The Deposition of a Lectin from Oreochromis niloticus on the Surface of Titanium Dioxide Nanotubes Improved the Cell Adhesion, Proliferation, and Osteogenic Activity of Osteoblast-like Cells

Titanium and its alloys are used as biomaterials for medical and dental applications, due to their mechanical and physical properties. Surface modifications of titanium with bioactive molecules can increase the osseointegration by improving the interface between the bone and implant. In this work, titanium dioxide nanotubes (TiO2NTs) were functionalized with a lectin from the plasma of the fish Oreochromis niloticus aiming to favor the adhesion and proliferation of osteoblast-like cells, improving its biocompatibility. The TiO2NTs were obtained by anodization of titanium and annealed at 400 °C for 3 h. The resulting TiO2NTs were characterized by high-resolution scanning electron microscopy. The successful incorporation of OniL on the surface of TiO2NTs, by spin coating, was demonstrated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIE), and attenuated total reflection-Fourier transform infrared spectrum (ATR-FTIR). Our results showed that TiO2NTs were successfully synthesized in a regular and well-distributed way. The modification of TiO2NTs with OniL favored adhesion, proliferation, and the osteogenic activity of osteoblast-like cells, suggesting its use to improve the quality and biocompatibility of titanium-based biomaterials.


Introduction
Titanium (Ti) and its alloys have been extensively applied in the fabrication of implants and prosthesis to repair and/or replace hard tissues, due to their physical characteristics such as: high mechanical strength, corrosion resistance, and good biocompatibility [1,2]. Although, TiO 2 presents several advantages, as low cost and improved biocompatibility over other biomaterials, therapeutic failure of TiO 2 -based implants and other medical devices may occur due to the ineffective bone formation and fixation, leading to bacterial infection and the implant loss. In this regard, the long-term success of titanium alloy implants is reliant on its stable fixation to the surrounding bone which, in turn, depends on the osseointegration-i.e., the formation of a direct interface between an implant and bone-without intervening soft tissue [3]. However, titanium per se lacks biological activity was purchased from ATCC Co. (Manassas, VA, USA). The kits for detection of alkaline phosphatase activity and calcium were purchased from LabTest Ltd. (São Paulo, Brazil).

Sample Preparation
Titanium samples (99.6% purity) with 0.5 mm thickness were prepared as square sheets sized 1cm 2 . The metal surface was polished utilizing a 400-grit emery paper down to 1200-grit emery paper, followed by wet polishing in a 15 µm alumina slurry. Next, the titanium samples were washed with distilled water, cleaned up with neutral detergent and sonicated for 10 min in isopropyl alcohol. Then, the samples were rinsed with deionized water and dried in a nitrogen stream. TiO 2 NTs were obtained by anodization of titanium samples by using a solution containing 89.3% ethylene glycol/0.7% ammonium fluoride in distilled water, as the electrolyte. The anodization was performed at a potential of 30V for 30 min at 2A. The time-dependent anodization current was recorded with a computer controlled Minipa ET-2076A multimeter (Minipa Co., Joinville, Brazil). After the electrochemical treatment, the samples were rinsed with deionized water for acid replacement and dried in a nitrogen flux. To crystallize the amorphous anodized TiO 2 NTs arrays into crystalline anatase phase, the samples were annealed in a furnace at 400 • C for 3 h under air atmosphere.

Coating of TiO 2 with OniL
OniL was purified from the plasma of Nile tilapia Oreochromis niloticus and characterized as previously described [14]. For the lectin adsorption, the TiO 2 NTs were preincubated for 10 min in a 10% NaOH ethanolic solution to confer a negative charge to NTs surface (Neg-TiO 2 NTs). The OniL lectin, was then diluted at 100 and 200 µg/mL in PBS. Neg-TiO 2 NTs were coated with the lectin by spin coating, using a WS-650 Mz-23 NPPPB Spin Coater (Laurell Technology Co.,North Wales, PA, USA), operating at initial spin and final speed of 500 rpm and 2000 rpm, respectively, for 6 min.

