Proteome analysis of human serum proteins adsorbed onto different titanium surfaces used in dental implants

Abstract Titanium dental implants are commonly used due to their biocompatibility and biochemical properties; blasted acid-etched Ti is used more frequently than smooth Ti surfaces. In this study, physico-chemical characterisation revealed important differences in roughness, chemical composition and hydrophilicity, but no differences were found in cellular in vitro studies (proliferation and mineralization). However, the deposition of proteins onto the implant surface might affect in vivo osseointegration. To test that hypothesis, protein layers formed on discs of both surface type after incubation with human serum were analysed. Using mass spectrometry (LC/MS/MS), 218 proteins were identified, 30 of which were associated with bone metabolism. Interestingly, Apo E, antithrombin and protein C adsorbed mostly onto blasted and acid-etched Ti, whereas the proteins of the complement system (C3) were found predominantly on smooth Ti surfaces. These results suggest that physico-chemical characteristics could be responsible for the differences observed in the adsorbed protein layer.


Introduction
Titanium dental implants are commonly used due to their biocompatibility and biochemical properties (Lemons & Lucas 1986;Smith 1993;Nakajima & Okabe 1996). Blood plasma is the main biological fluid interacting with these implants (Park & Davies 2000). The first event that takes place at the biomaterial-tissue interface is the interaction of water molecules and salt ions with the surface of the implant. Shortly after the formation of a hydration layer, a variety of blood proteins adsorbs onto implant surfaces. This occurs within seconds or minutes after implantation (Puleo 1999;MacDonald et al. 2002). The resulting protein film mediates all subsequent biological interactions between the material and the surrounding environment; the cells are unlikely ever to interact directly with the native material surfaces. The concentration, composition and conformation of the protein layer on a biomaterial surface may vary. These characteristics of the protein layer are important for synergistic interactions promoting either favourable or adverse cellular and tissue responses such as attachment to material surfaces, proliferation, and phenotypic changes (Molino et al. 2012;Fernández-Montes Moraleda et al. 2013).
Rough and blasted acid-etched Ti have replaced smooth Ti after reports of a positive correlation between surface roughness and bone integration (Wennerberg & Albrektsson 2010). Moreover, rough Ti surfaces adsorb more proteins than smooth Ti due to the increased surface area (Sela et al. 2007;Rockwell et al. 2012).
Protein adsorption is a dynamic process involving non-covalent interactions such as hydrophobic interactions, electrostatic forces, hydrogen bonding and Van der Waals forces (Andrade & Hlady 1987). Non-covalent interactions are controlled by many protein parameters such as protein size, pI and secondary and tertiary structures (Haynes & Norde 1994;Rabe et al. 2011). The specific physicochemical properties of the biomaterial surface such as its chemistry, wettability, charge and surface morphology also affect the protein adsorption process (Schmidt et al. 2009).
For these reasons, researchers have focused on the elucidation of the mechanisms governing protein interactions with various biomaterials including polymers, metals and ceramics (Wehmeyer et al. 2010). A number of surface-sensitive techniques have been used for the quantification of protein adsorption, viz. surface plasmon with in vitro test outcomes was performed. Therefore, this work aimed to establish a correlation between protein deposition and in vitro outcomes when testing surfaces, such as those currently used in commercial dental implants.

Surface disc preparation
Ti discs (12 mm in diameter, 1-mm thick) were fabricated from a bar of commercially available, pure, grade-4 Ti (Ilerimplant SL, Lleida, Spain). Some of the discs, sandblasted acid-etched (SAE) Ti, were abraded with 4-μm aluminium oxide particles and acid-etched by submersion in sulfuric acid for 1 h to obtain a moderately rough implant surface. All discs were then washed in acetone, ethanol and 18.2 Ω purified water (for 20 min in each liquid) in an ultrasonic bath and dried under vacuum. Finally, all Ti discs were sterilised using UV radiation.

