Titanium-hydroxyapatite composites sintered at low temperature for tissue engineering: in vitro cell support and biocompatibility

Preparation of composites

Ti-HA composites were prepared by PM from TiH₂ 98% pure (Sigma) mesh-325 and bovine HA (Inbiomed S.A., Argentina). TiH₂ powder was mixed 20% HA powder (Ti-20HA), and the mixture was homogenized for 30 minutes. The powders were compacted at 390 MPa without addition of lubricant to obtain samples of size 8-mm diameter and 2-mm height. Then, these were sintered in an argon atmosphere at atmospheric pressure at 800°C for 2 hours. This inert atmosphere is achieved with argon preflow for 30 minutes. Hydrogen from TiH₂ was released at the beginning of the sintering step by heating samples at 500°C for 30 minutes. Discs of pure Ti (Ti) and porous Ti (Ti-Por) were prepared in the same way, with Ti-Por being obtained from a mixture of Ti with 20% in NH₄HCO₃. All composites were ultrasonically cleaned in 100% acetone, then with distilled water for 10 minutes at each step, and then were sterilized by dry heat at 140°C for 2 hours.

Surface characterization and in vitro bioactivity of Ti-HA composites

To investigate the chemical and structural composition of the samples, X-ray diffraction (XRD) analyses in a Philips PW1800/10 diffractometer (LAMARX-UNC) were carried out. The surface topography of composites was examined by scanning electron microscopy (SEM) using a FE-SEM ΣIGMA microscope at 12 kV (LAMARX-UNC). The samples were mounted on stubs and sputtered coated with chrome. To analyze their bioactivity, Ti-HA composites were immersed in simulated body fluid (SBF) (30) and kept at 37°C. After 5 and 10 days, surface composites were examined by SEM.

Preparation of composite extracts

Extracts of all composites were prepared to assess the hazard potential of samples. Extraction conditions were selected in accordance with ISO 10993-12 (31). The sterilized composites were put into sterile, chemically inert and closed containers. Dulbecco`s modified Eagle’s medium (DMEM) (Gibco) was added at a ratio of 0.1 g/mL (mass of disc to volume of DMEM). Then, samples were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C for 72 hours. The discs were removed from the media, and a 100% extract (100-extract) was obtained after addition of fetal bovine serum (FBS) (PAA) (10% v/v). For the cytotoxicity assay, 75%, 50% and 25% extracts (75-, 50-, 25-extracts) were obtained by dilution in supplemented DMEM of 100-extract.

Cell culture

Experiments were performed using the epithelial Vero cell line and fibroblastic NIH3T3 cells (provided by CIQUIBIC-Argentina).The Vero cell line is mandatory for cell cytotoxicity and cell–substratum interaction studies in biomaterial research (32). In addition, the NIH3T3 cells were also used here because they share a common origin with osteoblastic cells. Both cell lines were cultured in DMEM with 10% FBS, 4 mM of L-glutamine (Gibco), 4 mM of sodium pyruvate (Sigma) and 10,000 IU/mL of penicillin with 10,000 µg/mL of streptomycin in a humidified 5% CO₂ atmosphere at 37°C. Cells were seeded on 96-well plates at a density of 10,000 cells per well and incubated in supplemented DMEM. The culture medium was replaced every 3 days.

Cytotoxicity assay

Cytotoxicity of extract composites was performed by the MTT assay (33), according to ISO 10993-5 (32). Vero or NIH3T3 cells were seeded as described above for 24 hours, after which media were removed, and extracts from different composites were added. After 24 hours, cells were rinsed with phosphate-buffered saline (PBS) twice, and 50 µL of 1 mg/mL MTT in DMEM was added to the appropriate wells and incubated at 37°C for 1.5 hours. The formazan crystals formed were dissolved by adding isopropyl alcohol in each well. Absorbance was quantified at 595 nm using a Multiskan Spectrum microplate reader (Thermo Scientific). High-density polyethylene (HDPE) 100-extract (ratio of 6 cm²/mL, surface of HDPE to volume of DMEM) was used as negative control, and 0.2% phenol solution was assayed as positive control. The percentage of cell viability was calculated according to the optical density measured for each incubation condition with respect to the optical density determined for cells grown in supplemented DMEM ×100. All experiments were carried out in sextuplicate.

Cell morphological analysis

The morphology of Vero and NIH3T3 cells grown in the presence of extracts of composite was observed by microscopy (34). The morphology and relative density of Vero cells cultured in the presence of 100-extract or supplemented DMEM were examined using SEM (JEOL JSM 6480 Brand LV; Iniqui, Argentina). Cells were plated at a density of 10,000 cells/cm² on glass coverslips, and incubated in the presence of 100-extracts or supplemented DMEM for 4 days. After incubation, samples were washed with PBS and fixed overnight in 4% glutaraldehyde buffered in PBS at 4°C. Next, the samples were washed 3 times with PBS and dehydrated in graded ethanol (10%-100%) for 10 minutes each step. Finally, after critical point drying, the samples were mounted on stubs and were sputtered coated with gold.

NIH3T3 cell morphology was analyzed by light microscopy. NIH3T3 cells were suspended in 0.5 mL of supplemented DMEM and seeded in 24-well plates (10,000 cells/well). After incubation for 24 hours, the media were removed, and 100-extract from each composite was added. The culturing was stopped at days 2 and 5 of culture, and cell images were captured digitally (Eclipse TE2000-U microscope, Digital Sight DS-U1 camera, Nikon).

