N a t C T a b a A R R 2 A A K B B N H I O A S 1 i s t u I P h 0 Colloids and Surfaces B: Biointerfaces 163 (2018) 321–328 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces j o ur nal ho me pa ge: www.elsev ier .com/ locate /co lsur fb ano hydroxyapatite-blasted titanium surface creates a biointerface ble to govern Src-dependent osteoblast metabolism as prerequisite o ECM remodeling élio J.C. Fernandesa,1, Fábio Bezerraa,1, Marcel R. Ferreiraa,1, Amanda F.C. Andradea, hais Silva Pintoa, Willian F. Zambuzzia,b,∗ Department of Chemistry and Biochemistry, Bioscience Institute, São Paulo State University, UNESP, Campus Botucatu, Botucatu, São Paulo, Brazil Electron Microscopy Center, IBB, UNESP, Botucatu, SP, Brazil r t i c l e i n f o rticle history: eceived 21 September 2017 eceived in revised form 1 December 2017 ccepted 27 December 2017 vailable online 28 December 2017 eywords: iointerfaces iotechnology anotechnology ydroxyapatite mplants steoblast dhesion ignal transduction a b s t r a c t Over the last several years, we have focused on the importance of intracellular signaling pathways in dynamically governing the biointerface between pre-osteoblast and surface of biomaterial. Thus, this study investigates the molecular hallmarks involved in the pre-osteoblast relationship with different topography considering Machined (Mc), Dual Acid-Etching (DAE), and nano hydroxyapatite-blasted (nHA) groups. There was substantial differences in topography of titanium surface, considering Atomic Force Microscopy and water contact angle (Mc = 81.41 ± 0.01; DAE = 97.18 ± 0.01; nHA = 40.95 ± 0.02). Later, to investigate their topography differences on biological responses, pre-osteoblast was seeded on the different surfaces and biological samples were collected after 24 h (to consider adhesion signaling) and 10 days (to consider differentiation signaling). Preliminary results evidenced significant differences in morphological changes of pre-osteoblasts mainly resulting from the interaction with the DAE and nHA, distinguishing cellular adaptation. These results pushed us to analyze activation of specific genes by exploring qPCR technology. In sequence, we showed that Src performs crucial roles during cell adhesion and later differentiation of the pre-osteoblast in relationship with titanium-based biomaterials, as our results confirmed strong feedback of the Src activity on the integrin-based pathway, because integrin-ß1 (∼5-fold changes), FAK (∼12-fold changes), and Src (∼3.5-fold changes) were significantly up-expressed when Src was chemically inhibited by PP1 (5 �M). Moreover, ECM-related genes were rigorously repro- grammed in response to the different surfaces, resulting on Matrix Metalloproteinase (MMP) activities concomitant to a significant decrease of MMP inhibitors. In parallel, we showed PP1-based Src inhibition promotes a significant increase of MMP activity. Taking all our results into account, we showed for the first time nano hydroxyapatite-blasted titanium surface creates a biointerface able to govern Src-dependent osteoblast metabolism as pre-requisite to ECM remodeling © 2017 Elsevier B.V. All rights reserved. . Introduction Biomedical implants are one of the most common therapies used n case of bone lesions. Although several materials have been con- idered to produce these metallic devices, titanium is considered he gold standard [1]. Mechanistically, the success of the therapy sing these biomaterials can be evaluated by the profile of osteoin- ∗ Corresponding author at: Dept. of Chemistry and Biochemistry Biosciences nstitute/IBB−UNESP, P.O. Box: 510, Zip Code: 18618−970 Rubião Jr, Botucatu, São aulo, Brazil. E-mail address: wzambuzzi@ibb.unesp.br (W.F. Zambuzzi). 