lipidic and ionic microenvironment on NPP1's phosphohydrolase and phosphodiesterase activities.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PPi) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation. Atomic force microscopy (AFM) revealed that DPPC-liposomes had spherical and smooth surface. The presence of SM and Chol elicited rough and smooth surface, respectively. NPP1 insertion produced protrusions in all the liposome surface. Maximum phosphodiesterase activity emerged at 0.082 M ionic strength, whereas maximum phosphomonohydrolase activity arose at low ionic strength. Phosphoserine-Calcium Phosphate Complex (PS-CPLX) and amorphous calcium-phosphate (ACP) induced mineral propagation in DPPC- and DPPC:SM-liposomes and in DPPC:Chol-liposomes, respectively. Mineral characterization revealed the presence of bands assigned to HAp in the mineral propagated by NPP1 harbored in DPPC-liposomes without nucleators or in DPPC:Chol-liposomes with ACP nucleators. These data show that studying how the ionic and lipidic environment affects NPP1 properties is important, especially for HAp obtained under controlled conditions in vitro.

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.

Strontium-driven physiological to pathological transition of bone-like architecture: A dose-dependent investigation.

Whilst strontium (Sr2+) is widely investigated for treating osteoporosis, it is also related to mineralization disorders such as rickets and osteomalacia. In order to clarify the physiological and pathological effects of Sr2+ on bone biomineralization , we performed a dose-dependent investigation in bone components using a 3D scaffold that displays the hallmark features of bone tissue in terms of composition (osteoblast, collagen, carbonated apatite) and architecture (mineralized collagen fibrils hierarchically assembled into a twisted plywood geometry). As the level of Sr2+ is increased from physiological-like to excess, both the mineral and the collagen fibrils assembly are destabilized, leading to a drop in the Young modulus, with strong implications on pre-osteoblastic cell proliferation. Furthermore, the microstructural and mechanical changes reported here correlate with that observed in bone-weakening disorders induced by Sr2+ accumulation, which may clarify the paradoxical effects of Sr2+ in bone mineralization. More generally, our results provide physicochemical insights into the possible effects of inorganic ions on the assembly of bone extracellular matrix and may contribute to the design of safer therapies for treating osteoporosis. STATEMENT OF SIGNIFICANCE: Physiological-like (10% Sr2+) and excess accumulation-like (50% Sr2+) doses of Sr2+ are investigated in 3D biomimetic assemblies possessing the high degree of organization found in the extracellular of bone. Above the physiological dose, the organic and inorganic components of the bone-like scaffold are destabilized, resulting in impaired cellular activity, which correlates with bone-weakening disorders induced by Sr2+.

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.

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.

Do Media Extracellular Vesicles and Extracellular Vesicles Bound to the Extracellular Matrix Represent Distinct Types of Vesicles?

Mineralization-competent cells, including hypertrophic chondrocytes, mature osteoblasts, and osteogenic-differentiated smooth muscle cells secrete media extracellular vesicles (media vesicles) and extracellular vesicles bound to the extracellular matrix (matrix vesicles). Media vesicles are purified directly from the extracellular medium. On the other hand, matrix vesicles are purified after discarding the extracellular medium and subjecting the cells embedded in the extracellular matrix or bone or cartilage tissues to an enzymatic treatment. Several pieces of experimental evidence indicated that matrix vesicles and media vesicles isolated from the same types of mineralizing cells have distinct lipid and protein composition as well as functions. These findings support the view that matrix vesicles and media vesicles released by mineralizing cells have different functions in mineralized tissues due to their location, which is anchored to the extracellular matrix versus free-floating.

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.

Strontium Carbonate and Strontium-Substituted Calcium Carbonate Nanoparticles Form Protective Deposits on Dentin Surface and Enhance Human Dental Pulp Stem Cells Mineralization.

