Porous titanium scaffolds modified with Zeolitic Imidazolate Framework (ZIF-8) with enhanced osteogenic activity for the prevention of implant-associated infections

Abstract

In this study, zeolitic imidazolate framework 8 (ZIF-8) was coated on porous Ti6Al4V scaffolds, either bare or previously modified using hydroxyapatite (HA) or HA and gelatin (HAgel), via a growing single-step method in aqueous media using two contact times at 6 h and 24 h. The coated scaffolds termed ZIF-8@Ti, ZIF-8@HA/Ti, and ZIF-8@HAgel/Ti were characterized via scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), and molecular plasma-atomic emission spectroscopy (MP-AES). In order to assess the cell proliferation rate, the cytocompatibility of the scaffolds was evaluated in primary osteoblasts (hOBs) using alamarBlue assay, while the osteoconductivity was analyzed in hOBs using a real-time approach, evaluating the expression of secreted phosphoprotein 1 (SPP1). Osteopontin, which is the protein encoded by this gene, represents the major non-collagenous bone protein that binds tightly to HA. The scaffolds were shown to be non-cytotoxic based on hOB proliferation at all time points of analysis (24 h and 72 h). In hOB cultures, the scaffolds induced the upregulation of SPP1 with different fold changes. Some selected scaffolds were assayed in vitro for their antibacterial potential against Staphylococcus epidermidis; the scaffolds coated with ZIF-8 crystals, regardless of the presence of HA and gelatin, strongly inhibited bacterial adhesion to the materials and reduced bacterial proliferation in the culture medium, demonstrating the suitable release of ZIF-8 in a bioactive form. These experiments suggest that the innovative scaffolds, tested herein, provide a good microenvironment for hOB adhesion, viability, and osteoconduction with effective prevention of S. epidermidis adhesion.

Keywords: anti-adhesion properties; bacterial infections; gelatin-A; hydroxyapatite; osteoblasts; titanium scaffolds; zeolitic imidazolate framework-8 (ZIF-8).

FIGURE 1

SEM images obtained on the samples listed in Table 1 (scale bar = 10 µm).

FIGURE 2

Comparison between PXRD patterns obtained from ZIF-8@Ti-1_24h (black) and ZIF-8@Ti-2_24h (green).

FIGURE 3

Schematization of the processes used for the preparation of the modified titanium scaffolds. Left: schematization (made with “BioRender”) of the processes used for the preparation of the modified titanium scaffolds; all samples were prepared in triplicate. Right: Zn content determined by MP-AES analyses; the wt% is referred to the weight of the whole scaffold.

FIGURE 4

ATR-FTIR spectra of the ZIF-8@Ti_24h (black), ZIF-8@HA/Ti_24h (red), and ZIF-8@HAgel/Ti_24h (blue) powders. The dotted lines represent the most significant bands.

FIGURE 5

PXRD patterns of the ZIF-8@Ti_24h (black), ZIF-8@HA/Ti_24h (red), and ZIF-8@HAgel/Ti_24h (blue) samples.

FIGURE 6

SEM images of different samples after 6 and 24 h contact time (scale bar = 10 µm).

FIGURE 7

Evaluation of hOB proliferation seeded on materials: i) Ti, ii) ZIF-8@Ti_6h, iii) ZIF-8@Ti_24h, iv) ZIF-8@HA/Ti_6h, v) ZIF-8@HA/Ti_24h, vi) ZIF-8@HAgel/Ti_6h, and vii) ZIF-8@HAgel/Ti_24h, following a time-course of 24 h (T1) and 72 h (T2) (*p < 0.05; **p < 0.005; and ***p < 0.0001; ANOVA test and Tukey’s multiple-comparison test).

FIGURE 8

TaqMan real-time PCR was employed to evaluate the expression of the SPP1 gene in hOBs grown on scaffolds: i) Ti, used as control, ii) ZIF-8@Ti_6h, iii) ZIF-8@Ti_24h, iv) ZIF-8@HA/Ti_6h, v) ZIF-8@HA/Ti_24h, vi) ZIF-8@HAgel/Ti_6h, and vii) ZIF-8@HAgel/Ti_24h after 72 h (*p < 0.05; **p < 0.001; and ***p < 0.0001; ANOVA test and Tukey’s multiple-comparison test).

FIGURE 9

Anti-adhesion assay. Antibacterial performance of the Ti scaffolds incubated for 4 h with a Staphylococcus epidermidis culture. Histograms are the OD values at 600 nm, measuring bacterial proliferation in the culture medium (A) and at 550/630 nm measuring the metabolic activity of the adhered bacterial cells on the surface of the materials. (B) (***p < 0.0001; ANOVA test considered the bare Ti scaffolds as the reference control, and negative samples are aliquots of the culture media).

FIGURE 10

SEM images of S. epidermidis attached to the Ti scaffolds. The arrow indicates the corrugated surface of the bacteria onto a layer of HA (a SEM image of the Ti scaffold coated with only HA is reported in Supplementary Figure S6), which is suggestive of the loss of the membrane integrity.

FIGURE 11

Bacterial growth curves of S. epidermidis cultures incubated with the Ti scaffolds and in regular medium as a reference control. A threshold indicating growth above the background level was set at OD_600nm = 0.1.

FIGURE 12

Evaluation of the Zn²⁺ ion-releasing profiles (%) via MP-AES analyses carried out on the harvested solutions of the bacterial growth medium at discrete time points. Histograms are cumulative Zn²⁺ percentages referred to the total amount of released zinc ions.

FIGURE 13

Antibiofilm assay. Values are percentages of the biomass of S. epidermidis within the biofilm produced on the tested Ti scaffolds and relative to the plain material. Differences were statistically different for ZIF-8@Ti and ZIF-8@HAgel/Ti (***p < 0.0001; ANOVA test considering the bare Ti scaffolds as the reference control).