1 These authors contributed equally in this work. ttps://doi.org/10.1016/j.colsurfb.2017.12.049 927-7765/© 2017 Elsevier B.V. All rights reserved. tegration promoted by the osteoprogenitor cells in response to them. It is known that the physico-chemical properties of the bio- material surfaces decisively trigger biological phenomena able to modulate cellular response and later impact the time and quality of osseointegration [2–4]. Therefore, a special look at cellular trans- ducers that govern cell-biomaterial response becomes necessary. Intracellular signaling pathways triggered upon integrin acti- vation are classical biomarkers of the eukaryotic cell adhesion, and focal adhesion kinase (FAK) and product of the transforming gene of Rous sarcoma virus, Src, are important intracellular pro- teins to link integrin activation with cytoskeleton rearrangement [5–7]. In detail, integrins are transmembrane proteins responsible for cell anchoring in substrata, such as extracellular matrix compo- https://doi.org/10.1016/j.colsurfb.2017.12.049 http://www.sciencedirect.com/science/journal/09277765 http://www.elsevier.com/locate/colsurfb http://crossmark.crossref.org/dialog/?doi=10.1016/j.colsurfb.2017.12.049&domain=pdf mailto:wzambuzzi@ibb.unesp.br https://doi.org/10.1016/j.colsurfb.2017.12.049 3 rfaces n l r m T i e t [ t E [ t K a i t a p a b o ( c i r r S 2 i M n P e b f s t i S n w S 2 2 t t y ( ( i s a o g 22 C.J.C. Fernandes et al. / Colloids and Su ents [8]. When activated, integrins recruit FAK and Src molecules eading to a specific intracellular pathway culmination in different esponses such as cytoskeletal rearrangement, cell proliferation, otility, differentiation, and survival, as discussed elsewhere [9]. hus, understanding the biomaterial biointerface able to favor cell nteractions is an interesting strategy to contribute to biomedical ngineering and biotechnological business. It is already known that for a complete biomaterial osseointegra- ion a well-orchestrated ECM remodeling peri-implant is necessary 10,11]. As important proteases in this context, matrix metallopro- einases (MMPs) are proteins responsible for degradation of the CM components and are fundamental for adequate remodeling 12]. On the other hand, tissue inhibitor of matrix metallopro- einases (TIMP’s) and reversion-inducing cysteine rich protein with azal motifs (RECK) are important modulators of MMP activities, nd are required to orchestrate ECM remodeling [13,14]. Summarizing, we investigated the molecular hallmarks nvolved with the pre-osteoblast relationship with different opography considering Machined (Mc), Dual acid-etching (DAE), nd nano hydroxyapatite-blasted (nHA) groups. We showed Src erforms crucial roles during cell adhesion and later differenti- tion of the pre-osteoblast in relationship with titanium-based iomaterials, because our results confirmed a strong feedback f the Src activity on the integrin-based pathway as integrin-ß1 ∼5-fold changes), FAK (∼12-fold changes), and Src (∼3,5-fold hanges) were significantly up-expressed when Src was chemically nhibited by PP1. Moreover, ECM remodeling-related genes were igorously reprogrammed in response to the different surfaces, esulting on Matrix Metalloproteinase (MMPs) activities inverse to rc expression, suggesting Src as a suppressor of MMP processing. . Material and methods Materials Three different titanium surfaces (discs) were investigated n this study, distinguishing by the surface properties, called achined (Mc; control), Dual Acid-Etched (DAE), and acid-etched anoHA-blasted (nHA). The nHA was obtained by using the romimic HAnano-method, a detailed description can be found lsewhere [15–17]. Briefly, the samples were dipped into a sta- le particle suspension containing 10 nm in diameter HA particles ollowed by a heat treatment at 550 ◦C for 5 min in nitrogen atmo- phere. The surfactant-mediated process allows better control of he chemical composition of the coating [16]. The primers used n this study were purchased from Exxtend company (Campinas, ão Paulo, Brazil). The antibodies were purchased from Cell Sig- alling Technology (Boston, MA, USA). All the titanium materials ere sterilized by exposure to Gamma irradiation and donated by .I.N. – Sistema Nacional de Implantes (São