Physico-chemical characterisation of Ti discs
The surface topography of the Ti discs was characterised using atomic force microscopy (AFM, Bruker Multimode, Billerica, MA, USA) under dry conditions. Images were taken at different amplitudes. Measurements at scan sizes of 60 and 1 μm, with a scan rate of 1 and 0.3 Hz, respectively, were carried out (n = 3). The results were analysed using the NanoScope Analysis software (http://nanoscaleworld.bruker-axs.com/nanoscaleworld/media/p/775. aspx). Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX, Leica-Zeiss LEO, Wetzlar, Germany) was used to study these surfaces under vacuum. Platinum sputtering was employed to make the samples more conductive for SEM examination. SEM micrographs were analysed by image processing using Image J software (https://imagej.nih.gov/ij/).
The roughness of the samples was determined using a mechanical Dektak 6 M profilometer (Veeco, Plainview, NY, USA). Two samples of each material were tested, with three measurements for each sample to obtain the average values of the parameters R a and R t .
Wettability was evaluated by measuring the contact angle using an automatic contact-angle meter (DataPhysics OCA 20, DataPhysics Instruments GmbH, Filderstadt, Germany) after depositing 10 μl of ultrapure water W04 on the Ti surface at room temperature. The drops were formed at the dosing rate of 27.5 μl s −1 and the angles were determined using SCA 20 software (http://www.dataphysics.de/startseite/produkte/software-module/). Five discs of each material were studied after depositing two drops on each sample. resonance, optical waveguide lightmode spectroscopy, ellipsometry, quartz crystal microbalance with dissipation and total internal reflection fluorescence spectroscopy (Malmström et al. 2007).
Many studies evaluating the kinetics of protein adsorption onto Ti have focused on the exposure of Ti to single protein solutions or protein mixtures (Sousa et al. 2008;Imamura et al. 2008;Pei et al. 2011;Pegueroles et al. 2012;Kohavi et al. 2013). However, the protein adsorption process is a complex phenomenon depending on many parameters, some of which are not considered in these studies. For instance, in multi-protein systems such as blood plasma/serum, increasing the protein concentration and/or the number of small molecules improves their diffusion and accelerates displacement; thus, they are the first to be adsorbed onto the surface. With time, molecules with greater affinity for the surface but slower rates of diffusion (due to their low concentration or large size) replace the smaller molecules. This is known as the Vroman effect (Dee et al. 2003;Wang et al. 2012).
A study using mass spectrometric techniques identified fibronectin, albumin, fibrinogen, IgG and complement C3 adsorbed on a modified Ti surface incubated in human plasma for 24 h (Sela et al. 2007). The same study showed that the adsorption of plasma proteins depends on the roughness of the surface. Recently, label-free quantitative proteomics has been used in a study of the composition and function of adsorbed protein layers (Montoya et al. 2011). Dodo et al. (2013) characterised the proteome of the protein layer adsorbed onto a rough Ti surface, after exposure to human blood plasma. The study has shown that the layer adsorbed on this surface is composed mainly of proteins associated with cell adhesion, molecular transportation and coagulation processes. This layer creates a polar and hydrophilic interface for subsequent interactions with host cells (Dodo et al. 2013).
At present, the biological evaluation of medical devices includes a battery of standardised tests, as defined in ISO 10993, highly accepted in the biomaterials research field. Typical tests for biocompatibility of biomaterials involve cytotoxicity, cell attachment, cell proliferation and mineralization assays. However, a lack of correlation between in vitro and in vivo results has been observed in many instances. Since the first step before cell attachment on the material surface is protein adsorption, the use of proteomics is proposed to further the understanding of material biocompatibility.
Thus the aim of this study was to compare the protein layers adsorbed onto two types of Ti surfaces, smooth Ti and blasted acid-etched Ti, after incubation in the serum. To achieve this goal, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was employed. Furthermore, a comparison of the more relevant results
Cells were cultured (at a concentration of 1 × 10 4 cells well −1 ) with the Ti discs in 24-well culture plates (Thermo Scientific ® , Waltham, MA, USA) at 37°C in a humidified (95%) atmosphere of 5% CO 2 . The Ti discs were not exposed to blood serum before cell culture. The culture medium was changed every 48 h. In each plate, wells with the same concentration of cells, but no Ti discs, were used as a control of culture conditions.

