Fibroblast Cell Responses to Vanadium and Niobium Titanium Alloys: A Biocompatibility Study

The interactions of a biomaterial with tissues must be determined for the material to be fully compatible with the body for a long time. The tissue and environment where the material is implanted are highly affected by its content. Titanium-6Aluminum-4Vanadium is widely used in orthopedics and dentistry. Recently, Titanium-6Aluminum-7Niobium alloys have been studied because of Titanium-6Aluminum-4Vanadium toxicity, which may be caused by vanadium. The aim of this study was to determine whether Titanium-6Aluminum-4Vanadium and Titanium-6Aluminum-7Niobium affect fibroblast cell proliferation, mineralization, and collagen production and whether they change the expression of type 1 collagen and fibronectin genes. It was determined that the niobium-containing alloy increased cell proliferation and calcium mineralization compared with the vanadium-containing alloy (p < 0.05). However, the alloys did not cause changes in the expression of collagen type 1 or fibronectin in cells. The collagen content of the cells on the niobium-containing alloy was lower than that on both the vanadium-containing alloy and tissue culture plate surface (p < 0.05). The niobium-containing alloy was found to be superior to the vanadium-containing alloy in terms of cell proliferation and calcium mineralization. Furthermore, neither vanadium-containing alloy nor niobium-containing alloy implant materials altered gene expression. Although both alloys are considered compatible with bone tissue, it should be considered whether they are also biocompatible with fibroblast cells.


INTRODUCTION
Titanium and its alloys are commonly used in orthopedics and dentistry and are typically expected to have a long-lasting life.However, implantation fails when it is not sufficiently biointegrated with the bone and/or surrounding tissues. 1 After placing a biomaterial in the body, its interaction with the surrounding cells/tissues needs to be evaluated to determine whether the biomaterial is biocompatible and/or whether the healing process is successful. 2,3he biocompatibility of a material is determined by a number of functions such as proliferation, adhesion, and tissue formation of host body cells in contact with the material. 4,5Calcium, which is involved in many cellular functions, is a secondary messenger molecule that regulates cell proliferation, migration and cell death. 6,7Concurrently, the extracellular level of calcium is also important, as it is the main component of the extracellular matrix (ECM), mineralization, and osseointegration. 8Although many studies on orthopedic implants have focused on osseointegration before fibro integration 2,4,9 soft-tissue formation around the implant is as critical as osseointegration.Collagen and fibronectin, which are large components of the ECM, also play a role in soft tissue formation and are produced by fibroblast cells. 10−13 Vanadium and its derivatives are ultratrace elements that are structural analogues of phosphates and are thought to be essential for living organisms. 14Although vanadium-containing Titanium-6Aluminum-4Vanadium (Ti6Al4V) is the most commonly used alloy for medical implants, there are still many unresolved questions regarding the effects of its components on living tissue.Despite the excellent corrosion properties and mechanical strength of Ti6Al4V, the released metal ions may cause long-term problems, 15−18 and also, titanium alloys with V content are more expensive than those without V. 17 Recently, niobium-containing Ti alloys have been proposed as alternatives to Ti6Al4V. 18−20 However, there is limited information on the biological responses of the Ti6Al7Nb alloy. 21n the present study, fibroblasts were cultured on Ti6Al4V and Ti6Al7Nb surfaces in vitro, and the effects of Ti alloys on cell proliferation, calcium mineralization, and total collagen content were investigated.Furthermore, type 1 collagen (COL1) and fibronectin (FN) gene expressions were investigated to understand the functionalities of the cells.