Paulo, SP, Brazil). .1. Surface characterization of the materials .1.1. Atomic force microscopy (AFM) Ti-modified surfaces were first processed by AFM, in which AFM ip acts as a “nano profilometer” and is able to generate informa- ion on the micro and nano-dimensional aspects of the surface (x, , and z axis). The images were acquired by using Bioscope Catalyst Bruker Coorp) equipment, and AFM cantilevers, also from Bruker, “RTESPA”, k = 20 to 80 N/m) were used. The equipment worked in ntermittent contact mode and images were taken from 3 different amples (Machined, Double-Acid Etched, and nano Hydroxyap- tite) and 3 different regions on each sample (border, middle, and pposite border). Scans of 3.3 × 3.3 �m2 were taken to check homo- eneity. B: Biointerfaces 163 (2018) 321–328 2.1.2. Water contact angle An automated goniometer (Ramé Hart, 100-00) was used to evaluate water wettability through contact angle measurements. Deionized water and diiodomethane were used as probe liquids and the presented results correspond to the average of 10 mea- surements. 2.1.3. Cell culture MC3T3-E1 (subclone 4), mouse pre-osteoblastic cells, was used in this study. Pre-osteoblasts were cultured in �-MEM supple- mented with 10% Fetal Bovine Serum (FBS) at 37 ◦C and 5% CO2. Sub-confluent passages were tripsinized and used in all exper- iments. For the collect samples, the cells were seeded on the titanium surfaces (direct contact) or treated with the titanium- enriched medium (prepared according to the ISO 10993: 2016) up to 24 h (for the adhesion evaluation) and up to 14 days (for the osteogenic phenotype). During this time, the cells were maintained at 37 ◦C in conditioned medium. The culture medium was changed every 3 days to provide an adequate concentration of nutrients to the cells. To understand whether Src was affecting the feedback of adhesion-related genes, pre-osteoblast was treated up to 24 h with PP1 (5 �M), when the total RNA and protein extract were collected. The efficiency of PP1 was determined by immunoblot- ting. Briefly, the cells were lysed (50 mM Tris [tris(hydroxymethyl)aminomethane]–HCl [pH 7.4], 1% (vol/vol) Tween 20, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM O-Vanadate, 1 mM NaF, plus protease inhibitors [1 �g/mL aprotinin, 10 �g/mL leupeptin, and 1 mM 4-(2-amino-ethyl)-benzolsulfonyl-fluor-hydrochloride] and kept on ice for 2 h. After clearing the lysate by centrifugation, the amount of protein obtained was determined using Lowry’s method. Afterwards, an equal volume of 2× sodium dodecyl sulfate (SDS) gel loading buffer (100 mM Tris-HCl [pH 6.8], 200 mM dithiothreitol [DTT], 4% SDS, 0.1% bromophenol blue, and 20% glycerol) was added to samples, which were then boiled for 5 min. Proteins were resolved by SDS-polyacrylamide gel elec- trophoresis (SDS-PAGE) and transferred to PVDF membranes. Then, membranes were blocked with either 1% fat-free dried milk or bovine serum albumen (2.5%) in 1× Tris-buffered saline (TBS)–Tween 20 (0.05%, vol/vol) and incubated overnight at 4 ◦C with appropriate primary antibody at 1:1000 dilutions. After washing in 3× TBS-Tween 20, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgGs antibodies, at 1:2000 dilutions (in all immunoblotting assays), in blocking buffer for 1 h. After washing in 3 x TBS, the detection was performed by using enhanced chemiluminescence (ECL) [29]. 2.2. Scanning electron microscopy (SEM) Pre-osteoblasts were seeded on different titanium surfaces at the density of 5 × 104 cells/disc. After 4 h, cells were fixed with 2.5% of glutaraldehyde in 0.1 M phosphate buffer pH 7.3 for 24 h. They were immersed in osmium tetroxide 0.5% for 40 min, dehydrated with a series of alcohols, dried at a critical point, and finally met- allized. Samples were studied using a Quanta 200 – FEI Company scanning electron microscope at an accelerating voltage of 12.5 kV. 2.3. Quantitative PCR assay (qPCR) Cells were directly cultured on different texturized titanium sur- faces and after 24 h or 14 days, the total RNA