INTRODUCTION

The generation of functional tissue through tissue engineering has a high impact in various areas of regenerative medicine, among which is skeletal tissue. The first implants were used in the field of medicine, in 1909, when Kirschner wires and Steinman nails were developed for the fixation of bone fractures, where stainless steel was used. Over the years, steel has been improved, making it more resistant to corrosion and not causing harmful effects on the human body. In 1940, the study of titanium (Ti) began as a biomaterial for bone implantation¹.

The phase change determines the change in the crystalline structure of the material when subjected to temperature changes. Titanium’s allotropic transformation occurs at 882ºC and goes from an a phase, which has a compact and hexagonal structure (HCP), little deformable and resistant at room temperature, to a ß phase characterized by a cubic structure centered on the body (BCC), which is easily deformable and allows for carrying out heat treatments to optimize the material’s properties².

Among the characteristics that make titanium one of the best materials for bone implants, its greater specific resistance (resistance/density), its high ductility and lower Young's modulus³ compared to other elements such as, for example, stainless steel are noteworthy. At the same time, it is a non-ferromagnetic material, which does not present inconveniences when the patient with a Ti implant undergoes magnetic resonance imaging. Anchorage to bone tissue is possible because of the oxide layer formed in the material when passivated⁴.

Pure commercial Ti (c.p.) and other alloys such as Ti6Al-4V are currently used mainly for bone implants⁵. However, both elements present a high elastic modulus (E: 100-112 GPa) compared to the elastic modulus of cortical bone (18.6-20.7 GPa) and trabecular (10.4-14.8 GPa) (Table 1) what produces the stress-shielding effect or stress-shielding⁶. This phenomenon is due to the stiffness of the bone implant material being greater than the stiffness of the bone, which places the entire load on the bone implant. Bone remodeling is largely regulated by the mechanical loads to which it is subjected, so that the presence of loads stimulates its bone formation, and the absence of them increases resorption. As a consequence of the bone’s reduced load supported, it decreases its density in the area near the implant and, therefore, both premature fracture of the implant and loosening of the implant may occur⁵.

At present, to eliminate the voltage shielding phenomenon, reducing the elastic modulus is sought, with several available solutions. One of them, reduce the density of the material used, through porosity. However, as the porosity increases, the mechanical resistance decreases. Another approach would be to search for face-centered cubic structure Ti alloys (BCC) or β-Ti alloys, which are low in elastic modulus and do not show a decrease in mechanical strength. For the stabilization of the ß phase, ß-stabilizing elements are required: Mo, V, Ta, Nb and Zr as β-isomorphic elements and Cr, Co, Cu, Fe and Ni as β-eutectoid elements. The advantage of β-isomorphic elements is their high amount of substitute solid solution and their inability to form intermetallic compounds of Ti, which have high E. Therefore, it has been shown that elements such as Nb and Ta have a high level of biocompatibility and ability to prevent the increase of particles, which would avoid a bad cohesion interface⁷.

Niobium (Nb) and tantalum (Ta) are two transition metals widely used in alloys; in particular, Nb is used in the formation of steel. Until 1866, it was thought that both elements were the same, since they have very similar physicochemical characteristics. The TiNbTa alloy stands out for its high stabilization of behavior, due to the absence of β phase, which allows a decrease in the elastic modulus (49 + 3 GPa), its excellent elastic resistance (sy>1860 MPa) and its high biocompatibility^7,8. Therefore, a material such as TiNbTa, with a good combination of high strength and low Young's modulus close to that of bone, could be used to prevent loosening of implants to avoid revision surgery (Table 1).