Cell Adhesion, Proliferation, and Viability Assays
Cells cultured on the modified TiO 2 NTs, coated or not with OniL-for 24 to 72 hwere assayed for adhesion and proliferation. For this, the samples were stained with rhodamine-phalloidin fluorescent probe for F-actin and counterstained with 4 ,6-Diamidine-2 -phenylindole dihydrochloride (DAPI) following the manufacturer's instructions with minor modifications. Briefly, the cells were washed twice in prewarmed phosphate-buffer saline, pH 7.4, fixed in 3.7% formaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min, at room temperature. After permeabilization, the Biomolecules 2021, 11, 1748 4 of 13 cells were washed in PBS and staining with 5 µL of rhodamine-phalloidin methanolic stock solution diluted into 200 µL PBS/sample, for 20 min at room temperature. After the incubation time, the cells were washed twice in PBS, and counterstained with 300 nM of 4 ,6-Diamidine-2 -phenylindole dihydrochloride (DAPI) diluted in PBS, for 3 min at room temperature. After incubation time, the samples were washed in PBS and visualized in a ZEISS Observer Z.1.apoTome microscope (Zeiss Co., Oberkochen, German). The number of adhered cells was estimated by counting the DAPI labeled nuclei in 10 randomly chosen field/sample (10X objective), using ImageJ 1.4.v software.

Osteogenic Potential
Cells cultured on the TiO 2 NTs were evaluated for its osteogenic potential. For this, after 24, 48, and 72 h of cultivation, the samples were washed three times in PBS and transferred for a new 24-well culture plates. The cells were lysed in a 0.5% Triton X-100 solution in PBS by three freezing/thawing cycles. The samples were collected, centrifuged at 10,000× g for 5 min and tested for ALP activity assay. ALP activity was quantified by the colorimetric alkaline phosphatase kit according to manufacturer's instructions using paranitrophenol phosphate as substrate. For calcium quantification the cells were cultured for 72 h, lysed as described above and submitted to calcium detection using calcium liquiform kit according to the manufacturer's instructions. The absorbance of the samples was read at 590 nm for alkaline phosphatase and 570 nm for calcium detection, using a Multiskan GO spectrophotometer (Thermo Co., Walthan, MA, USA).

Statistical Analysis
The data are expressed as mean ± SD of two independent experiments in triplicate and analyzed by analysis of variance (ANOVA) test followed by Bonferroni post-test or Student t-test. p < 0.05 was considered statistically significant. The statistical analyses were performed using the GraphPad Prisma 5.0.

Results and Discussion
Nowadays, modifications on the surface of titanium implants and its further functionalization with biomolecules have been investigated to improve the quality and biocompatibility of medical devices as prosthesis, and implants [6,7,21,22]. In this work, the anodization followed by thermal treatment of titanium surface efficiently produced a self-organized and homogeneous layer of TiO 2 NTs with a mean diameter 73.8 ± 8.2 nm, as confirmed by the EDS-SEM analysis ( Figure 1a). Several studies reported that the viability, proliferation, migration, and differentiation of mesenchymal and hematopoietic stem cells [22,[24][25][26][27], as well as the behavior of osteoblasts and osteoclasts [28,29] are strongly affected by the nanometric scale of TiO 2 NTs. The diameter of TiO 2 NTs obtained in our study was able to promote cell adhesion and proliferation, even in the absence of any functionalization of its surface. Our data corroborated previous studies that demonstrated an increase in the biocompatibility of TiO 2 NTs in 60-80 nm size range [25,30]. The annealing treatment after anodization changed the structure of TiO 2 NTs to the anatase, as observed by XRD analysis, according to the JCPDS file no. 21-1272. This crystallographic form of TiO 2 NTs has been reported as a highly organized structure that favors the nucleation of hydroxyapatite, the inorganic component of bone tissue, supporting the osseointegration process [7,[31][32][33][34][35].