MATERIALS AND METHODS
2.1.Sample Preparation, Cell Culture, and Proliferation Assay.The commercially available Ti alloys used in this study are Ti6Al4V and Ti6Al7Nb in hot rolled and annealed discs with 10 mm diameter and 2 mm thickness.The chemical compositions (wt %) of materials provided by their manufacturers are given in Table 1.Specimens were ground with 220, 400, 800, 1200, and 2000 grade silicon carbide emery papers to obtain clean and homogeneous surfaces.The obtained average surface roughness (Ra) of materials were 0.48 ± 0.04 μm for Ti6Al4V and 0.51 ± 0.06 μm for Ti6Al7Nb.The discs were sterilized by autoclaving at 121 °C for 2 h.
The mouse fibroblast cell line-L929 (ATCC CCL-1) was cultured in Dulbecco's Modified Eagle's Medium (Gibco,USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA), 2 mM glutamine (Sigma Chemical Co., St. Louis, MO, USA), and 1% penicillin/streptomycin solution (Gibco,USA) in a humidified atmosphere containing 5% CO 2 at 37 °C.Fibroblasts were seeded on 24-well tissue culture plate (Sigma-Aldrich, St Louis, MO, USA) surfaces (TCPS) and Ti alloy discs at a density of 40,000 cells/well.The cytotoxicity assays were performed in six separate experiments (n = 6).Well plates containing the cells were incubated for 24, 48, and 72 h.TCPS was used in all experiments as control samples.Cell proliferation was measured using an Alamar blue assay.After each incubation period, the medium was replaced with fresh medium supplemented with 10% PBS containing resazurin sodium salt (R7017, Sigma, USA).After 3 h of incubation at 37 °C in a humidified atmosphere, the optical density of the medium was read at 570 nm using an automated microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Mineralization Assay.
Plates were washed with cold PBS and fixed for 1 h at 4 °C in cold 70% ethanol (sample size n = 9).After washing twice with sterile water, alizarin red solution (40 mM, pH 4.2) (Chembio, Germany) was added, and the cells were stained for 30 min. 22After staining, the wells were rinsed with PBS and a solution containing 15% acetic acid and 20% methanol was added to solubilize the precipitate.The alizarin red S should solubilize into the extraction buffer, resulting in a color change from red to yellow.The optical density of the solution was measured at 405 nm wavelength. 23.3.Total Collagen Quantification Assay.The collagen content in cells (sample size n = 6) was measured using a Sirius Red/Fast Green collagen staining kit (Chondrex Inc., USA).The medium was removed, and the wells were washed with 1× PBS.One milliliter of Kahle fixative (60 mL of distilled water, 28 mL of 96% ethanol, 10 mL of 37% formaldehyde, and 2 mL of glacial acetic acid) was added, and the cells were incubated for 10 min at room temperature.A dye solution (0.2−0.3 mL) was added and incubated for 30 min at room temperature.The dye solution was aspirated, and the wells were rinsed with distilled water (0.5 mL of distilled water.A dye extraction buffer (1 mL) was added to each sample and mixed gently by pipetting until the color was eluted.The OD values were read at 540 and 605 nm using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
2.4.Type 1 Collagen and Fibronectin Gene Expressions.RNA was isolated from cells (n = 6) treated with alloys using a PureLink RNA Mini Kit (Thermo Fisher Scientific, USA).The purity and concentration of the isolated RNAs were determined using a spectrophotometer (Thermo MultiskanGo, Drop Plate, USA).Complementary DNA (cDNA) was obtained using the RT2 HT First Strand kit (Qiagen) using 100 ng/μL of RNA in cell groups.
cDNA synthesis was carried out using a PCR (Techne) device by incubating at 42 °C for 5 min, 42 °C for 15 min, and 95 °C for 5 min.Expression levels of type 1 collagen (COL1A1) and fibronectin genes were determined using a Magnetic Induction Cycler (Mic qPCR) device with an RT2 SYBR Green FAST Mastermix kit.
The qRT-PCR conditions were as follows: 1 cycle at 95 °C for 10 min, 40 cycles at 95 °C for 15 s, 55 °C for 35 s, and 72 °C for 30 s.The qRT-PCR reaction was prepared using 12.5 μL of Mastermix, 1.25 μL of forward primer (10 μM), 1.25 μL of reverse primer (10 μM), 2 μL of cDNA, and 8 μL of nucleasefree water.F-R-primer sequences were used for fibronectin, while F-R-primer sequences were used to determine collagen gene expression.
The GAPDH gene was selected as the housekeeping gene, and F-R-sequences were used.Target genes were normalized to GAPDH gene and gene expression levels, and Cq values were calculated according to the formula 2 −ΔΔCt = 2^(−(treated Ct target gene − treated Ct housekeeping gene) − (untreated Ct target gene − untreated Ct housekeeping gene)). 24he control group that was not incubated with the alloy was used as the calibrator, and the gene expression level was calculated according to this group.No template control (NTC) was read that did not contain the cDNA template in each read performed by qRT-PCR.