was extracted from cells with Ambion TRIzol Reagent (Life Sciences − Fisher Scientific Inc, Walthan, MA, USA) and treated with DNase I (Invitrogen, Carls- band, CA, USA). cDNA synthesis was performed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. qPCR was C.J.C. Fernandes et al. / Colloids and Surfaces B: Biointerfaces 163 (2018) 321–328 323 Table 1 Expression primers sequences and qPCR cycle conditions. Gene Primer 5’-3’ Sequence Reactions Condition MMP2 Forward AACTTTGAGAAGGATGGCAAGT 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse TGCCACCCATGGTAAACAA MMP9 Forward TGTGCCCTGGAACTCACACGAC 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse ACGTCGTCCACCTGGTTCACCT TIMP1 Forward ATCCTCTTGTTGCTATCACTG 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse GGTCTCGTTGATTTCTGGG TIMP2 Forward GCAACAGGCGTTTTGCAATG 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse CGGAATCCACCTCCTTCTCG RECK Forward CCTCAGTGAGCACAGTTCAGA 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse CCTGTGGCATCCACGAAACT FAK Forward TCC ACC AAA GAA ACC ACC TC 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse ACG GCT TGA CAC CCT CAT T Src Forward TCGTGAGGGAGAGTGAGAC 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sReverse GCGGGAGGTGATGTAGAAAC Integrin �1 Forward TATCCTCCTGAGCGCCTTT 95 ◦C – 15 min; 95 ◦C – 15 s; 60 ◦C – 30 s; 72 ◦C – 20 sGAAT ATCC TGTC c M 0 w a t 2 p m f t i s t ( ( l g 1 T g w s 2 c s o S 3 d u ( f ( Reverse TGGCCTTTTGAA �-actin Forward TCTTGGGTATGGA Reverse AGGTCTTTACGGA arried out in a total of 10 �l, containing PowerUpTM SYBRTM Green aster Mix 2× (5 �l) (Applied Biosystems, Foster City, CA, USA), .4 �M of each primer, 50 ng of cDNA and nuclease free H2O. Results ere expressed as relative amounts of the target gene using �- ctin as inner reference gene (housekeeping gene), using the cycle hreshold (Ct) method. Primers and details are described in Table 1. .4. Activities of MMP were determined by a gelatin roteolysis-based zymography The proteolytic activities of MMP-2 and MMP-9 in conditioned edium were assayed by gelatin-based zymography, widely used or this end. Conditioned medium was collected in response to the itanium surfaces or by cells in response to PP1 (5 �M), and later t was clarified by centrifugation 13,200g for 15 min at 4 ◦C, and tored at −20 ◦C. Samples were quantified using the Lowry pro- ein assay (Lowry et al., 1956) and diluted in non-reducing buffer 0.1 M Tris–HCl, pH 6.8, 20% (v/v) glycerol, 1% (w/v) SDS, and 0.001% w/v) bromophenol blue). Equal amounts of protein (75 �g) were oaded onto SDS–polyacrylamide gel (10% (w/v)) and 4% (w/v) elatin. MMPs renaturation was performed in 2% (v/v) Triton X- 00 for 40 min followed by incubation in incubation buffer [50 mM ris–HCl and 10 mM CaCl2 (pH 7.4)] at 37 ◦C for 18 h. Afterwards, els were stained with 0.5% (w/v) coomassie blue G 250 for 30 min, ashed in a 30% (v/v) methanol and 10% (v/v) glacial acetic acid olution and then analyzed using ImageJ software. .5. Statistical analysis Mean values and standard deviation obtained for each test were alculated, and one-way ANOVA was performed (alpha error type et to 0.05) when appropriate, with Bonferroni corrected post-test, r non-parametric analysis, using GraphPad Prysm 5 (GraphPad oftware, USA). . Results As reported previously elsewhere [17], there are considerable ifferences in the topography of the titanium-based surfaces eval- ated in this study, mainly documented by atomic force microscopy insert in Fig. 1). The topography modifications were decisive or wettability, which significantly impacts water contact angle Mc = 81.41 ± 0.01; DAE = 97.18 ± 0.01; nHA = 40.95 ± 0.02). CCAA TGTG 95 ◦C – 15 min; 95 ◦C – 15 s; 60◦C – 30 s; 72 ◦C – 20 sAACG 3.1. Biointerface between pre-osteoblast and titanium-modified surfaces requires reprogramming of dynamic adhesion-related genes Thereafter, we started with the biological characterization by seeding pre-osteoblast on all of those surfaces and after 24 h of seeding. The pre-osteoblast