Although the electrochemical anodization is a useful approach to improve the biocompatibility and osteogenesis, as it provides an appropriate microenvironment for the fixation of bone cells, previous studies have demonstrated the great advantages of TiO 2 surface modification with biocompatible and bioactive molecules [7,[36][37][38][39]. This procedure has been shown to reduce the postoperative infection and improve the biocompatibility and osseointegration of the implant [7]. In this work, we used the lectin OniL to coat the surface of TiO 2 NTs. This lectin is a 17 kDa protein consisting of two subunits of 11 and 6.6 kDa. This protein presents a high affinity for methyl-α-D-mannopyranoside and D-mannose [14]. In mammals, mannose-binding lectins (MBLs) constitute a fundamental link between the innate immune system and other functions, such as coagulation, homeostasis after injury and defense against microorganisms [40]. As the success of the implant and prosthesis is intrinsically associated with the above-mentioned processes, we hypothesized whether the functionalization of TiO 2 NTs with OniL could improve the cell adhesion and proliferation, compared to the bare TiO 2 NTs. For this, we first incubated TiO 2 NTs with NaOH solution (pH 13) to charge them negatively improving the adsorption of OniL on its surface. According to Bavykin et al. [41] negatively charged nanotubes (Neg-TiO 2 NTs) promote electrostatic interactions with cations, presenting an excellent matrix for protein binding [42]. After being negatively charged, the samples were subjected to spin coating to immobilize OniL on the surface of Neg-TiO 2 NTs. The spin coating is a useful technique to fast and easily create a homogenous film with desired and well-controlled thickness [43]. As observed for the bare TiO 2 NTs the SEM analysis showed that the deposition of OniL on the surface of these nanotubes did not alter its morphology (Figure 1b). The elucidation of the chemical composition of these TiO 2 NTs by EDS showed the presence of Ti and O, demonstrating the successful anodization process, as well as the absence of sample contaminants ( Figure 1c). procedure has been shown to reduce the postoperative infection and improve the biocompatibility and osseointegration of the implant [7]. In this work, we used the lectin OniL to coat the surface of TiO2NTs. This lectin is a 17 kDa protein consisting of two subunits of 11 and 6.6 kDa. This protein presents a high affinity for methyl-α-D-mannopyranoside and D-mannose [14]. In mammals, mannose-binding lectins (MBLs) constitute a fundamental link between the innate immune system and other functions, such as coagulation, homeostasis after injury and defense against microorganisms [40]. As the success of the implant and prosthesis is intrinsically associated with the above-mentioned processes, we hypothesized whether the functionalization of TiO2NTs with OniL could improve the cell adhesion and proliferation, compared to the bare TiO2NTs. For this, we first incubated TiO2NTs with NaOH solution (pH 13) to charge them negatively improving the adsorption of OniL on its surface. According to Bavykin et al. [41] negatively charged nanotubes (Neg-TiO2NTs) promote electrostatic interactions with cations, presenting an excellent matrix for protein binding [42]. After being negatively charged, the samples were subjected to spin coating to immobilize OniL on the surface of Neg-TiO2NTs. The spin coating is a useful technique to fast and easily create a homogenous film with desired and wellcontrolled thickness [43]. As observed for the bare TiO2NTs the SEM analysis showed that the deposition of OniL on the surface of these nanotubes did not alter its morphology (Figure 1b). The elucidation of the chemical composition of these TiO2NTs by EDS showed the presence of Ti and O, demonstrating the successful anodization process, as well as the absence of sample contaminants (Figure 1c). . This result demonstrates that the lectin was efficiently immobilized on the surface of the samples. The bare TiO2NTs showed a Rct value of 1.7 kΩ. For negatively charged nanotubes, this resistance increased to 3.2 kΩ. The adsorption of OniL to TiO2NTs substantially increased the Rct value to 24.2 kΩ, whereas causing a simultaneous decrease in Cd to 6.89 μF. The impedance parameters, adjusted to the Randles equivalent circuit, are shown in Table 1. The adsorption of OniL on the TiO 2 surface was monitored by electrochemical impedance spectroscopy (EIS) using K4 [Fe(CN) 6 ]/K 3 [Fe(CN) 6 ] (1:1) as redox pair. The Figure 2 shows that each step of lectin immobilization generates a blockage in the transfer of electrons, increasing the resistance value (Rct). This result demonstrates that the lectin was efficiently immobilized on the surface of the samples. The bare TiO 2 NTs showed a Rct value of 1.7 kΩ. For negatively charged nanotubes, this resistance increased to 3.2 kΩ. The adsorption of OniL to TiO 2 NTs substantially increased the Rct value to 24.2 kΩ, whereas causing a simultaneous decrease in Cd to 6.89 µF. The impedance parameters, adjusted to the Randles equivalent circuit, are shown in Table 1.
