Cytotoxicity

Cytotoxicity was assessed according to the International Organization for Standardization

(ISO) 10993-5 (Biological evaluation of medical devices-Part 5: Tests for in vitro cytotoxicity)
specifications. The cell lines NIH3T3 and L929 were used for the test. Cell viability was
considered potential cytotoxicity if it dropped below 70% (vs. RC). Parameters and procedures
are in full compliance with ISO 10993-5 specifications.

Liu CF et al, obtained cell viability results among cell lines L929 and NIH3T3 when co-
cultured in media containing specimen extracts of TiOH, TiG, and TiGC. The positive control
group showed an important cytotoxic response, as indicated by low cell viability. The reagent
control and negative control groups showed a potentially non-cytotoxic (negative) response, as
indicated by good cell viability. L929 cells incubated in extract-containing media exhibited the
same viability as cells incubated in extract-free medium (reagent control). Cell viability in the
NIH3T3 cell line was approximately 82-91% (vs. control reagent) when co-cultured with any
of the extract-containing media. Therefore, the results show that none of the test samples have
indicated potential cytotoxicity

Cell adhesion and migration

Cell adhesion was monitored using human bone marrow mesenchymal stem cells (hMSCs)
transduced using the green fluorescent protein (GFP) gene via retroviral delivery. Fluorescence
microscopy was used to observe in situ cell migration at various time points (0, 6, and 12 h).

The initial attachment of osteogenic cells to the surface of the biomaterial is believed to be an
important step in material-cells interaction, with profound effects on migration, proliferation,
and differentiation. In the initial 2 h incubation period, there was no significant difference in
the number of attached cells. However, after 24 h, the cells on the surface of the unmodified
Titanium were spindle-shaped and grew parallel along the grooves of the implant surface,
whereas the cells on modified Titanium surface, particularly TiGC surface, showed a well
spreading morphology and cell–cell interactions.

Research by rezvanian et al, showed that cell adhesion on biomaterials can be controlled by
surface roughness or collagen coating. The most obvious cell dispersion and intercellular
interactions were observed in the TiGC specimens, indicating that a nano/submicron-scale
porous surface combination with moderate roughness (Ra ~ 0.1m), high hydrophilicity
(contact angle <22º), and type I collagen immobilized were highly suited to hMSCs adhesion.

Differences in distribution of vinculin (focal adhesion complex related protein) and nuclei on
unmodified implants and surface-modified Ti specimens after 2 h of incubation could be
observed along the cell edges on unmodified Ti and TiGC surfaces, but vinculin expression
was not widely observed on the TiOH or TiG surface. In terms of cytoskeletal arrangement,
presents that expression of F-actin (cytoskeletal protein) was more widespread on Ti and TiGC
surfaces than on TiOH and TiG surfaces, after incubation for 2 h. After incubation for 6 hours,
unidirectional extension of F-actin was observed on unmodified Ti surface, but nondirectional
attachment on the TiOH, TiG, and TiGC surfaces. Particularly, the F-actin arrangement showed
a better extended morphology on the TiGC surface.

Research by Kado T and formetin et al reported that the Ti surface immobilized by type I
collagen facilitates even cell growth while increasing vinculin expression and cell proliferation.
Type I collagen has also been shown to benefit the surface modification of other implant
materials (e.g, anodic alumina or silicon) in terms of cell adhesion, morphology, and
proliferation. Good cell adhesion has been linked to cell communication, cell regulation, and
tissue maintenance.

The study of Liu CF et al showed the average migration rate of GFP-labeled hMSC cells on
TiGC surface was approximately 10m/h, which was far exceeds those on other test surfaces
(<5m/h).

Cell proliferation and mineralization

Cell proliferation during 7 days of incubation was analyzed using MTT test. The results
revealed a significant differences among titanium implant specimens. Cell proliferation in
TiOH and TiG specimens was significantly lower than Ti and TiGC specimens. Overall, cell
proliferation was higher in the unmodified Ti specimens than in the other test specimens. The
results analysis of Alizarin red S staining for cell mineralization were used to detect calcium
components after incubation for 7 and 14 days. After 7 days incubation in osteogenic culture,
cell mineralization was much more pronounced in TiGC specimens than in others. This seems
to indicate that the slowdown of cell proliferation on the TiGC surface indicates the cells move
more rapidly to the next growth stage (i.e. mineralization) in the region of inhibited cell
proliferation, osteogenic markers tend to initiate mineralization. Similar results have been
reported in another study that type I collagen immobilized surface would progress more rapidly
to the cell mineralization stage, despite a slight lack of surface cell proliferation.

Reference

1. Liu CF, Chang KC, Sun YS, Nguyen DT, Huang HH. Immobilizing type I collagen via
natural cross-linker genipin to enhance the osteogenic responses to titanium implant
surface. J Mater Res Technol 2021;15:885-900.
2. Rezvanian P, Daza R, Lopez PA, Ramos M, Gonzalez-Nieto D, Elices M, et al.
Enhanced biological response of AVS- functionalized Ti-6Al-4V alloy through covalent
immobilization of collagen. Sci Rep 2018;8:3337.
3. Kado T, Aita H, Ichioka Y, Endo K, Furuichi Y. Chemical modification of pure
titanium surfaces to enhance the cytocompatibility and differentiation of human
mesenchymal stem cells. Dent Mater J 2019;38:1026e35.
4. Formentin P, Catalan U, Pol L, Fernandez-Castillejo S, Sola R, Marsal LF.
Collagen and fibronectin surface modification of nanoporous anodic
alumina and macroporous silicon for endothelial cell cultures. J Biol Eng
2018;12:21.