morphology was evaluated to predict the quality of the pre-osteoblast/surface interactions (Fig. 1b,d,f). Pre-osteoblast performed better on DAE or nHA at this stage, as they completely spread on the both surfaces (Fig. 1d,f). To inves- tigate whether the integrin-downstream pathway (Fig. 2a) was required, the pre-osteoblast was seed on the surfaces, and 24 h later the biological samples were harvested in order to study specific genes. Curiously, there was a significant decrease on the integrin- ß1 expression in response to DAE and nHA (Fig. 2b). A similar profile was also found for FAK and Src genes (Fig. 2c,d). Elsewhere, we reported a significant activation of Src and FAK in response to the same surfaces [17] and suggested the down-modulation of those genes might be regulated by a negative feedback. To validate this hypothesis, we treated the pre-osteoblast with a well-documented Src inhibitor (PP1), which promoted a significant down-regulation of Src at Y416 (Fig. 2e). Our results confirmed a strong feedback of the Src activity on the integrin-based pathway, because integrin- ß1 (∼5-fold changes), FAK (∼12-fold changes), and Src (∼3.5-fold changes) were significantly up-expressed when Src was chemi- cally inhibited (Fig. 2f–h). Here, cells not treated with PP1, but with vehicle, were considered the control. 3.2. Titanium-modified surfaces differentially orchestrate ECM remodeling-related genes The titanium-related surfaces modulated the cell morphology and adhesion up to 24 h; therefore, we decided to explore their relationship with the surfaces. MMPs and TIMPs were significantly involved in the establishment of the adequate biointerface for the osteoblast phenotype (Fig. 3a–e). Our results found increasing of MMP activities in response to both DAE and nHA (Fig. 3f–j), and this biological effect is guaranteed by the significant decrease of tissue- inhibitor matrix metaloproteinases: TIMP1, TIMP2, and RECK (Fig. 3c–e, respectively). As suggested previously for osteoblast adhesion, when MMPs-2 and −9 activities increase in the extra- cellular compartment, ECM remodeling-related genes seems to establish negative feedback on their respective MMP gene activa- tion because both of them significantly decreased in response to DAE and nHA. 324 C.J.C. Fernandes et al. / Colloids and Surfaces B: Biointerfaces 163 (2018) 321–328 Fig. 1. Ti-texturing surface properties and their impact on morphological changes of pre-osteoblast. The titanium topographies were investigated by atomic force microscopy (AFM; three-dimensional perspective image showing a 3 × 3 (m 2-field representation of Ti-texturing surfaces: Mc; DAE; and nHA). A fine roughness (nanometric scale) covers t he dua o ning E b s to c 3 d p a he surface of nHA and goes along with the micrometric roughness promoted by t n the surfaces and 24 h after seeding, the cell morphology was acquired by Scan ehavior on those titanium-texturized surfaces. (For interpretation of the reference .3. Src and ECM remodeling-related genes are hallmarks of irect and indirect effects of titanium-modified surfaces on re-osteoblast behavior during osteogenic phenotype In bone, it has been reported that ECM remodeling and Src re decisive during osteoblast differentiation [18,19]. Previously, l acid-etching treatment (inserts in a; b; c). Thereafter, pre-osteoblasts were seed lectron Microscopy (yellow arrows; b,d,f). Note the significant differences in c ell olour in this figure legend, the reader is referred to the web version of this article.) we showed titanium-modified surfaces stimulate osteoblast dif- ferentiation in a direct contact manner. By exploring the same biological model (Fig. 4a), we found here a significant increase of Src gene expression (Fig. 4b), suggesting a direct feedback of the Src activity, which decreased as a pre-requisite for osteogenic phenotype [20]. On the other hand, in an indirect contact man- C.J.C. Fernandes et al. / Colloids and Surfaces B: Biointerfaces 163 (2018) 321–328 325 Fig. 