The deposition of a lectin from Oreochromis niloticus on the surface of titanium dioxide nanotubes improve the cell adhesion, proliferation, and osteogenic activity of osteoblast-like cells

Titanium and its alloys are used biomaterials for medical and dental applications, due to their mechanical and physical properties. The surface modifications of titanium with bioactive molecules can increase the osseointegration by improving the interface between the bone and implant. Titanium dioxide nanotubes (TiO 2 NTs) have excellent bioactivity inducing cell adhesion, spreading, growth and differentiation. In this work, TiO 2 NTs were functionalized with a lectin from the plasma of the fish Oreochromis niloticus aiming to favour the adhesion and proliferation of osteoblast-like cells, improving its biocompatibility. The TiO 2 NTs were obtained by anodization of titanium and annealed at 400 °C for 3 h. The resulting TiO 2 NTs were characterized by high-resolution scanning electron microscopy. The successfully incorporation of OniL on the surface of TiO 2 NTs 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 TiO 2 -NTs were successfully synthesized in a regular and well-distributed way. The functionalization of TiO 2 -NTs with OniL favoured 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, TiO2 presents several advantages, as low cost and improved biocompatibility over other biomaterials, therapeutic failure of TiO2-based implants and other medical devices may occur due to 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]. Furthermore, titanium per se lack biological activity and cannot significantly promote the cell adhesion and tissue healing. There is a consensus that the surface topography, morphology, chemical composition, and surface energy of titanium play a critical role on cell adhesion, proliferation, production, and maintenance of extracellular matrix during the osseointegration process [4].
Therefore, physical, and chemical modifications of the titanium surfaces have been developed to create more suitable interfacial microenvironment, promoting cell-material interactions and osseointegration [5,6]. Another way to improve the osseointegration can be achieved throughout the functionalization of implant material surfaces with biomolecules [7].
Lectins, a non-immune proteins, which bind reversibly and in a highly specific manner to simple or complex carbohydrates, have been widely investigated due to its prominent role in several physiological and pathological processes, including immune response, inflammation, cell-cell communication, recognition, and differentiation [8]. It has been demonstrated that lectins from plants and animals have mitogenic [9,10], antibacterial [11,12], immunomodulatory [13,14] antithrombotic [15] and healing [16] activities. Some of these activities are remarkably interesting from the point of view of osseointegration and tissue repair.
The lectin OniL, from the plasma of Nile tilapia Oreochromis niloticus, is a mannose recognition lectin with mitogenic and immunomodulatory activities already proven in the literature [17]. These features make OniL a promising functionalizer of TiO2 surfaces. OniL is a C-type lectin that depends on Ca 2+ for carbohydrate recognition. This class of lectins has demonstrated important roles on the bone biology and pathogenesis. Clec11a, also known as the stem cell growth factor (SCGF) or osteolectin 1, for example, has been identified as a growth factor able to promote the proliferation and differentiation of hematopoietic stem/progenitor cells. The messenger RNA for this protein is abundantly expressed in proliferating chondrocytes, the primary ossification centre, perichondrium and periosteum [18]. Furthermore, it has been shown that secreted blood mannose-binding lectins (MBLs) of fishes, as well as other organisms, are important components of innate immunity, playing a crucial role in the body defence [17]. MBL is a constituent of the lectin pathway of complement system activation, one of main components of innate immunity. This lectin plays an essential role in the defence against infectious microorganisms and maintenance of bone the homeostasis [19] and participates in different stages of bone healing.
Previous studies have demonstrated that a lectin from seed of leguminous plant Cratylia mollis was efficiently immobilized on the nanotubular surface of TiO2 nanotubes enhancing the adhesion of osteoblast-like cells [20]. In the present study we explore, for the first time, the potential of an animal-derived lectin with mannose-binding specificity, as a functionalizer agent of TiO2 nanotubes. For this the TiO2 nanotubes are produced by anodic oxidation followed by thermal treatment [21,22]. The TiO2NTs were then negatively charged to improve the binding of OniL. The deposition of OniL on the surface of TiO2NTs was performed by spin coating.
This technique is a solution-based process developed for low-cost deposition of thin films of molecules over a substrate surface [23]. The functionalized TiO2 nanotubes were characterized and investigated for adhesion and osteogenic activity in osteoblast-like cells.

Materials and methods
2.1 Sample preparation -Titanium samples (99.6% purity, Realum Ltd.) with 0.5 mm thickness were prepared as square sheets sized 1 × 1 cm. The metal surface was polished utilizing a 400grit 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. Then, the samples were rinsed with deionized water and dried in a nitrogen stream. TiO2 nanotubes (TiO2NTs) were obtained by anodization of titanium samples by using a solution containing 89.3% ethylene glycol / 0.7% ammonium fluoride /10% 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. 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 TiO2NTs arrays into crystalline anatase phase, the samples were annealed in a furnace at 400 °C for 3 h under air atmosphere.   2.5. Osteogenic potential-Cells cultured on the TiO2NTs were evaluated for its osteogenic potential. For this, after 24, 48 and 72 hours 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 solution in PBS by three freezing/thawing cycles. The samples were collected, centrifuged at 10,000 x g for 5 min and tested for ALP activity assay. ALP activity was quantified by the colorimetric alkaline phosphatase kit (LabTest, Brazil) according to manufacturer's instructions using para-nitrophenol phosphate as substrate. For calcium quantification the cells were cultured for 72 hours, lysed as described above and submitted to calcium detection using Calcium liquiform kit (LabTest, Brazil) according to 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 Scientific, USA).