Cell proliferation
For measuring cell proliferation, the commercial cell viability assay alamarBlue ® (Invitrogen) was used. This kit measures cell viability based on a redox reaction with resazurin. The cells were cultured in wells with the discs (three replicates per treatment) and examined following the manufacturer's protocol after 24, 72 and 120 h. The percentage of reduced resazurin was used to evaluate cell proliferation.

ALP activity
ALP activity was assayed by measuring the conversion from p-nitrophenyl phosphate (p-NPP) to p-nitrophenol, and the specific activity of the enzyme was calculated.
Aliquots (0.1 ml) of the solution used for measuring the protein content were assayed for ALP activity. To each aliquot, 100 μl of p-NPP (1 mg ml −1 ) in substrate buffer (50 mM glycine, 1 mM MgCl 2 , pH 10.5) were added. After incubation for 2 h in the dark (37°C, 5% CO 2 ), absorbance was spectrophotometrically measured at 405 nm using a microplate reader. ALP activity was acquired from a standard curve obtained using various concentrations of p-nitrophenol in 0.02 mM sodium hydroxide. The results were calculated in mmols of p-nitrophenol h −1 (mM PNP h −1 ) and the data were expressed as ALP activity normalised to the total protein content after 14 and 21 days.

Statistical analysis
Data were submitted for analysis of variance (ANOVA) and a Newman-Keuls multiple comparison test, when appropriate. Differences at p ≤ 0.05 were considered statistically significant.

Total protein
Total protein content was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) for colorimetric protein quantitation based on copper reduction. The culture medium was removed from the wells, the wells were washed three times with 1 × DPBS, and 100 μl of lysis buffer (0. 2% Triton X-100, 10 mM Tris-HCl, pH 7.2) were added to each. After 10 min, the lysate was sonicated and centrifuged for 7 min at 13,300 rpm and 4°C. Twenty μl of the supernatant were used for colorimetric measurement of BCA at 570 nm on a microplate reader Multiskan FC ® (Thermo Scientific ® ). The total protein content was calculated from a standard curve for bovine albumin and expressed as μg μl −1 . These data were used to normalise the alkaline phosphatase (ALP) activity after 14 and 21 days.

Formation of the protein layer
Each disc was incubated in a well of a 24-well plate (Thermo Scientific®) with 2 ml of human blood serum from male AB plasma (Sigma-Aldrich, St Louis, MO, USA) for 180 min (37°C, 5% CO 2 ). The use of blood serum may deplete some very high abundant proteins, such as fibrin-related proteins. Then, the serum was removed and the discs were subjected to five consecutive washes with 200 μl of double-distilled water and a final wash with 100 mM NaCl in 50 mM Tris-HCl, at pH 7.1, to remove unadsorbed proteins. The final eluate was obtained by submerging the discs in a solution containing 4% SDS, 100 mM DTT and 0.5 M TEAB according to a method was based on previous studies (Kaneko et al. 2011). Three elutions were performed for each surface treatment; each eluate was obtained from four separate discs. The total protein of the serum was quantified using the method described above (Pierce™ BCA Protein Assay Kit), yielding a concentration of 51 mg ml −1 .