The capacitive behavior observed in the Nyquist plots (Figure 2) was demonstrated by the presence of a double electrochemical layer at the electrode-solution interface, and the dielectric nature of TiO 2 [44].
The ATR-FTIR analysis of TiO 2 and Neg-TiO 2 NTs corroborated our electrochemical data. Our results revealed the presence of one absorption band peak characteristic of Ti-O vibration in the region of 400-800 cm −1 . The presence of OniL lectin can be confirmed by the appearance of the two main stretches, corresponding to the lectin amide groups in 1643 cm −1 [45]/1025 cm −1 and 1456 cm −1 /1010 cm −1 [46] (Figure 3). The secondary protein structures are usually identified by analyzing the vibration of amide I (1700-1600 cm −1 ), mainly due to the C=O elongation and amide II (1600-1500 cm −1 ), with minor contributions from C-N elongation and N-H [45,46].  The capacitive behavior observed in the Nyquist plots (Figure 2) was demonstrated by the presence of a double electrochemical layer at the electrode-solution interface, and the dielectric nature of TiO2 [44].
The ATR-FTIR analysis of TiO2 and Neg-TiO2NTs corroborated our electrochemical data. Our results revealed the presence of one absorption band peak characteristic of Ti-O vibration in the region of 400-800 cm -1 . The presence of OniL lectin can be confirmed by the appearance of the two main stretches, corresponding to the lectin amide groups in 1643 cm -1 [45]/1025cm -1 and 1456 cm -1 /1010 cm -1 [46] (Figure 3). The secondary protein structures are usually identified by analyzing the vibration of amide I (1700-1600 cm −1 ), mainly due to the C=O elongation and amide II (1600-1500 cm -1 ), with minor contributions from C-N elongation and N-H [45,46].
In a previous work, the lectin Cramoll from seeds of Cratylia mollis bean was efficiently immobilized on the surface of anodized TiO2 nanotubes using layer-by-layer (LbL) technique [21]. This technique consists in the growth of alternated layers of poly (allylamine hydrochloride) (PAH) and poly(acrylic)acid (PAA). This self-assembling process occurs by the adsorption of oppositely charged polyelectrolytes on the surface of TiO2NTs [21]. Although layer-by-layer was proved to be useful to adsorb Cramoll lectin on the surface of TiO2NTs, this technique should be more expensive and time-consuming. In the present work, the lectin OniL was directly bound onto Neg-TiO2NTs without the need of any additional functionalization step, remaining strongly attached to the surface of negative charged TiO2NTs.   The behavior of osteosarcoma cells in response to the OniL-decorated nanotubes was investigated (Figure 4). This osteoblast-derived lineage is widely used as a cell model to investigate the osseointegration on the surface of nanomaterials in vitro [47,48]. To evaluate the human osteosarcoma cells' attachment on the TiO2-modified nanotubes, the cells were labeled with rhodamine-phalloidin, a fluorescent probe for actin, emitting fluorescence at the red channel. The quantification of the adhered cells was performed by count- In a previous work, the lectin Cramoll from seeds of Cratylia mollis bean was efficiently immobilized on the surface of anodized TiO 2 nanotubes using layer-by-layer (LbL) technique [21]. This technique consists in the growth of alternated layers of poly (allylamine hydrochloride) (PAH) and poly(acrylic)acid (PAA). This self-assembling process occurs by the adsorption of oppositely charged polyelectrolytes on the surface of TiO 2 NTs [21]. Although layer-by-layer was proved to be useful to adsorb Cramoll lectin on the surface of TiO 2 NTs, this technique should be more expensive and time-consuming. In the present work, the lectin OniL was directly bound onto Neg-TiO 2 NTs without the need of any additional functionalization step, remaining strongly attached to the surface of negative charged TiO 2 NTs.
