In Vitro Evaluation of Ag- and Sr-Doped Hydroxyapatite Coatings for Medical Applications

Abstract

Osseointegration plays the most important role in the success of an implant. One of the applications of hydroxyapatite (HAp) is as a coating for metallic implants due to its bioactive nature, which improves osteoconduction. The purpose of this research was to assess the in vitro behavior of HAp undoped and doped with Ag and/or Sr obtained by galvanostatic pulsed electrochemical deposition. The coatings were investigated in terms of chemical bonds, contact angle and surface free energy, electrochemical behavior, in vitro biomineralization in acellular media (SBF and PBS), and biocompatibility with preosteoblasts cells (MC3T3-E1 cell line). The obtained results highlighted the beneficial impact of Ag and/or Sr on the HAp. The FTIR spectra confirmed the presence of hydroxyapatite within all coatings, while in terms of wettability, the contact angle and surface free energy investigations showed that all surfaces were hydrophilic. The in vitro behavior of MC3T3-E1 indicated that the presence of Sr in the HAp coatings as a unique doping agent or in combination with Ag elicited improved cytocompatibility in terms of cell proliferation and osteogenic differentiation. Therefore, the composite HAp-based coatings showed promising potential for bone regeneration applications.

Keywords: bioactivity; biocompatibility; electrochemical deposition; hydroxyapatite; osteogenic differentiation; silver; strontium.

Figure 1

Schematic illustration of the obtained coatings and surface features.

Figure 2

Representative profiles used to measure the coating thickness (a) and the deposition rate evolution for each coating (b).

Figure 3

FTIR spectra of undoped and doped HAp-based coatings.

Figure 4

Surface free energy (a) and contact angle with SBF (b) for the investigated surfaces.

Figure 5

Open-circuit potential (a) and Tafel (b) plots of the investigated surfaces.

Figure 6

The mass evolution for cp-Ti and HAp-based coatings exposed in SBF.

Figure 7

The mass evolution for cp-Ti and HAp-based coatings exposed to PBS.

Figure 8

Fluorescence microscopy images of MC3T3-E1 preosteoblasts grown in direct contact with the bare cp-Ti substrate and the developed coatings for 1 and 3 days. LIVE/DEAD viability assay: live cells (green fluorescence); dead cells (red fluorescence, arrows and area delimited by the dashed line).

Figure 9

CCK-8 assay showing the proliferative capacity of cells grown for 1 and 3 days in contact with the developed HAp-based coatings and the bare cp-Ti (*** p < 0.001 vs. 1 day; * p < 0.05 for H vs. Ti coatings at 1 day; ●● p < 0.01 for H and H-Sr vs. Ti at 3 days).

Figure 10

Morphology of MC3T3-E1 preosteoblasts grown in direct contact with HAp-based coatings and the control support (cp-Ti), as revealed by the Alexa Fluor 488-coupled phalloidin labeling of actin filaments (actin—green, nucleus—blue).

Figure 11

Differentiation potential of MC3T3-E1 preosteoblasts grown in contact with bare and HAp-coated cp-Ti: (a) specific enzyme activity of the intracellular ALP at 7 and 14 days after seeding (* p < 0.05 for H-Sr vs. Ti sample and ●● p < 0.01 for H-Ag vs. Ti at 14 days); (b) the level of collagen synthesis and extracellular matrix deposition after 3 weeks of culture (●●● p < 0.001 vs. 2 weeks; * p < 0.05 for H-Sr and H-Sr-Ag vs. Ti at 4 weeks); (c) the level of matrix mineralization as quantified by Alizarin Red staining at 4 weeks post-seeding (*** p < 0.001 for H-Sr and H-Sr-Ag vs. Ti, ** p < 0.01 for H-Ag vs. Ti).