Statistical Analysis.
One-way analysis of variance (ANOVA) was applied, and a Tukey-b test was used for multiple comparisons.Statistical calculations were conducted using the IBM SPSS 26 program (SPSS Inc., Chicago, IL, USA).Values were normalized to the tissue culture polystyrene surface control and represented as a percentage of the mean ± standard deviation (SD).The mean difference was significant at the level of 0.05.

RESULTS
An Alamar blue assay was used to measure the effect of surfaces on fibroblasts, and the proliferation on surfaces is presented in Figure 1.Although the alloys used did not support cell proliferation at 24 h, they promoted proliferation at 48 h compared to the control.At 72 h, the niobium-containing alloy surface TCPS had a significantly greater proliferative effect than the vanadium-containing Ti alloy (p < 0.05).
Alizarin red quantification indicated that the calcium mineralization of the cells on Ti6Al4V was statistically lower at the 48th and 72nd hours compared to Ti6Al7Nb and TCPS (p < 0.05).In contrast, the mineralization of cells on TCPS and niobium-containing surfaces was similar (Figure 2).
When the total collagen secreted by fibroblast cells was examined, it was determined that the niobium-containing Ti alloy had less collagen content than the TCPS and vanadiumcontaining Ti alloy at all time points (Figure 3).
As shown in Figure 4, there was no significant difference in the expression of COL1 and FN at 72 h between the groups.