The behavior of osteosarcoma cells in response to the OniL-decorated nanotubes was investigated (Figure 4). This osteoblast-derived lineage is widely used as a cell model to investigate the osseointegration on the surface of nanomaterials in vitro [47,48]. To evaluate the human osteosarcoma cells' attachment on the TiO 2 -modified nanotubes, the cells were labeled with rhodamine-phalloidin, a fluorescent probe for actin, emitting fluorescence at the red channel. The quantification of the adhered cells was performed by counting the cell nuclei labeled with DAPI, which specifically binds to nucleic acids, emitting fluorescence at the blue channel. Our results showed that the deposition of OniL on the surface of TiO 2 NTs did not exhibited cytotoxicity and were able to significantly improve the cell percentage of adhered cells on the NTs. The osteosarcoma cells cultured on both the bare TiO 2 NTs and OniL-TiO 2 NTs (at 100 and 200 µg/mL) for 24 h showed a typical distribution of the actin filaments with the formation of focal adhesion points, an essential characteristic to maintain the shape, migration, and proliferation of the cells on the substrate [49]. At this time, there is a predominance of cells with spindle morphology and cell-cell interactions could be easily observed. However, the existence of empty spaces in the bare TiO 2 NT samples indicates a low rates of cell proliferation. After 48 and 72 h a confluent monolayer was observed in all TiO 2 NT preparations.
In the OniL-TiO 2 NTs samples, it was possible to observe that cells presented a more flattened extended phenotype and an increased spreading on the TiO 2 surface. Interestingly, by 48 h of cultivation, the cells on the TiO 2 NTs decorated with 200 µg/mL of OniL begun to orient themselves in a more organized concentric manner in comparison to the those cultivated on the bare TiO 2 NTs (Figure 4a). Violin et al. [50], using the lectinhistochemistry methodology, evaluated the differential expression of surface glycoconjugates in a rabbit's tibia implanted with microporous biphasic ceramic material. These authors showed that the lectin binding pattern during bone formation changed, corroborating the role of differential expression of glycoconjugates and its putative recognition by lectins during the osseointegration process. After 72 h of cultivation, the cell morphology remained preserved and strict cell-cell contacts could be observed in all modified TiO 2 NTs. The orientated organization of osteosarcoma cells observed on the OniL-treated surfaces, compared to bare TiO 2 NTs, may reflect what happens in vivo, showing the importance of carbohydrate recognition during osteogenesis on the surface of implants and prothesis.