Strontium acetate is applied for dental hypersensitivity treatment; however, the use of strontium carbonates for this purpose has not been described. The use of Sr-carbonate nanoparticles takes advantage of both the benefits of strontium on dentin mineralization and the abrasive properties of carbonates. Here in, we aimed to synthesize strontium carbonate and strontium-substituted calcium carbonate nanoparticles and test them as potential compounds in active dentifrices for treating dental hypersensitivity. For this, SrCO3, Sr0.5Ca0.5CO3, and CaCO3 nanoparticles were precipitated using Na2CO3, SrCl2, and/or CaCl2 as precursors. Their morphology and crystallinity were evaluated by electron microscopy (SEM) and X-ray diffraction, respectively. The nanoparticles were added to a poly (vinyl alcohol) gel and used to brush dentin surfaces isolated from human third molars. Dentin chemical composition before and after brushing was investigated by infrared spectroscopy (FTIR) and X-ray dispersive energy spectroscopy. Dentin tubule morphology, obliteration, and resistance of the coatings to acid attack were investigated by SEM and EDS. The cytotoxicity and ability of the particles to trigger the mineralization of hDPSCs in vitro were studied. Dentin brushed with the nanoparticles was coated by a mineral layer that was also able to penetrate the tubules, while CaCO3 remained as individual particles on the surface. FTIR bands related to carbonate groups were intensified after brushing with either SrCO3 or Sr0.5Ca0.5CO3. The shift of the phosphate-related FTIR band to a lower wavenumber indicated that strontium replaced calcium on the dentin structure after treatment. The coating promoted by SrCO3 or Sr0.5Ca0.5CO3 resisted the acid attack, while calcium and phosphorus were removed from the top of the dentin surface. The nanoparticles were not toxic to hDPSCs and elicited mineralization of the cells, as revealed by increased mineral nodule formation and enhanced expression of COL1, ALP, and RUNX2. Adding Sr0.5Ca0.5CO3 as an active ingredient in dentifrices formulations may be commercially advantageous since this compound combines the well-known abrasive properties of calcium carbonate with the mineralization ability of strontium, while the final cost remains between the cost of CaCO3 and SrCO3. The novel Sr0.5Ca0.5CO3 nanoparticles might emerge as an alternative for the treatment of dental hypersensitivity.

The Effect of Octenidine on Proliferation, Migration, and Osteogenic Differentiation of Human Dental Pulp and Apical Papilla Stem Cells.

INTRODUCTION: The research for alternative irrigating solutions is ongoing, since no "ideal" solution has yet been found. Octenidine dihydrochloride (OCT) has been indicated as an endodontic irrigant because it has adequate antimicrobial and biological properties. The present study aimed to assess the effects of OCT on proliferation, migration, and induction of the osteogenic phenotype of stem cells from human dental pulp and apical papilla. METHODS: Cells were collected from human third molars and exposed to different doses of OCT, chlorhexidine (CHX), sodium hypochlorite (NaOCl), and ethylenediaminetetraacetic acid (EDTA) to determine cell viability by alamarBlue assay; proliferation by bromodeoxyuridine incorporation; migration by the Transwell assay; alkaline phosphatase activity by thymolphthalein release; and production of mineralized nodules by alizarin red staining. The results were analyzed by 1- or 2-way analysis of variance and Tukey (α = .05). RESULTS: CHX promoted lower cell viability, followed by OCT, NaOCl, and EDTA, especially at intermediate doses (P < .05). Cells exposed to CHX had less proliferation than the other groups (P < .05). The Transwell assay revealed no differences among OCT, EDTA, and culture medium (control group) (P > .05). OCT and EDTA induced greater migration than CHX and NaOCl (P < .05). OCT and EDTA induced higher alkaline phosphatase activity than NaOCl and CHX (P < .05). No difference was detected among the groups using alizarin red staining (P > .05). CONCLUSIONS: OCT induced high migration, proliferation, and alkaline phosphatase activity of stem cells from human dental pulp and apical papilla, which could be advantageous for regenerative endodontic procedures.

Trypanosoma cruzi nitroreductase: Structural features and interaction with biological membranes.

Due to its severe burden and geographic distribution, Chagas disease (CD) has a significant social and economic impact on low-income countries. Benznidazole and nifurtimox are currently the only drugs available for CD. These are prodrugs activated by reducing the nitro group, a reaction catalyzed by nitroreductase type I enzyme from Trypanosoma cruzi (TcNTR), with no homolog in the human host. The three-dimensional structure of TcNTR, and the molecular and chemical bases of the selective activation of nitro drugs, are still unknown. To understand the role of TcNTR in the basic parasite biology, investigate its potential as a drug target, and contribute to the fight against neglected tropical diseases, a combined approach using multiple biophysical and biochemical methods together with in silico studies was employed in the characterization of TcNTR. For the first time, the interaction of TcNTR with membranes was demonstrated, with a preference for those containing cardiolipin, a unique dimeric phospholipid that exists almost exclusively in the inner mitochondrial membrane in eukaryotic cells. Prediction of TcNTR's 3D structure suggests that a 23-residue long insertion (199 to 222), absent in the homologous bacterial protein and identified as conserved in protozoan sequences, mediates enzyme specificity, and is involved in protein-membrane interaction.