2. Biointerface between pre-osteoblast and titanium-modified surfaces requires reprogramming of dynamic adhesion-related genes. a. Schematic depiction of the intracellular pathway evaluated in this study. Cells were seeded on different Ti-related surfaces. After 24 h the total mRNA was collected and adhesion-related genes evaluated. Note the decrease of integrin-B1 (b), FAK (c) and Src (d). Previously, we reported the relevance of Src and FAK activations in response to titanium, and suggested a n ated t ctivity a . n a a t w a u t r c T ( p w o s e w 9 c egative feedback, down-modulating both genes. To validate this hypothesis, we tre he effect of PP1 by reporting a down-phosphorylation of Src at Y416 (e). The Src a ctivations were evaluated by qPCR. The differences were significant when p < 0.05 er, Src gene expression significantly decreased in response to DAE nd nHA (Fig. 4c). Next, we investigated ECM-related gene MMPs nd their tissue inhibitors in both direct and indirect manner up o 14 days in vitro (Fig. 4). Curiously, all of the genes investigated ere significantly up-modulated in response to DAE and nHA in direct contact manner – specifically, MMP-2 and MMP-9 were p-modulated ∼4 and ∼10-fold changes, respectively, in response o DAE (Fig. 4d,e) and ∼2 and ∼2.5-fold changes, respectively, in esponse to nHA (Fig. 4d,e); while TIMP1 had ∼10 and ∼2.5-fold hanges, in response to DAE (Fig. 4f) and nHA (Fig. 4f), respectively. IMP2 had ∼20 and ∼10-fold changes, in response to DAE and nHA Fig. 4g), respectively. In addition, RECK, a membrane GPI-anchored rotein, increased ∼2.5-fold changes in response to DAE (Fig. 4h), hile it significantly decreased in response to nHA (Fig. 4h). All f these analyses were made in comparison with Machined (Mc) urfaces. Considering the indirect contact, we also found a decreased xpression of the gene MMP-2 in response to both DAE and nHA, hen compared with Mc (Fig. 4i). In addition, modulation of MMP- depended on material properties; significantly increased (∼2-fold hanges) in response to DAE and significantly decreased in response the pre-osteoblast with a well-documented Src inhibitor-PP1 (5 �M). We validated is dependent on modulating integrin-B1 (f), FAK (g), and Src (h). All of those gene to nHA (Fig. 4j). TIMP1 slightly but not significantly increased (Fig. 4k). However, TIMP2 was significantly up-modulated, reach- ing 20-fold change in response to DAE, and had an almost 10-fold increased in response to nHA (Fig. 4l); while RECK was significantly decreased (Fig. 4m). Importantly, we showed that PP1-inhibition of Src promoted a significant increase of both MMP expression (Fig. 5a,b) and activity (c,f). 4. Discussion Over the last 10 years, we have searched for alternative meth- ods to understand the molecular biocompatibility of gold-standard biomaterials to guide biomedical engineering and reduce the num- ber of experimental animals. In this sense, we have proposed cell signaling as a dynamic field to be considered [21,22]. In this work, three different surfaces were assayed: Machined (Mc), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA) groups. The nHA was obtained by adsorbing nanometer-scaled hydroxyapatite on the DAE-modified titanium surfaces [17,23]. First, the topography differences were characterized by atomic 326 C.J.C. Fernandes et al. / Colloids and Surfaces B: Biointerfaces 163 (2018) 321–328 Fig. 3. Titanium-modified surfaces differentially orchestrate ECM remodeling-related genes. First, we investigated whether the titanium-texturized surfaces were also able t nd MM e of tho a n p < f b t T b c i t s b r T d n r i s w t T b e a a S i r a h o modulate MMP expressions, and found a down-regulation of both MMP-2 (a) a valuated matrix metaloproteinase inhibitors – TIMPs −1, −2, and RECK – and all ctivation very similar between DAE and nHA. The differences were significant whe orce microscopy and corroborated with those obtained previously y Bezerra et al. (2017). Thereafter, we investigated whether