The quantification of DAPI-labeled nuclei demonstrated a significant increase in the cell adhered to TiO 2 NTs in the samples treated with OniL for 24 and 48 h compared to the bare TiO 2 NTs (Figure 4b). The deposition of OniL on the surface of TiO 2 NTs was able not only to significantly increase the percentage of adhered cells by approximately 50%, but also stimulate their proliferation and differentiation on the TiO 2 NTs, in both concentrations tested, compared to the control group (TiO 2 NTs). After 72 h of cultivation, the percentage of adhered cells decreases in all samples at levels compared to the TiO 2 NTs (Figure 4b). This behavior is also observed in the human osteosarcoma cultures maintained in culture plates under standard conditions for up 48 h (data not shown). The decrease of percentage of adhered cells can be explained by the osteosarcoma cells commitment to osteogenesis rather than to the proliferation process. also stimulate their proliferation and differentiation on the TiO2NTs, in both concentrations tested, compared to the control group (TiO2NTs). After 72 h of cultivation, the percentage of adhered cells decreases in all samples at levels compared to the TiO2NTs (Figure 4b). This behavior is also observed in the human osteosarcoma cultures maintained in culture plates under standard conditions for up 48 h (data not shown). The decrease of percentage of adhered cells can be explained by the osteosarcoma cells commitment to osteogenesis rather than to the proliferation process. To investigate whether the deposition of OniL on TiO 2 NTs was able to induce the osteogenesis, we examined the activity of alkaline phosphatase (ALP) (Figure 5a). Herein, we clearly demonstrated that the coating of NTs with OniL favored the rapid colonization of the substrate, allowing cell proliferation and osteogenic activity. Osteosarcoma cells cultured on OniL decorated nanotubes, showed a significant increase in the ALP activity as compared with TiO 2 NTs, mainly at 200 µg/mL of OniL. The highest increase was observed for OniL group after 48 h with values of ALP activity of 1.5 U/L (TiO 2 NTs), 3.2 U/L (OniL100 µg/mL), and 5.0 U/L (OniL200 µg/mL) (Figure 5a). A study by Ikeda et al. [51] showed that HOS cells cultivated on plastic culture plates presented ALP activity only after three weeks of cultivation. On the other hand, Min et al. [52] have demonstrated that the coating of TiO 2 NTs with laminin-derived functional peptides promotes HOS adhesion and ALP activity within the first 24 h of cultivation. Accordingly, we also showed that the modification of TiO 2 NTs with OniL was able to early trigger the osteogenesis processes in HOS cells. Taken together, these results showed that the adsorption of active biomolecules on the surfaces of biomaterials plays an important role in the induction of the osteogenesis process. Calcium, one of the main elements involved in the bone tissue remodeling, was also quantified after 72 h (Figure 5b). All the samples from the OniL groups presented a significant increase in the amount of calcium compared to the bare TiO 2 NTs samples. No statically significant differences could be observed between the lectin treatments ( Figure 5b).
To investigate whether the deposition of OniL on TiO2NTs was able to induce the osteogenesis, we examined the activity of alkaline phosphatase (ALP) (Figure 5a). Herein, we clearly demonstrated that the coating of NTs with OniL favored the rapid colonization of the substrate, allowing cell proliferation and osteogenic activity. Osteosarcoma cells cultured on OniL decorated nanotubes, showed a significant increase in the ALP activity as compared with TiO2NTs, mainly at 200 μg/mL of OniL. The highest increase was observed for OniL group after 48 h with values of ALP activity of 1.5 U/L (TiO2NTs), 3.2 U/L (OniL100 μg/mL), and 5.0 U/L (OniL200 μg/mL) (Figure 5a). A study by Ikeda et al. [51] showed that HOS cells cultivated on plastic culture plates presented ALP activity only after three weeks of cultivation. On the other hand, Min et al. [52] have demonstrated that the coating of TiO2NTs with laminin-derived functional peptides promotes HOS adhesion and ALP activity within the first 24 h of cultivation. Accordingly, we also showed that the modification of TiO2NTs with OniL was able to early trigger the osteogenesis processes in HOS cells. Taken together, these results showed that the adsorption of active biomolecules on the surfaces of biomaterials plays an important role in the induction of the osteogenesis process. Calcium, one of the main elements involved in the bone tissue remodeling, was also quantified after 72 h (Figure 