Proteomic analysis
Each eluted protein sample was resolved in 10% polyacrylamide gels, using a Mini-Protean II electrophoresis cell (Bio-Rad, Hercules, CA, USA). A constant voltage of 150 V was applied for 45 min. The gel was then stained using SYPRO Ruby stain (Bio-Rad) following the manufacturer's instructions. The gel was then washed, and each lane was cut into four slices. Each of these slices was digested with trypsin following a standard protocol (Anitua et al. 2015).
Progenesis LC-MS software (Nonlinear Dynamics, Newcastle-upon-Tyne, UK) was used for differential protein expression analysis. Raw files were imported into the programme and one of the samples was selected for a reference run to which the precursor masses in all the other samples were aligned. The abundance ratios between the run to be aligned and the reference run were calculated for all features at given retention times. These values were then logarithmised; the program, based on the analysis of the distribution of all ratios, automatically calculated a global scaling factor. Once normalised, the samples were grouped into the appropriate experimental categories and compared. Differences between groups were only considered for peptide abundances with an ANOVA p-value < 0.05 and a ratio > 1.5 in either direction. A peak list containing the differing peptides was generated for each comparison and searched against a Swiss Prot database using the Mascot Search engine (www.matrixscience. com). Proteins with ANOVA p < 0.05 and a ratio higher than 1.3 in either direction were considered different. Figure 1 shows SEM images of smooth Ti and SAE Ti surfaces. The different topographies can be clearly seen. The particles on the titanium surface ( Figure 1b) are visible in the image. EDX results indicated that these were alumina (Al 2 O 3 ) particles that may have been encrusted in the material after the sandblasting process ( Figure 2). The area of the disc covered by alumina particles comprises ~13.84% of the disc surface area. AFM images in Figure 3, with a scan size of 60 μm, were analysed and an increase on the surface area was detected after aluminium oxide blasting acid-etching treatment. The untreated discs
A SYNAPT G2-Si ESI Q-Mobility-TOF spectrometer (Waters) equipped with an ion mobility chamber (T-Wave-IMS) for high-definition data acquisition analysis was used for the analysis of the peptides. All analyses were performed using electrospray ionisation (ESI) in a positive ion mode. Data were post-acquisition lock-mass corrected using the double charged monoisotopic ion of [Glu 1 ]-fibrinopeptide B. Accurate LC-MS data were collected in HDDA mode, which enhances signal intensities using ion mobility separation. Searches were performed using the Mascot search engine (Matrix Science, London, UK) in the Proteome Discoverer v.1.4 software (Thermo, Waltham, MA, USA). Mascot generic files (MGF) files were generated from the original SYNAPT RAW files using ProteinLynx Global Server 3.0.2 (PLGS, Waters) and further processed using Proteome Discoverer. A peptide mass tolerance of 10 ppm and a fragment mass tolerance of 0.2 Da were used as parameters for the searches. Carbamidomethylation of cysteines was selected as the fixed modification and oxidation of methionine as a variable modification for tryptic peptides. Proteins identified with at least one peptide with an FDR < 1% were kept for further examination.   discs was significantly higher than the roughness of the untreated samples.
Contact angle measurements were carried out to determine the wettability of the surface. Significantly (p<0.05) lower contact angles were observed for blasted acid-etched Ti surfaces than for the untreated discs, namely, 85.70 ± 2.83° and 94.53 ± 2.59°, respectively. Thus, the treated discs showed greater hydrophilicity.

In vitro cultures
Analysis of cell proliferation (Figure 4) clearly showed that disc treatment had no significant effect on cellular growth. Cells proliferated at equal rates on both types of discs during the five-day protocol. A threefold increase in cell numbers was observed between 24 h and three days in culture. Proliferation slowed down between three and five days incubation, showing a plateau and a reduction in proliferation.
ALP enzyme activity ( Figure 5) was not affected by disc topography after 14 and 21 days (ANOVA, p > 0.05). Moreover, between these time points, there was a slight decrease in the ALP activity, as expected. These in vitro data indicate that the disc topographies examined in this study did not affect the metabolic and division processes of MC3T3-E1 cells, related to mineralization.