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.

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.

Ultrasensitive Diamond Microelectrode Application in the Detection of Ca2+ Transport by AnnexinA5-Containing Nanostructured Liposomes.

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca2+ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca2+ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.

Curcumin-loaded carrageenan nanoparticles: Fabrication, characterization, and assessment of the effects on osteoblasts mineralization.

The use of Curcumin (CR) as a bioactive molecule to prevent and treat inflammation- related diseases is widespread. However, the high hydrophobicity hinders the in vivo bioavailability of CR, reducing its therapeutic index. In the present study, we described the use of nanoparticles (NPs) made of kappa-carrageenan (κ-Carr), a sulphated polysaccharide, as cost-effective, biodegradable and biocompatible CR carriers. CR-loaded κ-Carr nanoparticles (CR@Carr NPs) were prepared by mixing a κ-Carr aqueous solution with a CR ethanolic solution. The final suspension was centrifuged and re-suspended in phosphate buffer solution. The NPs' size was tuned by changing the concentration of the polysaccharide. CR@CarrNPs displayed high CR incorporation efficiency (~80 wt%) and a double-exponential curve of CR release at physiological conditions (37 °C and pH 7.4) with a cumulative drug release of 32 wt% after 24 h for the smaller NP. Our results also showed that CR@CarrNPs were not cytotoxic to osteoblasts at concentrations up to 1 µM. Confocal microscopy images revealed the internalization of CR by the cells guided by the NPs. Cells treated with CR@CarrNPs exhibited higher activity of alkaline phosphatase and higher expression of the main osteogenic genes (Sp7, Col1 and Runx2), and mineralized the extracellular matrix in a higher extent compared to the cells cultivated in absence of the NPs. We posited that these effects were related to the NP-driven internalization of CR by osteoblasts. Our study sheds light on the possible use of CR@CarrNPs as efficient and safe therapeutic tools for the treatment of bone-related diseases.

Fabrication and characterization of a bioactive polymethylmethacrylate-based porous cement loaded with strontium/calcium apatite nanoparticles.

Polymethylmethacrylate (PMMA)-based cements are used for bone reparation due to their biocompatibility, suitable mechanical properties, and mouldability. However, these materials suffer from high exothermic polymerization and poor bioactivity, which can cause the formation of fibrous tissue around the implant and aseptic loosening. Herein, we tackled these problems by adding Sr2+ -substituted hydroxyapatite nanoparticles (NPs) and a porogenic compound to the formulations, thus creating a microenvironment suitable for the proliferation of osteoblasts. The NPs resembled the structure of the bone's apatite and enabled the controlled release of Sr2+ . Trends in the X-ray patterns and infrared spectra confirmed that Sr2+ replaced Ca2+ in the whole composition range of the NPs. The inclusion of an effervescent additive reduced the polymerization temperature and lead to the formation of highly porous cement exhibiting mechanical properties comparable to the trabecular bone. The formation of an opened and interconnected matrix allowed osteoblasts to penetrate the cement structure. Most importantly, the gas formation confined the NPs at the surface of the pores, guaranteeing the controlled delivery of Sr2+ within a concentration sufficient to maintain osteoblast viability. Additionally, the cement was able to form apatite when immersed into simulated body fluids, further increasing its bioactivity. Therefore, we offer a formulation of PMMA cement with improved in vitro performance supported by enhanced bioactivity, increased osteoblast viability and deposition of mineralized matrix assigned to the loading with Sr2+ -substituted hydroxyapatite NPs and the creation of an interconnected porous structure. Altogether, our results hold promise for enhanced bone reparation guided by PMMA cements.

Three-dimensional cell-laden collagen scaffolds: From biochemistry to bone bioengineering.