5b). All the samples from the OniL groups presented a significant increase in the amount of calcium compared to the bare TiO2NTs samples. No statically significant differences could be observed between the lectin treatments ( Figure  5b). The osteoblasts cells are responsible for the synthesis, deposition, and mineralization of the bone extracellular matrix. In this process, the production of alkaline phosphatase is one of the parameters used to assess the effects of a biomaterial on the bone tissue activity [5]. The increased activity of this enzyme indicated the induction of the biomineralization The osteoblasts cells are responsible for the synthesis, deposition, and mineralization of the bone extracellular matrix. In this process, the production of alkaline phosphatase is one of the parameters used to assess the effects of a biomaterial on the bone tissue activity [5]. The increased activity of this enzyme indicated the induction of the biomineralization process. In addition, during the growth and remodeling of adult bone, osteoblasts secrete calcium-rich vesicles to the calcifying osteoid [53,54]. As expected, the increase in ALP activity (Figure 5a) was followed by the simultaneous enhancement in the calcium deposition ( Figure 5b) [54,55]. Both ALP activity and calcium deposition, observed in our study, are indicative of metabolically active and viable cells. Previous studies showed that mannose-binding lectins as Lens culinaris (lentil) lectin (LcL) and Narcissus pseudonarcissus (daffodil) lectin (NpL), a α-d-mannose-binding protein, did not elicit potent cytotoxicity against osteosarcoma cells [56]. Furthermore, a study by da Silva et al. [57] showed that OniL was able to favor the proliferation of Balb/c splenocytes without causing significant cytotoxicity to these cells. Furthermore, our fluorescence microscopy assay showed that the cells remained adhered to the surface of TiO 2 NTs, presenting a preserved morphology and nuclei integrity throughout the experiments. Although the improvement in the cell proliferation and adhesion on TiO 2 NTs seems to be a natural consequence of the ability of OniL to recognize carbohydrates on the surface of HOS, some issues-such as the speed of this process, the specificity of lectin, and mainly the fate of adhered cells after binding to the lectin-should be taken in account. Marty-Detraves et al. [58], for example, showed that the deposition of a lectin from the mushroom Xerocomus chrysenteron (XcL) inhibited the cell-substrate adhesion and proliferation of the adherent cell lines NIH-3T3 and HeLa cells, but not of the non-adherent SF9 cells. Interesting, XcL did not interfere in the cell cycle or induced apoptosis to these cells. On the other hand, the lectin from Bauhinia forficata (BfL) inhibited integrin-mediated adhesion of MCF7 human breast cancer cells inducing the cells to death [59].
Besides its role in the adhesion and differentiation of osteosarcoma cells, the deposition of OniL on the surface of TiO 2 NTs may have other beneficial consequences. Due to its immunomodulatory role, this protein can regulate the local inflammatory response, assisting in the bone healing and regeneration. Lectins can bind to the cell surface carbohydrates and trigger various cell events, such as stimulation of cell proliferation. In our study, we used an inexpensive and faster methodology to absorb OniL on TiO 2 NTs without the need for intermediate polymers.

Conclusions
In this study, we successfully functionalized TiO 2 NTs with the lectin OniL by using the spin coating methodology. The osteosarcoma cells cultivated on the surface of OniLdecorated TiO 2 NTs presented an improved adhesion and proliferation. The OniL also promoted an increase in both the deposition of calcium and ALP activity, which is indicative of enhanced osteogenic activity compared to bare TiO 2 NTs. The rapid colonization of HOS on the surface of OniL-treated TiO 2 NTs can prevent bacteria from forming biofilm on its surface, improving the chances of implant success. Although further studies are still needed to better understand the nature of OniL-TiO 2 NTs/osteoblast interactions, our results indicate that OniL could enhance the biocompatibility of TiO 2 NTs-based medical devices, assisting in the osseointegration between the bone and TiO 2 NTs surfaces.

Acknowledgments:
We would like to thank the Centro de Tecnologias Estratégicas do Nordeste (CETENE) for the use of their microscopy facilities.

Conflicts of Interest:
The authors declare that they have no known competing financial interest or personal relationship that could have appeared to influence the work reported herein.