An in vivo method for measuring the adsorption of plasma proteins to titanium in humans

A novel method of collecting in vivo plasma proteins of humans from osteotomies prepared during insertion of an oral implant is described. A rod containing a collecting portion with a predetermined surface is introduced into the osteomy, removed, and transferred for enzyme-linked immunosorbent assay analysis. Two experiments were used to examine the feasibility of the method. In the first, titanium (Ti) rods with different roughness were exposed for 10 min to the blood. Blasted and acid-etched surfaces adsorbed four times more and acid-etched surfaces adosorbed two times more plasma proteins as compared to machined surfaces. In the second experiment, blasted and acid-etched rods were wetted for 10 s prior to the insertion. The adsorption for fibronectin, albumin, fibrinogen, and IgG was enhanced significantly compared with nonwetted rods. These results are discussed in the light of previous methods used in studies on adsorption. Thus, use of the collecting instrument enables aspects of human plasma–implant interface to be studied in a more realistic manner.     

Proteome analysis of the plasma protein layer adsorbed to a rough titanium surface

In this study a label-free proteomic approach was used to investigate the composition of the layer of protein adsorbed to rough titanium (Ti) after exposure to human blood plasma. The influence of the protein layer on the surface free energy (SFE) of the Ti was evaluated by contact angle measurements. Ti discs were incubated with blood plasma for 180?min at 37?°C, and the proteins recovered were subjected to liquid chromatography coupled to tandem mass spectrometry analysis. A total of 129 different peptides were identified and assigned to 25 distinct plasma proteins. The most abundant proteins were fibronectin, serum albumin, apolipoprotein A-I, and fibrinogen, comprising 74.54% of the total spectral counts. Moreover, the protein layer increased the SFE of the Ti (p?<?0.05). The layer adsorbed to the rough Ti surface was composed mainly of proteins related to cell adhesion, molecule transportation, and coagulation processes, creating a polar and hydrophilic interface for subsequent interactions with host cells.     

Proteome analysis of rat serum proteins adsorbed onto synthetic octacalcium phosphate crystals

The present study was designed to determine which proteins are selectively adsorbed onto two bone substitute materials, octacalcium phosphate (OCP) and hydroxyapatite (HA) crystals, from rat serum by proteome analysis. Ground crystals of synthetic OCP and commercially available sintered HA, with the same surface area, were incubated in rat serum proteins at 37 °C for 24 h. The proteins from the crystals extracted with guanidine–HCl–EDTA were listed on the basis of the results of liquid chromatography tandem mass spectrometry (LC/MS/MS). A total of 138 proteins were detected from OCP; 103 proteins were detected from HA. Forty-eight proteins were from both crystals. A quantitative analysis of the proteins detected was performed for the extracted two bone formation-related proteins apolipoprotein E (Apo E), a protein known to promote osteoblast differentiation, and complement 3 (C3). HA adsorbed C3 (3.98 ± 0.03 fmol/μg protein) more than OCP (1.81 ± 0.07 fmol/μg protein) did, while OCP adsorbed Apo E (2.42 ± 0.03 fmol/μg protein) more than HA (1.21 ± 0.01 fmol/μg protein) did even after deleting the high-abundance proteins, such as albumin. The results demonstrated that OCP exhibits a similar property but distinct capacity with HA in adsorbing bone formation-related proteins from the serum constituents.     

DNA affects the composition of lipoplex protein corona: a proteomics approach

The distribution of drug delivery systems into the body is affected by plasma proteins adsorbed onto their surface. Furthermore, an exact understanding of the structure and morphology of drug carriers is fundamental to understand their role as gene delivery systems. In this work, the adsorption of human plasma proteins bound to cationic liposomes and to their relative DNA lipoplexes was compared. A shotgun proteomics approach based on HPLC coupled to high resolution MS was used for an efficient identification of proteins adsorbed onto liposome and lipoplex surfaces. The distinct pattern of proteins adsorbed helps to better understand the DNA compaction process. The experimental evidence leads us to hypothesize that polyanionic DNA is associated to the lipoplex surface and can interact with basic plasma proteins. Such a finding is in agreement with recent results showing that lipoplexes are multilamellar DNA/lipid domains partially decorated with DNA at their surface. Proteomics experiments showed that the lipoplex corona is rich of biologically relevant proteins such as fibronectin, histones and complement proteins. Our results provide novel insights to understand how lipoplexes activate the immune system and why they are rapidly cleared from the blood stream. The differences in the protein adsorption data detected in the presented experiments could be the basis for the establishment of a correlation between protein adsorption pattern and in vivo fate of intravenously administered nanoparticles and will require some consideration in the future.     