DISCUSSION
Titanium alloys, which are frequently used in biomedical applications, have high biocompatibility, low density, corrosion resistance, and high-strength properties. 17,25,26In addition, metallic implants release potentially hazardous substances, thus diminishing the biocompatibility of the materials.The release of toxic ions into the body triggers an inflammatory reaction in the  surrounding tissue, which can lead to implant discoloration and loosening.−29 Although Ti6Al4V is the most commonly used implant, it may cause longterm toxicity and implant rejection because it contains vanadium. 14,30According to ISO 10993-5-2009, vanadiumcontaining Ti alloys are not considered cytotoxic as they do not cause more than 30% cell death. 31Commercially pure titanium, which was unsuccessful clinically, led to the development of Ti6Al4V.However, this alloy containing vanadium can also result in toxicity during prolonged usage.Therefore, the Ti6Al7Nb alloy was developed to enhance mechanical properties while avoiding such toxicity, but there is not enough information about their biological activity, and niobiumcontaining Ti alloys do not have a wide usage area. 32,33t is insufficient to evaluate the compatibility of a material to be implanted in hard tissue based only on bone tissue.Compatibility with fibroblast cells, which constitute a large part of the surrounding tissues, should also be considered.It was also reported that exposing cells to a higher level of extracellular calcium concentration or calcium-enriched materials would stimulate the recruitment, proliferation, differentiation, and bone-forming capacity of these cells. 34Based on our study, it is observed that the niobium-containing Ti alloy is more biocompatible compared to the vanadium-containing alloy as it stimulates higher fibroblast proliferation and calcium  mineralization.Similar to the results of the present study, it was found in different cell lines that surfaces containing Nb caused more preosteoblast cell proliferation 28 and stimulate the viability and calcium mineralization of mesenchymal stem cells. 31It has also been determined that niobium-containing different alloys such as Ti-Nb-Zr-Ta and Ti-Nb-Zr-Al lead to higher fibroblast cell viability compared to vanadium-containing Ti alloys. 41owever, contrary to these studies and our results, it has been reported that these two Ti alloy surfaces have no effect on proliferation of human osteosarcoma cells, 35−37 mesenchymal stem cells, 19 Saos-2 osteoblasts, and EA.hy-926 endothelial cells, 38 and even alloys containing Nb cause cytotoxicity in Saos cells. 39Of the two alloys containing nanotube films, it has been reported that alloys containing vanadium have better osteoblastic activities. 40Another study with titanium alloys containing molybdenum and chromium reported that these alloys caused slight cytotoxicity in fibroblast cells due to the release of elements into the environment.Particularly, prolonged exposure to molybdenum released from Ti-10Mo-10Cr resulted in reduced cell viability. 41The exposure duration is also important along with the elements contained in the titanium alloy.
There are 19 types of collagen, which is the main component of all connective tissues.Physical and chemical damage to tissue disrupts the organization of collagen. 42−44 Collagen production has been demonstrated in many cell types cultured on Ti alloys, 45−47 and total collagen, determined by Sirius red dye, which can bind to all collagen types, 42,43 was synthesized on both alloys.However, similar to the study by Lochner, 48 collagen synthesis was significantly lower on both alloys than on the TCP surface.Type 1 collagen (COL1), on the other hand, is a type of collagen synthesized by fibroblasts and osteoblasts, forming a large part of the total collagen. 49Niobium-containing alloys were found to be effective in stimulating COL1, osteopontin, and FN gene expression, 50,51 whereas vanadiumcontaining alloys did not affect COL 1 and osteopontin expression. 52However, the Ti alloys used in this study did not change the gene expression of the fibroblast cells.These conflicting results may be due to different cell types and incubation times 28,38,53 and different releases of vanadium and niobium into biological fluids (medium, calf serum, and PBS). 29,54

CONCLUSIONS
Most studies that investigated the biocompatibility of Ti alloys have focused on their cytotoxicity, adhesion, and proliferation effects.In this study, the amounts of calcium mineralization and total collagen were determined in addition to proliferation.Furthermore, the expression of the COL1 and FN genes was evaluated.While fibroblast cell proliferation and mineralization were stimulated by a niobium-containing Ti alloy, it was observed that the collagen amount was lower than the vanadium-containing Ti alloy.However, both alloys did not affect the gene expression levels.Although vanadium is a biological trace element, vanadium-containing Ti alloys may not have superior biological and functional properties.Additionally, niobium-containing Ti alloys can be considered as alternatives to Ti6Al4V for biomedical applications.Also, Ti6Al7Nb implants may be more effective than Ti6Al4V implants in terms of cell proliferation and mineralization.It is accepted that both implant materials are conducive to cell growth and are compatible with bone tissue.However, the effectiveness of fibroblasts, which constitute the surrounding tissues, in the biocompatibility process should also be known.It is important to consider the type of implant and the purpose of its application when making such comparisons.

Figure 1 .
Figure 1.Proliferation of fibroblasts on Ti alloys and TCPS for 24, 48, and 72 h (n = 6).Bars indicate the standard deviation.Groups at each hour with different letters are statistically different (p < 0.05).

Figure 2 .
Figure 2. Calcium mineralization of fibroblast cells on Ti alloys and TCPS after 48 and 72 h (n = 9).Bars indicate the standard deviation.Groups in each hour with the different letters are statistically different (p < 0.05).

Figure 3 .
Figure 3.Total collagen content of fibroblasts on Ti alloys and TCPS (n = 6).Bars indicate the standard deviation.Groups in each hour with the different letters are statistically different (p < 0.05).

Figure 4 .
Figure 4. COL1 and FN gene expression in fibroblasts on Ti alloys and TCPS for 72 h (n = 6).