Characterization of the in Vitro Osteogenic Response to Submicron TiO2 Particles of Varying Structure and Crystallinity.

Titanium oxide (TiO2) nano-/microparticles have been widely used in orthopedic and dental sciences because of their excellent mechanical properties, chemical stability, and ability to promote the osseointegration of implants. However, how the structure and crystallinity of TiO2 particles may affect their osteogenic activity remains elusive. Herein, we evaluated the osteogenic response to submicron amorphous, anatase, and rutile TiO2 particles with controlled size and morphology. First, the ability of TiO2 particles to precipitate apatite was assessed in an acellular medium by using a simulated body fluid (SBF). Three days after the addition to SBF, anatase and rutile TiO2 particles induced the precipitation of aggregates of nanoparticles with a platelike morphology, typical for biomimetic apatite. Conversely, amorphous TiO2 particles induced the precipitation of particles with poor Ca/P atomic ratio only after 14 days of exposure to SBF. Next, the osteogenic response to TiO2 particles was assessed in vitro by incubating MC3T3-E1 preosteoblasts with the particles. The viability and mineralization efficiency of osteoblastic cells were maintained in the presence of all the tested TiO2 particles despite the differences in the induction of apatite precipitation in SBF by TiO2 particles with different structures. Analysis of the particles' surface charge and of the proteins adsorbed onto the particles from the culture media suggested that all the tested TiO2 particles acquired a similar biological identity in the culture media. We posited that this phenomenon attenuated potential differences in osteoblast response to amorphous, anatase, and rutile particles. Our study provides an important insight into the complex relationship between the physicochemical properties and function of TiO2 particles and sheds light on their safe use in medicine.

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.

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.

Pendant-drop method coupled to ultraviolet-visible spectroscopy: A useful tool to investigate interfacial phenomena.

UV-vis spectroscopy is a powerful tool to investigate surface phenomena. Surface tension measurements coupled to spectroscopic techniques can help to elucidate how the interface organization influences the electronic properties of molecules. However, appreciable sample volumes are usually necessary to achieve strong signals during conduction of experiments. This study reports on the simultaneous acquisition of surface tension data and UV-vis spectra by axisymmetric drop shape analysis (ADSA) coupled to diffuse reflectance (DRUV) spectrophotometry using a pendant microliter-drop that requires small sample volumes and low analyte concentrations. Three example systems gave evidence of the applicability of this technique: (a) disaggregation of an organic dye driven by surfactant as a function of the surface tension and alterations in the UV-vis spectra, (b) activity of a glycosylphosphatidylinositol anchored enzyme estimated from formation of a colored product, and (c) interaction between this enzyme and biomimetic membrane systems consisting of dipalmitoylphosphaditylcholine and cholestenone. Apart from using smaller sample volume, this coupled technique allowed to investigate interfacial organization in the light of electronic spectra obtained in loco within a shorter acquisition time. This procedure provided precise interfacial information about static and dynamic systems. This has been the first study describing the kinetic activity of an enzyme in the presence of phospholipid monolayers through simultaneous determination of the surface tension and UV-vis spectra.

Proteoliposomes with the ability to transport Ca(2+) into the vesicles and hydrolyze phosphosubstrates on their surface.

We describe the production of stable DPPC and DPPC:DPPS-proteoliposomes harboring annexin V (AnxA5) and tissue-nonspecific alkaline phosphatase (TNAP) and their use to investigate whether the presence of AnxA5 impacts the kinetic parameters for hydrolysis of TNAP substrates at physiological pH. The best catalytic efficiency was achieved in DPPS 10%-proteoliposomes (molar ratio), conditions that also increased the specificity of TNAP hydrolysis of PPi. Melting behavior of liposomes and proteoliposomes was analyzed via differential scanning calorimetry. The presence of 10% DPPS in DPPC-liposomes causes a broadening of the transition peaks, with AnxA5 and TNAP promoting a decrease in ΔH values. AnxA5 was able to mediate Ca(2+)-influx into the DPPC and DPPC:DPPS 10%-vesicles at physiological Ca(2+) concentrations (∼2 mM). This process was not affected by the presence of TNAP in the proteoliposomes. However, AnxA5 significantly affects the hydrolysis of TNAP substrates. Studies with GUVs confirmed the functional reconstitution of AnxA5 in the mimetic systems. These proteoliposomes are useful as mimetics of mineralizing cell-derived matrix vesicles, known to be responsible for the initiation of endochondral ossification, as they successfully transport Ca(2+) and possess the ability to hydrolyze phosphosubstrates in the lipid-water interface.

Effects of pH on the production of phosphate and pyrophosphate by matrix vesicles' biomimetics.

During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (PPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP, and PPi was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph containing 1 mM ATP as substrate and amorphous calcium phosphate or calcium-phosphate-phosphatidylserine complexes as nucleators. Propagation of mineralization was significantly more efficient at pH 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP, and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.

Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics.

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5'-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5'-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5'-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP(i) were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP(i) by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase.

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture.

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.