Proteomic profile of the saliva and plasma protein layer adsorbed on Ti–Zr alloy: the effect of sandblasted and acid-etched surface treatment

Abstract

Titanium–zirconium (Ti–Zr) alloy has been widely used as a biomaterial for implant devices, and it is commonly treated by sandblasting followed by acid etching (SLA) to improve biological responses. Although protein adsorption is the first biological response, the effect of this SLA treatment on the proteomic profile of proteins adsorbed from saliva and blood plasma has not been tested. In this study, the proteomic profile was evaluated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). Streptococcus sanguinis was used to test whether the protein layer affects bacterial adhesion. SLA treatment affected the proteomic profile, showing exclusive proteins adsorbed from saliva (14) and plasma (3). However, both groups exhibited close patterns of intensity for common proteins, molecular functions and biological processes mediated by proteins. Interestingly, Ti–Zr_SLA showed higher bacterial adhesion (～1.9 fold over) for the surface coated with plasma proteins. Therefore, SLA treatment of Ti–Zr alloy changed the proteomic profile, which may affect bacterial adhesion.     

Chemisorption of proteins and their thiol derivatives onto gold surfaces: characterization based on electrochemical nonlinearity

This study was conducted to monitor the electrochemical responses of two proteins (bovine serum albumin (BSA) and gelatin) and their thiol derivatives adsorbed onto gold (Au) electrodes, which were analyzed by a "nonlinear" impedance method. A sinusoidal voltage is applied to a protein-containing aqueous solution and the waveform of the output current is analyzed by fast Fourier transformation (FFT). The intensities of the higher harmonics in the FFT varied with the species of protein and their thiol derivatives, and with time. From the higher harmonics, voltage-dependent capacitance and conductance were quantitatively evaluated to differentiate the state of adsorbed protein. Adsorption and desorption characteristics of BSA and its thiol derivative on the Au surface were continuously measured by a quartz crystal microbalance (QCM) in situ. The microscopic state of thiol-derivatized BSA adsorbed onto the Au surface was imaged by atomic force microscopy (AFM). In general, thiol-derivatized proteins were tightly adsorbed on the Au surface and showed no desorption. The present electrochemical measurements clearly differentiated adsorption characteristics of physically adsorbed (physisorbed) and chemically adsorbed (chemisorbed) proteins on Au surfaces.     

Salivary protein adsorption onto hydroxyapatite and sds‐mediated elution studied by in situ ellipsometry

Whole unstimulated saliva from two donors was investigated both with respect to adsorption characteristics and SDS‐induced elutability. Salivary protein adsorption onto hydroxyapatite (HA) discs was studied by means of in situ ellipsometry in the concentration range 0.1–20% saliva. The adsorbed amounts on HA were found to be similar to those on silica, but the rates of adsorption were lower. Protein adsorption was virtually unaffected by the presence of Na⁺, whereas Ca²⁺ induced nucleation of calcium phosphate at the surface, the deposition rate being influenced by the pellicle age but not by the presence of saliva in bulk solution. The SDS elutability of adsorbed pellicles was determined on HA as well as on silica surfaces. Desorption from both surfaces was found to occur in the same SDS concentration range, although a residual layer was observed on HA. The slight net positive charge and lower charge density of HA as compared to the strongly negatively charged silica, may, at least partly, account for this observation by causing a reduction in the repulsive force between protein‐surfactant complexes and the surface. Inter‐individual differences, observed in the adsorption as well as elution experiments, are thought to relate to the compositional differences observed by SDS‐PAGE.     

Bioactive surfaces via immobilization of self-assembling polymers onto hydrophobic materials.

Conjugation of proteins to copolymers from poly(acrylic acid) grafted onto PEO-PPO-PEO backbone (Pluronic-PAA) following adsorption of the conjugates onto hydrophobic surfaces is reported. Insulin-Pluronic-PAA conjugates show negligible internalization of insulin into human uterine smooth muscle cells as well as enhancement of mitogenic activity. Glucose-induced release of glycated albumin complexed with a Pluronic-PAA-concanavalin conjugate and adsorbed onto polystyrene nanospheres may provide a model for a glucose-responsive protein delivery system or a heterogeneous diagnostic device.     

Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems

A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich “protein corona” adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle–protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography–Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.     

Mechanisms of serum protein binding and cell anchorage to immobilized serotonin and indole analogs

Bovine aortal endothelial cells, bovine smooth muscle cells, chick embryo fibroblasts, and baby hamster kidney cells all attached and grew on immobilized tryptamine or L-tryptophan as successfully as on immobilized serotonin. A detailed investigation employing different serum compositions combined with cell blotting and immunoblotting techniques revealed that adhesion of cells to each of the immobilized indole analogs was mediated by vitronectin and fibronectin. Quantitative analyses revealed major differences in the variety of serum proteins adsorbed to each of the immobilized indole analogs and in particular major differences in the amounts of adsorbed vitronectin. However, similar levels of adsorbed fibronectin and fibronectin fragments were found on each of the immobilized indole analogs. The results indicate that (i) different composites of surface-adsorbed proteins may be directed by chemical differences between the immobilized indole analogs and (ii) mammalian cells may still populate chemically different surfaces with equal success despite differences in the surface profiles of adsorbed serum proteins.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.     

An in vitro study of initial adsorption from human parotid and submandibular/sublingual resting saliva at solid/liquid interfaces

The influence of saliva concentration, saliva total protein content and the wetting characteristics of exposed solids on in vitro film formation was studied by the technique of in situ ellipsometry. The rates and plateau values of adsorption (45 min) at solid/liquid interfaces (hydrophilic silica and hydrophobic methylated silica surfaces) were determinated for human parotid (HPS) and submandibular/sublingual (HSMSLS) resting saliva solutions (0.1 and 1.0%, (v/v), saliva in phosphate buffered saline). Adsorption rates were related to a model assuming mass transport through an unstirred layer adjacent to the surface. The results showed that the adsorption was rapid, concentration dependent and higher on hydrophobic than on hydrophilic surfaces. Analysis of the influence of protein concentration on the adsorbed amounts demonstrated an interaction between protein concentration and the two surfaces for HPS and HSMSLS, respectively. This may indicate differences in binding mode. Inter‐individual differences were found not to be significant at the 1% level of probability. Comparison of the observed adsorption and calculated diffusion rates suggest that on hydrophilic surfaces initial adsorption of proteins diffusing at rates corresponding to those of statherin and aPRPs takes place, whereas on hydrophobic surfaces lower molecular mass compounds appear to be involved.     

Proteomics characterization of protein adsorption onto hemodialysis membranes

Protein-adsorptive properties are a key feature of membranes used for hemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with structural feature of the membrane surfaces. Minidialyzers of identical structural characteristics composed of either cellulose diacetate or ethylenevinyl alcohol materials were employed to develop an ex vivo apparatus to investigate protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by 2-DE coupled to both MALDI-TOF mass spectrometry (MS) mass fingerprinting and fragmentation analysis on a nanoLC-MS/MS hybrid instrument. Membrane surface characterization included evaluation of roughness (atomic force microscopy), elemental chemical composition (X-ray-photoelectron-spectroscopy), and hydrophilicity (pulsed nuclear magnetic resonance). The present study identifies a number of different proteins as common or characteristic of filter material interaction, showing that proteomic techniques are a promising approach for the investigation of proteins surface-adsorbed onto hemodialysis membrane. Proteomic analysis enables the characterization of protein layers of unknown composition.     

Characterization of eukaryotic cell surfaces prior to and after serum protein adsorption by X-ray photoelectron spectroscopy

Elemental surface concentration ratios N/C, O/C, and P/C of fibroblasts, HELA epithelial cells, and smooth muscle cells, prior to and after washing in the absence or presence of serum proteins, were determined by X-ray photoelectron spectroscopy. Cell surfaces appeared to adsorb hardly any serum proteins, and the relatively high P/C, as compared to N/C and O/C, elemental surface concentration ratio indicated that the cell surfaces consisted mainly of the phospholipid bilayer, with little or no proteins present. The lack of adsorption of serum proteins to the cell surfaces seems at odds with the common notion that cells require adhesive proteins in order to adhere and spread. However, the adsorption behavior of cellularly produced proteins may be completely different, particularly since they seem to be able to displace adsorbed serum proteins from biomaterials surfaces. Interestingly, only HELA epithelial cells (a tumor cell line) appeared to adsorb a very small amount of proteins.     