Identification of proteins adsorbed onto the blasted acid-etched Ti and smooth Ti
LC-MS/MS analysis of the protein layers adsorbed to both Ti surfaces resulted in the identification of 218 different proteins, 30 of which related with bone metabolism (Table 1). Serum proteins involved in cell adhesion and showed a specific surface area of 0.69 ± 0.16%, while that of the blasted acid-etched discs was of 19.97 ± 1.40%.
Untreated titanium discs, with smoother topography (Figure 3a and c), showed a series of grooves due to the machining process. The change in the topography of Ti after the surface blasting and acid-etching treatment is clearly visible in Figure 3b and d. Machining grooves disappeared as a result of sandblasting and the roughness increased significantly (p < 0.05) when the surface was marked by alumina powder. As can be seen in Figure 3c and d, blasting and acid-etching resulted in larger irregularities but the surface was smoother in comparison with the untreated Ti. This can be attributed to the acid-etching treatment.
The mechanical profilometer revealed that for the smooth Ti surface, R a and R t parameters were 0.14 ± 0.04 and 1.28 ± 0.40 μm, respectively. After blasting and acid-etching, R a and R t were 0.93 ± 0.06 and 8.38 ± 0.99 μm, respectively. Thus, the surface roughness of the treated Figure 4. MC3T3-E1 cell proliferation on different treated discs: smooth-Ti (white circle), SAE-Ti (black semi-square with dotted line). Cells, on an empty well, without a disc were used as a control (black circle). no statistically significant differences were found between treatments.

Gene ontology analysis of the identified proteins
Proteomic analysis led to the identification of 181 and 162 proteins on smooth Ti and blasted acid-etched surfaces, respectively. Adsorbed proteins were classified using the PANTHER (Protein ANalysis THrough Evolutionary Re lationships) classification system (Figures 6 and 7). The results of protein classification according to biological processes were almost identical for the two types of surfaces (Figure 6a and b). However, classification of proteins according to the pathways in which they are involved extracellular matrix, important for implant integration, were also found: vitronectin (Salasznyk et al. 2004 (Kwiatkowski et al. 1989;Thouverey et al. 2011;Kim et al. 2013) and actin cytoplasmic 1 (Sen et al. 2015). LC-MS/MS analysis also revealed cellular and secreted proteins associated with bone homeostasis: peptidyl-prolyl cis-trans isomerase B (Pyott et al. 2011) and lysozyme C (Siebert et al. 1978;Briggs & Arinzeh 2014). Serum proteins involved in bone formation were also found to a certain degree: serum  (Kawada et al. 2006;Zarjou et al. 2010) minor proportion of proteins on the smooth Ti (4.69%) in comparison with the treated Ti surfaces. Proteins related to diseases such as Parkinson's and Alzheimer's and proteins related to CCKR signalling pathways were found on both disc types (a very small proportion of the total proteins). In addition to these categories, smooth Ti surfaces adsorbed a small percentage of proteins involved in apoptotic and plasminogen signalling pathways.

Specifically enriched proteins
To find the specifically enriched proteins adsorbed onto the two surface types that might reflect their different osteoinduction capabilities, a differential analysis was revealed differences between the two types of surfaces (Figure 7a and b). Interestingly, smooth Ti-adsorbed proteins were observed to participate in a wider range of pathways than those found on the blasted acid-etched Ti. Blood coagulation (43.35%), inflammation mediated by cytokines (17.34%) and integrin signalling (13.29%) were the three major process-classified protein categories found on the treated (blasted and acid-etched) Ti. For smooth Ti, blood coagulation (28.52%) and inflammation (11.91%) were the most significant categories. However, a major group of proteins related to glycolysis (11.91%) was adsorbed on smooth Ti, which is absent on SAE surfaces. Integrin signalling was only represented by a relatively  very active during osteoblast differentiation, decreased on both Ti surfaces with time with no statistically significant differences. In similar studies, no significant differences in either proliferation or mineralization were found (Yoshida et al. 2012). These results are supported by proteomic analysis of proteins adsorbed onto the different discs since the majority of proteins attach in a similar way to both surfaces. The extensive list of adsorbed proteins shows that at least 30 of these proteins are involved in bone homeostasis in a direct or indirect way (Table 1). However, the blasted and acid-etched Ti surfaces and smooth Ti surfaces showed different osteogenic properties in in vivo models (Wennerberg & Albrektsson 2009). Furthermore, Aparicio et al. (2011) showed that high R a values favour osseointegration of dental implants in comparison with smoother surfaces. This effect is attributed to a higher implant-bone contact interface as a consequence of increased roughness. Nevertheless, in this study chemical differences between treatments were also found. To test this premise a detailed analysis of the proteins adsorbed to the two surface types was performed. In order to isolate and identify these surface adsorbed proteins, a protocol was established where, following serum incubation, discs were washed and final protein elution was obtained with an SDS-containing buffer. This approach permitted washing of the surfaces thoroughly and getting a good protein yield for the characterisation of the differences between both surfaces. The procedure indicates that under the same regime of washes and the same elution strength, a number of different proteins bound more consistently to each of the surfaces at statistically significant different concentrations, revealing differences in surface-protein interactions. Although other approaches cannot be discarded, the present method has been shown to be useful for the intended purpose. On the other hand, the use of a harsher buffer could release proteins that might have remained attached after the SDS wash. However, it is believed that although the total list of proteins could increase, it should not affect the differential analysis results. There was an average of 181 proteins identified on the smooth Ti surface discs, and 162 proteins on the blasted and acid-etched Ti surface. This suggests that the differences observed performed (in triplicate) using the Progenesis QI software. This method identified nine proteins differentially enriched/associated with each surface (Table 2).
Proteins enriched on the blasted acid-etched Ti were apolipoproteins ApoA-I, ApoE, ApoA-IV, plectin, antithrombin III and vitamin K-dependent protein. The largest difference between the two surface types was found for ApoA-IV and plectin. It was also found that complement C3 and some immunoglobulins (Ig gamma and lambda chains) were significantly enriched on the smooth Ti but not on the blasted and acid-etched Ti discs.