Statistical analysis-
The data are expressed as mean ± SD of two independent experiments in triplicate and analysed using analysis of variance (ANOVA) test followed by Bonferroni post-test or t-Student 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 TiO2 nanotubes with a mean diameter 73.8 ± 8.2 nm, as confirmed by the EDS-SEM analysis (Fig 1a). Several studies reported that the viability, proliferation, migration, and differentiation of mesenchymal and hematopoietic stem cells, as well as the behaviour of osteoblasts and osteoclasts are strongly affected by the nanometre scale of TiO2NTs [21,22]. The diameter of TiO2-NTs obtained in our study was able to promote cell adhesion and proliferation, even without any functionalization of its surface.
Our data corroborated previous studies that demonstrated an increase in the biocompatibility of TiO2NTs in 60-80 nm size range [24,25]. The annealing treatment after anodization changed the structure of TiO2-NTs to the anatase, as observed by XRD analysis, according to the JCPDS file No. 21-1272. This crystallographic form of TiO2NTs has been reported as a highly organized structure that favours the nucleation of hydroxyapatite, the inorganic component of bone tissue, supporting the osseointegration process [7,[26][27][28][29][30]. 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 TiO2 surface functionalized with biocompatible and bioactive molecules [7,[31][32][33][34]. 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 functionalize the surface of TiO2NTs. This lectin is a 17 kDa protein consisting of two subunits of 11 and 6.6 kDa and presents a high affinity for methyl-α-Dmannopyranoside 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 defence against microorganisms [35]. As the success of the implant and prosthesis is intrinsically associated with the processes mentioned above, we hypothesized whether the functionalization of TiO2 with OniL could improve the cell adhesion and proliferation compared to the bare TiO2NTs. For this, we first incubated TiO2NTs with NaOH solution (pH 13,0) to charge them negatively improving the adsorption of OniL on its surface. According to Bavykin et al. [36] negatively charged nanotubes (Neg-TiO2NTs) promote electrostatic interactions with cations, presenting an excellent matrix for protein binding [37]. 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 well-controlled thickness [38]. 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. (Fig1b). 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 (Fig.1c).   Table 1.  In a previous work, the lectin Cramoll from seed of Cratylia mollis bean was efficiently immobilized on the surface of anodized TiO2 nanotubes using Layer-by-Layer (LbL) technique [20]. 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 adsorption of oppositely charged polyelectrolytes on the surface of TiO2NTs [20]. 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. In the OniL-TiO2NTs samples was possible to observe that cells presented a more flattened extended phenotype and an increased spread on the TiO2 surface. Interestingly, by 48 hours of cultivation, TiO2NTs decorated with 200 g/mL of OniL begun to orient themselves in a more organized concentric manner in comparison to the bare TiO2NTs (Fig. 4a). Violin et al [42], using the lectinhistochemistry methodology, evaluated the differential expression of surface glycoconjugates in 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  (Fig. 4b).
The functionalization of NTs with OniL was able not only to significantly increase the number Previous studies showed that mannose-binding lectins as Lens culinaris (lentil) lectin (LCL) and α-d-mannose-binding Narcissus pseudonarcissus (daffodil) lectin (NPA) did not elicit potent cytotoxicity against osteosarcoma cells [43]. To show that the adhered cells were metabolically active and viable, we quantified the amount of ATP produced by them (Fig. 4c).
All the samples actively produced ATP, indicating that the adhered cells were still viable over time. However, only in the cells cultured on OniL-TiO2NTs at 200 µg/mL there was a significant increase of cell viability compared to TiO2NTs. These data corroborate the fluorescence microscopy analysis which showed a peak in the percentage of adhered cells at this concentration at 48 hours of cell cultivation (Figure 4c). To investigate whether the functionalization of TiO2 with OniL was able to induce the osteogenesis, we examined the activity of alkaline phosphatase (ALP) (Fig.5a). Herein, we clearly demonstrated that the functionalization of NTs with OniL favoured 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g/mL of OniL. The highest increase was observed for OniL group after 48 hours with values of ALP activity of 1.5 U/L (TiO2NTs), 3.2 U/L (OniL100g/mL) and 5.0 U/L (OniL200g/mL) (Fig. 5a). Calcium, one of the main elements involved in the bone tissue remodelling was also quantified after 72 hours (Fig.5b). All the samples from the OniL group 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 (Fig.5b). The osteoblasts and osteoblastic 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 effect of biomaterial on bone tissue activity [5]. The increased activity of this enzyme indicated the induction of the biomineralization process. In addition, during growth and remodelling of adult bone, osteoblasts secrete calcium-rich vesicles to the calcifying osteoid [20,[44][45]. As expected, the increase in ALP activity observed (Fig 5a) was followed by the simultaneous enhancement in the calcium deposition (Fig. 5b) [45][46]. Besides its role in the adhesion and differentiation of osteosarcoma cells, the deposition of OniL on the surface of TiO2NTs 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 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 absorbed OniL on TiO2NTs without the need for intermediate polymers.

Conclusions
In this study we successfully functionalized TiO2NTs with the lectin OniL, by using spin coating methodology. Osteosarcoma cells cultivated on the surface of OniL-decorated TiO2NTs 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 an enhanced osteogenic activity compared to the bare TiO2NTs. Although further studies are still needed to better understand the nature of OniL-TiO2NTs/osteoblast interactions, our results suggest that OniL could enhance the biocompatibility of medical devices based on TiO2NTs, assisting in the osseointegration between the bone and TiO2NTs surfaces.

Declaration of Competing 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.