Microcolumn capture and digestion of proteins combined with mass spectrometry for protein identification

A procedure has been developed for protein identification using mass spectrometry (MS) that incorporates sample cleanup, preconcentration, and protein digestion in a single-stage system. The procedure involves the adsorption of a protein, or protein mixture, from solution onto a hydrophobic resin that is contained within a microcolumn. Sample loading is accomplished by flowing the protein solution through the microcolumn, where the protein adsorbs to the hydrophobic surface. The protein is digested while still bound to the hydrophobic surface by flowing a buffered trypsin solution through the column bed. The peptide fragments are subsequently eluted for detection by MALDI or ESI-MS. The procedure is demonstrated using dilute protein samples containing high concentrations of salt, urea, and modest amount of sodium dodecyl sulfate relative to protein. Peptide fragments are also detected by MS from a 500 nM bacteriorhodopsin solution digested in a microcolumn. In this case, a combined cyanogen bromide/trypsin digestion was performed in-column. The procedure is applied to the MALDI-MS/MS identification of proteins present in an individual fraction collected by ion exchange HPLC separation of E. coli total cell extract. An additional application is illustrated in the analysis of a human plasma fraction. A total of 14 proteins, which were present in the sample at sub-micromolar concentrations, were identified from ESI-MS/MS. The microcolumn digestion procedure represents the next step toward a system for fully automated protein analysis through capture and digestion of the adsorbed protein on hydrophobic surfaces.     

The leucine rich amelogenin protein (LRAP) adsorbs as monomers or dimers onto surfaces

Amelogenin is believed to be involved in controlling the formation of the highly anisotropic and ordered hydroxyapatite crystallites that form enamel. The adsorption behavior of amelogenin proteins onto substrates is very important because protein–surface interactions are critical to its function. We have previously used LRAP, a splice variant of amelogenin, as a model protein for the full-length amelogenin in solid-state NMR and neutron reflectivity studies at interfaces. In this work, we examined the adsorption behavior of LRAP in greater detail using model self-assembled monolayers containing COOH, CH₃, and NH₂ end groups as substrates. Dynamic light scattering (DLS) experiments indicated that LRAP in phosphate buffered saline and solutions containing low concentrations of calcium and phosphate consisted of aggregates of nanospheres. Null ellipsometry and atomic force microscopy (AFM) were used to study protein adsorption amounts and quaternary structures on the surfaces. Relatively high amounts of adsorption occurred onto the CH₃ and NH₂ surfaces from both buffer solutions. Adsorption was also promoted onto COOH surfaces only when calcium was present in the solutions suggesting an interaction that involves calcium bridging with the negatively charged C-terminus. The ellipsometry and AFM studies revealed that LRAP adsorbed onto the surfaces as small subnanosphere-sized structures such as monomers or dimers. We propose that the monomers/dimers were present in solution even though they were not detected by DLS or that they adsorbed onto the surfaces by disassembling or “shedding” from the nanospheres that are present in solution. This work reveals the importance of small subnanosphere-sized structures of LRAP at interfaces.     

The density and refractive index of adsorbing protein layers

The structure of the adsorbing layers of native and denatured proteins (fibrinogen, gamma-immunoglobulin, albumin, and lysozyme) was studied on hydrophilic TiO(2) and hydrophobic Teflon-AF surfaces using the quartz crystal microbalance with dissipation and optical waveguide lightmode spectroscopy techniques. The density and the refractive index of the adsorbing protein layers could be determined from the complementary information provided by the two in situ instruments. The observed density and refractive index changes during the protein-adsorption process indicated the presence of conformational changes (e.g., partial unfolding) in general, especially upon contact with the hydrophobic surface. The structure of the formed layers was found to depend on the size of the proteins and on the experimental conditions. On the TiO(2) surface smaller proteins formed a denser layer than larger ones and the layer of unfolded proteins was less dense than that adsorbed from the native conformation. The hydrophobic surface induced denaturation and resulted in the formation of thin compact protein films of albumin and lysozyme. A linear correlation was found between the quartz crystal microbalance measured dissipation factor and the total water content of the layer, suggesting the existence of a dissipative process that is related to the solvent molecules present inside the adsorbed protein layer. Our measurements indicated that water and solvent molecules not only influence the 3D structure of proteins in solution but also play a crucial role in their adsorption onto surfaces.     