The bones can be viewed as both an organ and a material. As an organ, the bones give structure to the body, facilitate skeletal movement, and provide protection to internal organs. As a material, the bones consist of a hybrid organic/inorganic three-dimensional (3D) matrix, composed mainly of collagen, noncollagenous proteins, and a calcium phosphate mineral phase, which is formed and regulated by the orchestrated action of a complex array of cells including chondrocytes, osteoblasts, osteocytes, and osteoclasts. The interactions between cells, proteins, and minerals are essential for the bone functions under physiological loading conditions, trauma, and fractures. The organization of the bone's organic and inorganic phases stands out for its mechanical and biological properties and has inspired materials research. The objective of this review is to fill the gaps between the physical and biological characteristics that must be achieved to fabricate scaffolds for bone tissue engineering with enhanced performance. We describe the organization of bone tissue highlighting the characteristics that have inspired the development of 3D cell-laden collagenous scaffolds aimed at replicating the mechanical and biological properties of bone after implantation. The role of noncollagenous macromolecules in the organization of the collagenous matrix and mineralization ability of entrapped cells has also been reviewed. Understanding the modulation of cell activity by the extracellular matrix will ultimately help to improve the biological performance of 3D cell-laden collagenous scaffolds used for bone regeneration and repair as well as for in vitro studies aimed at unravelling physiological and pathological processes occurring in the bone.

The functional role of soluble proteins acquired by extracellular vesicles.

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanosized particles released by all cell types during physiological as well as pathophysiological processes to carry out diverse biological functions, including acting as sources of cellular dumping, signalosomes and mineralisation nanoreactors. The ability of EVs to perform specific biological functions is due to their biochemical machinery. Among the components of the EVs' biochemical machinery, surface proteins are of critical functional significance as they mediate the interactions of EVs with components of the extracellular milieu, the extracellular matrix and neighbouring cells. Surface proteins are thought to be native, that is, pre-assembled on the EVs' surface by the parent cells before the vesicles are released. However, numerous pieces of evidence have suggested that soluble proteins are acquired by the EVs' surface from the extracellular milieu and further modulate the biological functions of EVs during innate and adaptive immune responses, autoimmune disorders, complement activation, coagulation, viral infection and biomineralisation. Herein, we will describe the methods currently used to identify the EVs' surface proteins and discuss recent knowledge on the functional relevance of the soluble proteins acquired by EVs.

Fluorescence evidence of annexin A6 translocation across membrane in model matrix vesicles during apatite formation.

Matrix vesicles (MVs) are 100-300 nm spherical structures released by mineralization competent cells to initiate formation of apatite, the mineral component in bones. Among proteins present in MVs, annexin A6 (AnxA6) is thought to be ubiquitously distributed in the MVs' lumen, on the surface of the internal and external leaflets of the membrane and also inserted in the lipid bilayer. To determine the molecular mechanism(s) that lead to the different locations of AnxA6, we hypothesized the occurrence of a pH drop during the mineralization. Such a change would induce the AnxA6 protonation, which in turn, and because of its isoelectric point of 5.41, would change the protein hydrophobicity facilitating its insertion into the MVs' bilayer. The various distributions of AnxA6 are likely to disturb membrane phospholipid organization. To examine this possibility, we used fluorescein as pH reporter, and established that pH decreased inside MVs during apatite formation. Then, 4-(14-phenyldibenzo[a,c]phenazin-9(14H)-yl)-phenol, a vibration-induced emission fluorescent probe, was used as a reporter of changes in membrane organization occurring with the varying mode of AnxA6 binding. Proteoliposomes containing AnxA6 and 1,2-Dimyristoyl-sn-glycero-3phosphocholine (DMPC) or 1,2-Dimyristoyl-sn-glycero-3phosphocholine: 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DMPC:DPPS 9:1), to mimic the external and internal MV membrane leaflet, respectively, served as biomimetic models to investigate the nature of AnxA6 binding. Addition of Anx6 to DMPC at pH 7.4 and 5.4, or DMPC:DPPS (9:1) at pH 7.4 induced a decrease in membrane fluidity, consistent with AnxA6 interactions with the bilayer surface. In contrast, AnxA6 addition to DMPC:DPPS (9:1) at pH 5.4 increased the fluidity of the membrane. This latest result was interpreted as reflecting the insertion of AnxA6 into the bilayer. Taken together, these findings point to a possible mechanism of AnxA6 translocation in MVs from the surface of the internal leaflet into the phospholipid bilayer stimulated upon acidification of the MVs' lumen during formation of apatite.