Discussion
The main part of this study characterised the protein layer adsorbed onto Ti discs with two different surface types: a SAE Ti and an untreated, smooth Ti. It is reasonable to assume that different surface characteristics will affect the adsorption of proteins.
Roughness is a key parameter in the assessment of the osseointegrative properties of material (Buser et al. 1991). The two surface types studied in this work have different topography, ie SAE Ti is rougher than the untreated Ti surface. These results are consistent with previous studies (Grassi et al. 2006). Moreover, the presence of alumina is also associated with a good bone response (Wennerberg et al. 1995) and a change in hydrophilicity affecting both chemical and physical composition of the surface. All these physico-chemical features affect the affinity of the protein layer formed on the material.
Ti surfaces are widely used in implants; techniques advancing osteogenesis are needed to improve the quality of health care and patient recovery. The surface types described here have been extensively used in orthopaedic implants with overall similar outcomes (Schwartz et al. 2008).
The in vitro experiments, using an osteosarcoma cell line, showed no differences between both samples either in proliferation or mineralization. Both surfaces showed very similar cell proliferation results with time, increasing gradually throughout the test period. Mineralization in cells, measured by ALP activity, an enzyme that becomes The most frequent ApoE allele is ApoƐ3, found with a frequency of 79%. ApoƐ2 is present in ~7% of the population, and ApoƐ4 in 14%. ApoƐ3 is also called the neutral allele because it is not associated with any of the human diseases. Apoe2 and 4 have been associated with an increased probability of developing arthrosclerosis and Alzheimer's disease (Eisenberg et al. 2010).
The method used to characterise the protein layer on Ti surfaces did not allow for the determination of the type of ApoE allele adsorbed. Moreover, is not clear whether physico-chemical properties of the surface discriminate between the allele types. It is tempting to hypothesise that blasted acidetched Ti has the ability to enrich the microenvironment of the implant with ApoE. However, this could only improve the osseointegration outcome if the patient carried the ApoƐ3 alleles. Following this line of thought might help to determine mechanisms of the variability in the outcomes of the same implant type in different patients. Kaneko et al. (2011) published a similar study using different surfaces, octacalcium phosphate (OCP) and hydroxyapatite crystals (HA). They found that ApoE and complement component 3 (C3) were among the proteins differentially associated with these surfaces. They observed that HA adsorbed more C3 than OCP, whereas OCP adsorbed more APoE.
Interestingly, in the present study, C3 was enriched on smooth Ti discs. C3 belongs to a family of proteins involved in immune and inflammatory responses (Sahu & Lambris 2001). Osteoclasts are bone macrophages derived from the myeloid lineage that requires complement C3 and C5 for optimal differentiation (Tu et al. 2010). Osteoclasts are necessary for bone resorption and the optimal balance between osteoblast and osteoclast differentiation must be reached to achieve healthy bone formation. It is not clear whether increased C3 adsorption on smooth Ti surfaces alters this balance.
