The in-vitro tests of biocompatibility and bioactivity of TiC coatings were studied on the adhesion, growth, maturation, viability, stress adaptation and potential immune activation of osteogenic cells in cultures on these materials. For the cell culture ex­periments, the samples have been sterilized with 70% ethanol, inserted into 24-well polystyrene cell culture plates (TPP, Switzerland; internal well diameter 15.6 mm). These studies were carried out on human osteoblast-like cell line MG-63 (European Collection of Cell Cultures, Salisbury, UK).

The cells have been cultured for 1, 3, and 7 days at 37 °C in a humidified air at­mosphere containing 5% CO2. For the cell culture experiments, glass slides and also the bottom of standard polystyrene cell culture dishes have been used as reference materials. The detailed description of cell seeding was described by Balazsi et al.56 57

The samples have been used for evaluation of cell number and their viability by the LIVE / DEAD viability/cytotoxicity kit for mammalian cells (Invitrogen, Mo­lecular Probes, USA) according to the manufacturer’s protocol. Briefly, nonfixed cells have been incubated for 5 to 10 min at room temperature in a mixture of two of the following probes: calcein AM, a marker of esterase activity in living cells, emitting green fluorescence (excitation/emission ~495/~515 nm), and ethidium homodimer-1, which penetrated into dead cells through their damaged membrane and produced red fluorescence (excitation/emission ~495/~635 nm).

TiC/A:C nanocomposite thin films did not lead to an increase in cell number after 1 day (Fig. 2.19a) in culture in comparison with the control microscopic glass coverslips 56. However, on days 3 and 7 after seeding, the cell numbers on TiC/A:C surface have been found to be similar to PS and significantly higher than that on the microscopic glass coverslips (Fig. 2.19). Harcuba et al. 58 investigated the biological properties of Ti-6Al-4 V alloy after surface treatment by the electric discharge machining (EDM) process using MG63 osteoblast cells. These results could be compared with our results and showed that samples modified by EDM provide a better substrate for the adhesion and growth of human bone-derived cells than the alloy plasma-sprayed withTiO258.

The cell-matrix adhesion involved cell adhesion with many integrin or other adhe­sion receptors of an appropriate type, associated with cell differentiation, and also the formation of a large number of mature cell-matrix adhesion sites.59 The microscopic investigations have been performed on cells stained by immunofluorescence staining. The pi-integrin adhesion receptors have been localized predominantly in the central region of the cells (Fig. 2.20). Vinculin, which formed dot-like structures, has been located on the whole surface of cells in the case of TiC/A:C (Fig. 2.4.7). However, on microscopic glass coverslips, vinculin has been situated mainly at the cell edges. The cells on TiC/A:C displayed larger and more numerous vinculin-containing focal adhe­sions than the cells on microscopic glass coverslips (Figs. 2.20 and 2.21).

Ti based implants are classified as bioinert. Bioinert refers to a material that retains its structure in the body after implantation and does not include any immunologic host reaction. Bioactive materials could be used for modification of the surface that occurs upon implantation. Bioactive refers to materials that direct chemical bonds with bone or even with soft tissue of a living organism. One of most used bioactive materials is a hydroxyapatite. The structure of bioinert TiC /A:C thin films covered by bioactive HAp is shown in Fig. 2.22.

2.5. CONCLUSION

Biomaterials used for implant should posses some important properties in order to long-term usage in the body without rejection. The creation of nanocomposites of ceramic materials with particle size few ten nanometers can significantly improve the bioactivity of the implant and enhance the osteoblast adhesion. The most used bioma­terials are hydroxyapatite, polymer and titanium (Ti).

Hydroxyapatite (HAp) prepared from eggshells are a good candidate as bioma­terial. Nanosized HAp prepared by attritor or ball milling had significantly higher bone formation than the unfilled control at 8 weeks after the operation. The histologic measurements showed that the remaining graft material was much lower in the HAp group. HAp from eggshells could be considered as an economic bone graft material. The results from this animal study cannot be extrapolated to clinical applications; sub­sequent toxicologic assessment and clinical trials would be necessary.

Polymer-HAp composites were successfully manufactured and applied in bone tissue engineering by employing the electrospinning technique. CA-HAp nano scaf­folds were proved to promote favorable adhesion and growth of osteoblasts as well as to stimulate the cells to exhibit functional activity of bone cells. Overall, our studies suggest that both the morphology and structure of the CA-HAp composite scaffolds play important roles in facilitating cell spreading and differentiation and enhance apa­tite mineralization.

The sputtered TiC/A:C nanocomposite thin films were prepared as potential bar­rier coating for interfering of Ti ions from pure Ti or Ti alloy implants. Columnar TiC crystallites with 15-20 nm width have been embedded in 5 nm thin amorphous carbon matrix. MG63 osteoblast cells have been used for in-vitro study of nanocomposites. The 7 day lasting tests showed a higher value of cells on TiC/A:C nanocomposite surface. On the other hand, the cells on TiC/A:C film showed amore spreading ten­dency than the cells on control. The distribution of osteocalcin on microscopic glass coverslip did not reach the intensity of osteocalcin in cells on TiC/A:C films. The initial adhesion, subsequent growth and viability of human osteoblast-like MG63 cells in cultures on different substrates have been studied. From these measurements, no significant differences in the viability of MG63 cells have been found concerning all tested materials.

Based on our observations, the nanosized HAp prepared from eggshells by mill­ing, electrospun CA-HAp nano scaffolds or TiC based thin films are considered as a promising candidate for bone tissue engineering application.