Table 1.  Physical characteristics of pure titanium, the different phases and bone: elastic modulus and elastic resistance 

In recent years, a whole series of techniques have been developed with the aim of obtaining porous materials that present a Young's modulus closer to that of cortical bone. Specifically, Chicardi et al. manufactured a TiNbTa alloy with physicochemical properties more similar to those of bone⁹. However, the improvement in the design of materials, destined to be used as bone grafts, must consider their osseointegration capacity, and in this sense, evaluating the cytotoxicity and the adhesion, proliferation and differentiation capacity of osteoblastic cells influenced by the material is required. Our main objective was to evaluate the osseointegrative characteristics of the TiNbTa alloy, with a Young's modulus similar to that of trabecular bone, and to compare them with pure Ti.

MATERIAL AND METHODS

RESULTS

The cell cultures on the Ti discs c.p. and in the alloy they had a similar cell growth. At 7 days of growth, the cells showed columnar and basophilic morphology under the light microscope (O.M.) compatible with the beginning of differentiation (Figure 1). The surface of the disc does not let light through, making it difficult to see cells clearly. However, in the highlighted parts, it could be observed that some cellular adhesion had occurred in the material, which would indicate that the alloy has good qualities to be used in implants.

Figure 1.  Inverted optical microscope photograph (Olympus CKX53) with a 40X medium magnification objective, of cells grown at 7 days in the TiNbTa discs 

Cell viability

Figure 2 represents the viability of the cell line as a function of the cell growth time (4 and 7 days) on the Ti discs c.p. and TiNbTa. At 4 days of culture, cell viability in the TiNbTa discs was similar to that of the Ti c.p. While at 7, we observed a slight increase in viability in the TiNbTa samples and a decrease in the Ti samples c.p., although without significant differences. In all cases, the percentage of viability was always higher than 150%. A toxic effect is considered when the viability is less than 75%, therefore, with the results obtained, it can be deduced that the cell culture is viable throughout the entire duration of the culture under all conditions.

Figure 2.  Cell viability of cells cultured in Ti discs c.p. or the TiNbTn alloy at 4 and 7 days of cell growth. Results presented as mean ± standard deviation 

Alkaline phosphatase activity

We quantified the alkaline phosphatase (ALP) activity values in MC3T3 cell cultures at 4 and 7 days. Figure 3 represents the mean and standard deviation of the results obtained in both culture conditions. In the Ti discs c.p., the results obtained showed a decrease in the enzymatic activity, the difference found between days 4 and 7 being statistically significant (p=0.001). In the TiNbTa discs, a decrease in activity was also observed with culture time and there was a significant difference between days 4 and 7 of culture (p=0.006). In both conditions, at 4 days, the maximum enzymatic activity was obtained, since there is a greater proliferation and cell growth, as the viability values corroborated, and from day 7, the cells presented a more differentiated phenotype, as observed in the SEM images, and ALP activity decreased.

Figure 3.  Activity of alkaline phosphatase in cell growth on Ti discs c.p. or TiNbTa for 4 and 7 days. *Ti c.p. 4 days vs. 7 days; ** TiNbTa 4 days vs. 7 days. P<0.05 

Cell morphology by scanning electron microscopy

A preliminary study was carried out to study the morphology and cell adhesion on the TiNbTa study material compared to the growth in Ti c.p. To do this, we visualized the samples of both types of disc at 4 and 7 days of culture. At 4 days, small cell clusters were observed on both types of surface and of similar density. At 7 days, the images showed a monolayer growth over the entire surface of the disk, being similar in both materials. Cell-cell and cell-biomaterial junctions were also observed. The cells adhered through filopodia (thin cell projections, yellow arrow) and lamelopodia (broader extensions, yellow asterisk), thus demonstrating the connection of the cells with the biomaterial. We began to observe the presence of small vesicles with a hexagonal structure, suggesting a possible nucleation of hydroxyapatite, on the cell surface, suggesting the beginning of the mineralization process development (Figure 4).