the opography could modulate pre-osteoblast morphological changes. he pre-osteoblast performed better on the nHA surfaces, maybe ecause this surface had a higher hydrophilicity than others, indi- ated by its smaller water contact angle. Another point to consider s the capacity of titanium-texturized surfaces to adsorb serum pro- ein, which is able to cause profound cell metabolic changes, as uggested by Zambuzzi’s group [22,24]. In sequence, we investigated the molecular fingerprint of the iointerfaces promoted by titanium-modified surfaces, mainly elated to cell adhesion (24 h) and differentiation (10 days). he adhesion-related gene reprogramming revealed a significant ecrease in integrin and Src expression in response to DAE and HA. A reasonable explanation is that Src activity is significantly equired during osteoblast adhesion on those surfaces and results n a negative feedback on Src expression. To obtain a better under- tanding of the participation of Src in this process, pre-osteoblasts ere treated up to 24 h with PP1, a chemical Src-inhibitor, and he gene expression of integrin, FAK, and Src were reevaluated. he significant increase of the Integrin, FAK, and Src expression y chemically Src-inhibited pre-osteoblasts validated our hypoth- sis. In addition, as integrin and FAK are required for later Src ctivation, this increase in gene expression can be understood as compensation mechanism for the maintenance of intracellular rc levels, because Src governs important intracellular pathways n osteoblast metabolism [7,20,25]. In addition, ECM remodeling- elated genes were also evaluated. Our data showed that MMP ctivity is a titanium surface-dependent, where nHA stimulated igher MMP-2 and −9 activities of the released MMPs by activate P-9 (b), although there is an increase on the both MMP activities (f-j). Then, we se genes were down-modulated (c, d, and e, respectively), with the profile of the 0.05. osteoblasts, while their genes were down-regulated in response to both DAE and nHA. According to the same reasonable explanation used earlier, the high activity of MMP might promote a compensa- tion feedback decreasing both MMP transcripts evaluated by qPCR technology. TIMPs were also down-regulated, favoring MMP activ- ity presented. A possible mechanism suggests the involvement of integrin-dependent Src function during osteoblast adhesion, which can be considered a prerequisite to ECM-related genes. Later, to analyze the possible involvement of Src and ECM remodeling process during the osteogenic phenotype acquisition, pre-osteoblastic cells were maintained in culture up to 10 days using two experimental strategies, direct and indirect contact. Src expression was higher in response to osteoblast under a direct adhesion on the titanium than those cells grown in a titanium- enriched medium, in indirect contact. Elsewhere we showed that titanium-based dental implants release considerable titanium amount when incubated in cell medium up to 24 h in CO2 incuba- tor [23]. This titanium-enriched medium promotes a considerable increase on Reactive Oxygen Species (ROS), culminating on phos- phorylation balance governed by the decrease of the PTP activities (since PTPs have cysteine in the enzymatic active-site and Cys- residue is very sensitive to oxidation) [23,26–28]. In addition, Fernandes et al. [26] found that ROS production is necessary dur- ing the first hours of pre-osteoblast adhesion and it guaranteed FAK and Src phosphorylations as an immediate consequence of the PTP oxidation. In addition, we have proposed Src as indispensable transduc- ers for osteoblast metabolism, governing their differentiation and osteogenic phenotype [7]. Previously, we found that Src activity decreases significantly (decrease of phosphoY416 and increase of C.J.C. Fernandes et al. / Colloids and Surfaces B: Biointerfaces 163 (2018) 321–328 327 Fig. 4. ECM remodeling-related genes and Src reprogramming are hallmarks of direct and indirect effects of titanium-modified surfaces