(B-75) Mechanical Stability of Funcationalized High-Performance Oxide Ceramic Surfaces for Implantation

Titanium alloy implants create debris particles over time, which can cause osteolysis: the degradation of peri-implant bone. Osteolysis can result in the failure of the implant fixation through aseptic loosening [1]. High performance oxide ceramics (HPOC) have lower wear rates than titanium, making them an ideal material for implant design; however, HPOC are biologically inert [2,3]. Therefore, HPOC can have poor integration with the surrounding bone, increasing the risk of failure and fibrotic encapsulation, further isolating the implant from fixation with surrounding tissue [3,4].

To counteract the biological inertness of ceramic materials and improve the success of cementless joint replacements, various implant surface modifications have been developed to increase cell adhesion resulting in greater osseointegration. Many of these functionalized coatings target integrin receptors with bioactive compounds rich in Arg-Gly-Asp (RGD) motifs including cyclic RGD (cRGD), laminin, and fibronectin [5,6].

Bone cement cannot be used in operations with this functionalization because it creates a barrier between bone and implant, inhibiting the functionalized surface from having its intended effect. Thus, this surface coating must be able to withstand the forces of press fitting, to have clinical viability. This force varies based on the number of hits and energy transmitted by the surgeon while hammering the implant; however, previous studies suggest that the average force of press fitting is 414 N [7]. This study aims to explore the stability of the cRGD functionalized surface coating under loading conditions mimicking that of press fitting to determine the viability of in vivo clinical translations.

Alumina toughened zirconia (ATZ) granulate was uniaxially dry pressed to create cylindrical green bodies that were subsequently sintered, grinded, and polished. The discs were subsequently cleaned and hydroxylated with piranha solution, a 3:1 solution concentrated sulfuric acid to 30% hydrogen peroxide. To improve the bioactivity of ATZ, the discs were subsequently functionalized with a multilayer coating comprised of a 3-aminopropyl(diisopropyl)ethoxysilane (ADPS) base, the bifunctional crosslinker bis(sulfosuccinimidyl)suberate (BS³), and the bioactive peptide cRGD.

To mimic the mechanical loading of press fitting, a novel scratch test was developed (Fig. 1). Aluminum, polypropylene, and bovine cortical bone cylinders (5mm diameter) were hammered across the ATZ surface while under 980 N (12.5 MPa) compression. The discs were then imaged with brightfield microscopy to explore any visible damage to the surface.

In addition to the three scratched experimental groups, two control groups were added: unscratched but functionalized discs and unfunctionalized, unscratched discs. Three discs from each sample were seeded with 1.0x10⁴ human mesenchymal stem cells (hMSCs)/cm² and allowed to incubate for 18 hours at 37°C. Live/dead staining was performed using Hoechst, fluorine diacetate, and propidium iodide; ImageJ was used for cell counting.

To examine cellular adhesion, an additional disc from each group was seeded with 1.5x10⁴ hMSCs/cm² and incubated for one hour. The samples were subsequently fixed with paraformaldehyde and stained for the nuclei (DAPI), F-actin, and phosphorylated Focal Adhesion Kinase (pFAK).

Results

All discs showed consistent growth across the surfaces with live/dead imaging, even within the scratched region (Fig 2). Analysis in ImageJ showed that all discs had at least 92% viability, with 12 of the 15 samples having over 97% living cells. The unfunctionalized control had the lowest mean survivorship, although it was only less than aluminum by a statistically significant amount. The nonfunctionalized control showed almost no pFAK signal, indicating a lack of surface adhesion. Comparatively, all functionalized surfaces showed high levels of pFAK within scratched regions (Fig 3).

Discussion

The goal of the scratch test is to mimic forces experienced by implant surfaces during press fitting. To ensure that the protocol was representative of most cases of press fitting, the load was set at 980 N.

The consistent growth across the surface of all samples in the live/dead staining suggests that the forces on the implant from press fitting does not negatively impact cell growth. Across experimental groups, the only statistically significant difference in viability was between the aluminum-scratched samples and the unfunctionalized control. As there was no statistical difference between any other functionalized surface and the untreated control, this is likely due to chance and limited sample size (n=3).

Cellular adhesion was investigated through the proxy marker pFAK, a downstream signaling molecule of integrin activation [8]. The high levels of pFAK signal across the functionalized surfaces suggest the cRGD coating successfully improved surface adhesion and remained functional after exposure to mechanical forces mimicking press fitting.

Conclusion

The ATZ surface functionalization with cRGD remained active after mechanical conditions simulating press fitting, with no observed side reactions inhibiting cellular growth and adhesion. Future studies should explore the in vivo efficacy of this coating and the translation of this coating to titanium implant surfaces.

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