To summarise, two types of surfaces, smooth and SAE, were studied by physico-chemical, in vitro and proteomic analysis. Al 2 O 3 was found in the SAE surface and only Ti in the smooth sample. Roughness and hydrophilicity were increased by SAE treatment. In this study, in accordance with published literature, no differences in in vitro tests (proliferation and mineralization) were found. Proteomic analysis of the proteins adsorbed onto both surfaces showed the presence of proteins related to bone generation. Proteins enriched on the SAE Ti were apolipoproteins ApoA-I, ApoE, ApoA-I, plectin, antithrombin III and vitamin K-dependent protein. The largest difference between the two surface types was found for ApoA-IV and plectin. It was also found that complement C3 and some immunoglobulins (Ig gamma and lambda chains) were significantly enriched on smooth Ti but not on the blasted and acid-etched Ti discs. between the surfaces is a result of differential binding of certain proteins and not from the total amount of protein.
The proteomics differential quantification analysis performed by Progenesis found some significant differences for plectin, antithrombin-III and several other apolipoproteins. Plectin is a cytoskeleton protein that links intermediate filaments to other cytoskeletal systems and anchors them to the membrane junction sites. It binds mostly to vimentin and is very important for preserving the mechanical integrity of the tissue (Burgstaller et al. 2010). Plectin is not a typical serum protein; therefore, its presence in the protein layer formed during incubation of Ti discs with serum was unexpected. Antithrombin (AT) is a glycoprotein that inactivates several enzymes of the coagulation system. Specifically, AT-III inactivates thrombin, which catalyses the formation of fibrin from fibrinogen. Fibrin architecture at the clot affects bone healing (Shiu et al. 2014). However, apolipoproteins are important serum proteins involved in lipid transport; different isoforms have different properties and activities. Apolipoprotein A-IV has antioxidant-like activity and is involved in the inhibition of lipid oxidation (Spaulding et al. 2006). It has been reported that patients with osteonecrosis, a skeletal pathology with intense bone degeneration, have lower levels of ApoA-IV in comparison with healthy individuals (Wu et al. 2008). Lipid metabolism and oxidative injury are important processes in the pathophysiology of the disease. Apo A-IV mutations are linked to corticosterone-induced osteonecrosis in patients with renal transplants (Hirata et al. 2007). In the present study, Apo A-IV level was significantly higher on blasted acid-etched Ti than on smooth Ti. This observation might account for a favourable osseointegration environment created by the treated discs as the protein acts as an antioxidant. Another important apolipoprotein, ApoA1, adsorbed to treated Ti at higher concentrations than to smooth Ti. ApoA1 is the main component of the high-density cholesterol complex but it has not been associated with bone formation or resorption. Interestingly, ApoE, which is involved in the regulation of bone metabolism, was also adsorbed to the blast acid-etched Ti at higher concentrations than to smooth Ti. Although still somewhat controversial, ApoE has been extensively reported to be involved in bone homeostasis (Niemeier et al. 2012), possibly via the promotion of vitamin K uptake into the osteoblasts (Newman et al. 2002). However, various ApoE alleles behave very differently in this process. ApoƐ2 is the allele with the least involvement in the transport of vitamin K (Saupe et al. 1993). The ApoƐ4 allele has been associated with a low bone mass in several studies in postmenopausal women (Shiraki et al. 1997;Sanada et al. 1998). More recently, epidemiological studies have confirmed that ApoƐ2 represents an increased risk for trabecular bone fracture (Dieckmann et al. 2013).
Although significant physico-chemical differences were found between samples (chemical composition, roughness and hydrophilicity), in vitro tests did not show any differences. Further work is needed to demonstrate that proteomic analysis can correlate with in vivo behaviour.