Cell adhesion assessment

NIH3T3 cells were seeded on the surface of the composites placed on 24-well plates at a density of 10,000 cells/well or on glass coverslips as controls, and then they were incubated in supplemented DMEM. NIH3T3 cell adhesion was observed using fluorescence microscopy through DAPI staining to determine the presence of nuclei. Cultures on all surfaces were stopped when the cells covered the entire glass coverslip. Then, they were washed several times with PBS, and cells were fixed with 4% paraformaldehyde solution, permeabilized with 0.1% Triton X-100 in PBS, and incubated with DAPI for 10 minutes at room temperature for each step. Subsequently, all samples were observed with a Nikon Eclipse TE2000-U microscope with a fluorescence filter (range 340-360 nm) for DAPI. The number of cells growing on composite surfaces was estimated using ImageJ software (n = 3 for each group).

The morphology of cells attached to the Ti-20HA surface was examined by SEM at 1 kV with variable magnification (×2,000 and ×5,000) (LAMARX-UNC). Cells adhered to the surface of composites were washed carefully with PBS and fixed overnight in 4% glutaraldehyde buffered in PBS at 4°C. Then, the samples were prepared as previously described.

Statistical analysis

Experimental data were expressed as means ± standard error of the mean (SEM). Results from the cytotoxicity and cell count were analyzed using 1-way ANOVA, and whenever the test indicated significant effects (p<0.05), a pairwise comparison (Newman-Keuls posttest) was also carried out. A p value <0.05 was considered to represent a significant difference in all cases.

Surface characterization and in vitro bioactivity of the Ti-HA composites

The XRD pattern of the Ti-20HA composite showed characteristic peaks of Ti and HA (Fig. 1A). HA and Ti mainly existed in their simple substance, suggesting that reactions between HA and Ti did not take place. HA decomposition products were not identified. Peaks characteristic of titanium (near 2θ 40°) and a broad halo in the range 31°-34°, due to crystalline HA, were observed.

SEM micrographs of composite surfaces before immersion in SBF showed micropores and macropores on all surfaces, with HA particles well integrated into the Ti matrix (Fig. 1B, A). After 5 days of composite immersion, calcium phosphate deposition was observed (Fig. 1B, b), and at 10 days postimmersion, depositions covered the whole surface, especially into the pores (Fig. 1B, d). An energy-dispersive spectroscopy (EDS) spectrum is presented in Figure 1C.

Cytotoxicity

The cytotoxicity of the Ti-20HA as well as Ti and Ti-Por composites was evaluated in vitro by measuring Vero and NIH3T3 cell viability after incubation with the composite extracts for 24 hours. One-way ANOVA revealed a significant effect of 100-extracts of the different composites on Vero cell viability (F_(4,25) =791.55, p<0.0001). Figure 2A shows that the percentages of viability for Ti-20HA (95.7% ± 0.81%; p = 0.397) and Ti (100.11% ± 1.35%; p = 0.155) were not significantly different from those of the negative control (98.50% ± 1.44%). Although viability of the cell exposed to Ti-Por 100-extract (88.83% ± 1.84%; p<0.02) was significantly lower than negative control, it was greater than 70%. According to the ISO 10993 standard, the samples are considered nontoxic whenever cell viability is greater than 70% (31). All groups (p<0.001) revealed percentages higher than that of the positive control (9.80% ± 0.83%).

In the same way, 1-way ANOVA showed a significant effect of 100-extracts of all composites on NIH3T3 cell viability (F_(4,25)=463.56, p<0.0001). The percentages of viability of NIH3T3 cells for the Ti-20HA, Ti and Ti-Por 100-extracts were 91.30% ± 2.38%, 92.30% ± 2.06% and 93.24% ± 1.64%, respectively, which were significantly different from the negative control group: 99.49% ± 1.52% (p<0.02) (Fig. 2B). Nevertheless, NIH3T3 viability was greater than 70% in all cases, thereby indicating a lack of extract composite toxicity to this cell line. Extract dilution did not show any effect on composite cytotoxicity (Fig. 2).

Cell morphological analysis

The Vero cell morphology did not reveal any differences between the cells incubated with supplemented DMEM medium or composite 100-extract (Fig. 3A, C, E). Moreover, the cell confluence reached on day 4 of cell culture was similar among Ti-20HA, Ti-Por and control. In addition, composite extracts did not affect the cell size (Fig. 3B, D, F). Similar effects of different composite extracts on cell morphology, cell size and density of NIH3T3 lineage were also observed. After 2 and 5 days of incubation in the presence of extracts from Ti-20HA, Ti and Ti-Por, the microphotographs showed the NIH3T3 cells to be well attached and with similar morphologies compared with the controls (Fig. 4A-D). Furthermore, these cells proliferated widely between days 2 and 5 of incubation and were almost confluent on day 5 (Fig. 4E-H), indicating that the extracts did not alter their growth ability or development.

Cell adhesion

The micrographs showed that NIH3T3 cells stained with DAPI were able to attach to all surfaces (Fig. 5A), reaching a similar density on surfaces of Ti-HA, Ti and Ti-Por (Fig. 5Ab, d, f). Cell number was not significantly different between the 3 surfaces (Fig. 5B) (p = 0.69). These results suggest that Ti-20HA as well as Ti-Por and Ti composites were harmless to the cells. Furthermore, on the Ti-20HA surface, cells adhered to HA of the composite (Fig. 6B) and grew by spreading out across the surface with extensions observed of up to 10 µm in length (Fig. 6A, B). Cells also grew into the pores of the composite (Fig. 6C), thus supporting a possible biological fixation.