Aluminium and phosphate uptake by Phragmites australis: the role of Fe,Mn and Al root plaques

Aluminium, a potentially phytotoxic metal, is an important constituent of many mine water discharges but has largely been neglected in the literature. The behaviour of this element in the rhizosphere of the wetland plant Phragmites australis was investigated in the laboratory in the presence and absence of Mn and Fe root plaques. Electron microscopy and chemical extraction techniques were utilized to determine the physico-chemical properties of the plaques and any association of Al. Both Mn and Fe plaques occurred as amorphous coatings on root surfaces with uneven distributions. Al was not adsorbed onto the surface of either plaque type but formed a separate phosphate deposit closely resembling the Fe and Mn plaques. Phosphorus was also found to be adsorbed to the surface of the Fe plaques (but not the Mn plaques). Both mechanisms were found to immobilize P at the root surface but this did not significantly reduce the concentration of P in aerial plant tissues that was sufficient to ensure adequate growth.     

Characterization of eukaryotic cell surfaces prior to and after serum protein adsorption by X-ray photoelectron spectroscopy. Fibroblasts, HELA epithelial, and smooth muscle cells.

Elemental surface concentration ratios N/C,O/C, and P/C of fibroblasts, HELA epithelial cells, and smooth muscle cells, prior to and after washing in the absence or presence of serum proteins, were determined by X-ray photoelectron spectroscopy. Cell surfaces appeared to adsorb hardly any serum proteins, and the relatively high P/C, as compared to N/C and O/C, elemental surface concentration ratio indicated that the cell surfaces consisted mainly of the phospholipid bilayer, with little or no proteins present. The lack of adsorption of serum proteins to the cell surfaces seems at odds with the common notion that cells require adhesive proteins in order to adhere and spread. However, the adsorption behavior of cellularly produced proteins may be completely different, particularly since they seem to be able to displace adsorbed serum proteins from biomaterials surfaces. Interestingly, only HELA epithelial cells (a tumor cell line) appeared to adsorb a very small amount of proteins.     

Inhibition de l'adhérence de Porphyromonas gingivalis sur la surface de titane greffé de poly(styrène sulfonate de sodium)

Dental implant-associated infections as peri-implantitis represent one of the major causes of osteointegration failures of oral implants. Adhesion of Porphyromonas gingivalis, one of the bacterial strains mainly involved in such infections, is tightly dependent on the topographical and/or physico-chemical properties of the implant surfaces. As a matter of fact, we showed that the grafting of one bioactive polymer such as poly(sodium styrene sulfonate) onto titanium implant surfaces allowed a sensitive decrease of Staphylococcus aureus adhesion (> 40%). The aim of the study consists in evaluating the adhesion of P. gingivalis onto titanium surfaces grafted with poly(sodium stryrene sulfonate) in order to elaborate implants exhibiting appropriate inhibiting properties towards the adhesion of periodontal pathogens. The grafting of poly(sodium stryrene sulfonate) onto titanium surfaces is carried out in two steps: chemical oxydation of titanium to initiate radical species then grafting of poly(sodium stryrene sulfonate) by radical polymerization. Chemical characterization of the surfaces is achieved by Fourier transformed infrared spectroscopy (FTIR). Bacterial adhesion was studied on grafted and non grafted (control) titanium surfaces, preadsorbed or not by plasmatic proteins. Protein adsorption as well as bacteria adhesion is followed by fluorescence spectroscopy by using proteins or bacteria previously labelled with fluorescence probes; the quantification of adsorption and bacteria adhesion are performed by image analysis. Results showed that protein adsorption is more important (~3 times) and that P. gingivalis adhesion is strongly inhibited (~73%) onto poly(sodium styrene sulfonate) grafted surfaces when compared to titanium control. Moreover, the inhibition of bacterial adhesion on grafted surfaces preadsorbed with plasma proteins is comparable to that observed on grafted surfaces preadsorbed with fibronectin. In conclusion, the obtained results evidenced that the grafting of titanium surface by poly(sodium styrene sulfonate) led to significant inhibition of P. gingivalis adhesion and that this inhibitory activity involved adsorbed proteins. Poly(sodium styrene sulfonate) grafted titanium surfaces present a high interest for the elaboration of oral implants in various clinical dental applications.