Nanostructured TiC Layer: a suitable surface for osteointegration

In recent years, the number of patients undergoing arthroplasty surgery for joint problems, such as osteoarthritis and accidental fractures, has grown considerably. Wide research has been conducted to study the possible use of biomaterials in orthopedic surgery that would provide bone fixation or able to induce new bone tissue formation and osteointegration. Titanium is the gold standard material used for permanent implants in contact with bone, thanks to its biocompatibility, resistance to corrosion and mechanical properties. In our laboratory, nanostructured titanium-derivative surfaces have been analysed with the aim to find a surface with the best osseointegration features. Titanium carbide (TiC) layer was produced by IPPA deposition directly on glass slides, obtaining surfaces with 25% light transmittance ability. This feature allows to perform several kinds of experiments on cells, retaining the good characteristics of nanostructured titanium. We studied the adhesion, proliferation, and morphology of cells on nanostructured TiC surfaces, comparing them to both cell-culture-treated polystyrene dishes and poly-d-lysinated glass slides (poly-d-Lys). For a more reliable investigation, we chose to use human primary cells, isolated from patients undergoing arthroplasty surgery. Three different types of cells were studied, dermal fibroblasts (FBs), human osteoblasts (hOBs) and human chondrocytes (HPCs). To study the effect of the different surfaces on cell adhesion and morphology, an immunofluorescence experiment was performed evaluating the actin filament organization. Very interestingly, the cells cultivated on TiC showed an actin structure more similar to the tissue disposition. The FBs were arranged in 3D structure, showing filaments disposed on different planes. HPCs formed a particular structure with the nucleus on one side and the cytoplasm on the other side and the hOBs showed a complex network, with a larger number of contact points among cells. These differences in adhesion are also confirmed by the Atomic Force Microscopy (AFM) images, where the cells grown on TiC substrates show well-defined actin filaments which are not evident on the membrane of cells grown on polystyrene and poly-d-Lys. Moreover, the presence of these stress fibers and the overall height of the cells over the substrate indicate that the cells have a better attachment on TiC. In order to analyse the effect of TiC surface on cellular metabolism involved factors release, an ELISA assay was performed. The amount of Fibroblast Growth Factor-2 (FGF-2), Bone Morphogenetic Protein-2 (BMP-2) and Osteocalcin (OC) was measured in cell culture medium of FBs, HPCs and hOBs, respectively. FBs cultivated on TiC produced a higher amount of FGF-2 compared to polystyrene and poly-d-Lys, while HPCs cultured on poly-d-Lys and TiC produced a higher amount of BMP-2 compared to polystyrene. Osteocalcin is used as a serum marker of bone formation and as indicator of the proliferative and differentiated state of osteoblasts in cell culture. In hOBs cultivated on TiC a higher amount of OC compared to polystyrene and poly-d-Lys was obtained. Considering the involvement of FBs, HPCs and hOBs in the production of extracellular matrix components in the in vivo tissues, the effect of TiC surface on Collagen protein expression was evaluate by immunofluorescence. In order to allow cells to produce a detectable amount of collagens, cells were cultivated for seven days before analysis. Collagen type I in hFBs and hOBs, and Collagen type II in HPCs resulted increased in cells seeded on TiC compared to the other two substrates. All these results suggest how TiC is an excellent additional layer to cell culture and that can be considered as a biomaterial useful for in vivo osteointegration. [1] Lopreiato M., Mariano A. and Cocchiola R., Condensed matter, 2020, 5, 29.