Figure 4.  Scanning electron microscope micrographs at 4 and 7 days of osteoblast cultures on the surface of Ti c.p. or TiNbTa. Cell morphology and proliferation is shown on the two surfaces tested. Cell-cell interactions through filopodia (yellow arrows) and cell-surface interactions through lamellipodia (yellow asterisk) 

DISCUSSION

Inflammatory and degenerative problems of the bones and joints affect millions of people around the world. In fact, they represent half of the chronic diseases in people over 50 years of age in developed countries¹⁰. These diseases often require surgery, including replacement of the entire joint in cases of deterioration. This fact, accompanied by the increase in life expectancy and the aging of the population, entails a great demand for healthcare derivatives, among which the development of surgical implants and materials with a longer useful life period stand out.

Biomaterials constitute one of the most important advances in current medicine, improving the patient’s quality of life and reducing the healing and convalescence time. In 2009, Bjursten highlighted the importance of increasing implant-bone bonding, given that the half-life of an implant is between 10 and 15 years^7,8, which implies an increase in the number of revision surgeries.

In our study, we have carried out tests to assess a new material, TiNbTa alloy, with a Young's modulus similar to that of trabecular bone. Numerous studies have shown the decrease in elastic modulus in alloys with Nb and Ta, such as Ti35Nb5Ta7Zr, which shows an elastic modulus of 55GPa¹¹. The advantage compared to the Ti c.p. or the Ti-6Al-4V alloy would be the lower Young's modulus of the alloy studied, which would considerably reduce the voltage shielding.

The percentage of viability is largely related to the biocompatible and cytotoxic properties of the material; various authors^12,13 have shown how high percentages of viability are optimal for considering a candidate material to be used as a bone implant in humans. Our cultures, at different study times, presented a high degree of biocompatibility, as well as an absence of toxicity. Positive results were obtained between the cell line and the material. It could be suggested that if this material were implanted, it would tend to synthesize the bone matrix, adhere well to the bone, and be biocompatible^14-15.

The viability values indicate that there are no differences in cell growth between the TiNbTa and Ti c.p alloy, and in all conditions the values are above 75%. Therefore, both materials would be non-cytotoxic and therefore viable. On the other hand, the adequate activity of cellular metabolism confirms that the alloy does not release toxic residues for our cells^15-18. Previous studies of animal implantation of materials such as Nb and Ta in soft and hard tissues of rats have shown the high biocompatibility of metals and osteogenesis^19,20.

We also quantify cellular activity by measuring ALP activity. Increased ALP activity is directly related to cell proliferation and is a marker of differentiation of the osteoblastic phenotype. In tissues such as bone and cartilage, ALP is expressed early in the calcification process and later in development, ALP expression is decreased. It has been shown that when ALP activity decreases, cell differentiation increases¹⁴.

Our values confirm that the culture on the discs with the new material presents ALP activity that varies according to the study time. At the beginning of the culture, at 4 days, the MC3T3 cells showed a higher cell proliferation in Ti c.p. than those grown on the alloy material, indicating a slower growth in the TiNbTa discs during the first days of culture. At 7 days, the ALP values were similar in both samples and lower than the initial ones, which shows that the culture is behaving with similar characteristics in terms of proliferation and differentiation. In the images obtained by SEM, we observed that at 7 days the cells covered the entire surface of the discs, cell growth occurred equally in both samples and they reached cell confluence, and the lowest activity was consistent with the images cells that are starting the mineralization process. In addition, we began to observe the secretion of material that will form the extracellular matrix by the osteoblasts, and an increase in molecules with the appearance of hydroxyapatite was observed on the surface of the cells²¹. On the other hand, osteoblasts presented filopodia and lamelopodia, essential cellular structures during the cell adhesion process, which indicates that the union to other cells and to the material is direct; other authors describe these junctions in MC3T3 cells and Ti discs c.p.^22,23.

In conclusion, our in vitro study allows us to conclude that the novel alloy that combines elements such as Nb and Ta with Ti, in addition to improving the mechanical properties of the material, is, in the short term, biocompatible with osteoblastic cells, behaving with characteristics of viability, proliferation, differentiation and adhesion capacity of osteoblastic cell lines, in a very similar way to that of pure Ti.