on pre-osteoblast behavior. We focused on understanding the effect of the direct or indirect strategies on ECM-related genes and Src gene reprograming up to 14 days (a). Curiously there was an inverse behavior of Src gene activation: when direct contact promoted an increase of the gene expression (b), it significantly decreased (c) in a titanium-enriched medium (indirect way). We also investigated whether ECM remodeling-related genes were modulated and found a similar profile in the direct contact among the groups; it increased in response to DAE, for all genes investigated (d-h), while nHA stimulated an increase in comparison with Mc. With indirect contact, when titanium-enriched medium was considered, the response is even more dynamic; nHA stimulated an increase of TIMPs-1 and −2 (k,l), while MMP expression decreased (i,j) and RECK was down-regulated in response to both DAE and nHA (m). The differences were significant when p < 0.05. Fig. 5. Src is a suppressor gene of MMP expression and activities. To analyze whether Src was able to modulate MMP processing, we subjected pre-osteoblast to PP1 (5 �M) for 24 h and later the zymography was resolved by using the conditioned medium and the cell lysate used to evaluate gene expression. We found that PP1 promoted an increase of both expression (a,b) and activities (c-f). The differences were significant when p < 0.05. 3 rfaces p d d d i d e l M o 5 t b a C A ( g A R [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [ [29] A. Marumoto, R. Milani, R.A. da Silva, C.J. da Costa Fernandes, J.M. Granjeiro, C.V. Ferreira, M.P. Peppelenbosch, W.F. Zambuzzi, Phosphoproteome analysis reveals a critical role for hedgehog signalling in osteoblast morphological transitions, Bone 103 (2017) 55–63, http://dx.doi.org/10.1016/j.bone.2017.06. 012. 28 C.J.C. Fernandes et al. / Colloids and Su hosphor-Y527) during osteoblast differentiation. Since osteoblast ifferentiation requires Src inhibition and nHA promotes osteoblast ifferentiation [17], we suggested the up-expression of the Src was ue to compensation feedback, as reported earlier for Src dur- ng osteoblast adhesion (up to 24 h) and MMPs. Importantly, we emonstrated that Src is necessary to govern cytoskeletal remod- ling during osteoblast morphological changes [29]. Altogether these results demonstrate the crucial role of intracel- ular Src in ECM remodeling, and maybe a prerequisite to processing MP. Thus, it is clear that Src performs a distinct role during steoblast adhesion and differentiation. . Conclusion This study showed that the molecular hallmarks involved with he pre-osteoblast relationship with different topography-based iointerface can govern Src-dependent osteoblast metabolism as pre-requisite to ECM remodeling. ompeting financial interests The authors declare no competing financial interests. cknowledgements We would like to thank FAPESP (grant 2014/22689-3) and CNPq Proc. Nr. 301966/2015-0) for the financial support. The authors are rateful to Dr. Giselle N. Fontes for her technical assistance with the FM images and SIN Implants Co. eferences [1] K. Hamad, M. Kon, T. Hanawa, K. Yokoyama, Y. Miyamoto, K. Asaoka, Hydrothermal modification of titanium surface in calcium solutions, Biomaterials 23 (2002) 2265–2272. [2] P.G. Coelho, R. Jimbo, Osseointegration of metallic devices: current trends based on implant hardware design, Arch. Biochem. Biophys. 561 (2014) 99–108, http://dx.doi.org/10.1016/j.abb.2014.06.033. [3] Y. Dang, L. Zhang, W. Song, B. Chang, T. Han, Y. 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analysis 3 Results 3.1 Biointerface between pre-osteoblast and titanium-modified surfaces requires reprogramming of dynamic adhesion-related ... 3.2 Titanium-modified surfaces differentially orchestrate ECM remodeling-related genes 3.3 Src and ECM remodeling-related genes are hallmarks of direct and indirect effects of titanium-modified surfaces on pre... 4 Discussion 5 Conclusion Competing financial interests Acknowledgements References