Multifunctional Properties of Quercitrin-Coated Porous Ti-6Al-4V Implants for Orthopaedic Applications Assessed In Vitro

Abstract

(1) One strategy to improve the outcome of orthopedic implants is to use porous implants with the addition of a coating with an antibacterial biomolecule. In this study, we aimed to produce and test the biocompatibility, the osteopromotive (both under normal conditions and under a bacterial challenge with lipopolysaccharide (LPS)) and antibacterial activities of a porous Ti-6Al-4V implant coated with the flavonoid quercitrin in vitro. (2) Porous Ti-6Al-4V implants were produced by 3D printing and further functionalized with quercitrin by wet chemistry. Implants were characterized in terms of porosity and mechanical testing, and the coating with quercitrin by fluorescence staining. Implant biocompatibility and bioactivity was tested using MC3T3-E1 preosteoblasts by analyzing cytotoxicity, cell adhesion, osteocalcin production, and alkaline phosphatase (ALP) activity under control and under bacterial challenging conditions using lipopolysaccharide (LPS). Finally, the antibacterial properties of the implants were studied using Staphylococcus epidermidis by measuring bacterial viability and adhesion. (3) Porous implants showed pore size of about 500 µm and a porosity of 52%. The coating was homogeneous over all the 3D surface and did not alter the mechanical properties of the Young modulus. Quercitrin-coated implants showed higher biocompatibility, cell adhesion, and osteocalcin production compared with control implants. Moreover, higher ALP activity was observed for the quercitrin group under both normal and bacterial challenging conditions. Finally, S. epidermidis live/dead ratio and adhesion after 4 h of incubation was lower on quercitrin implants compared with the control. (4) Quercitrin-functionalized porous Ti-6Al-4V implants present a great potential as an orthopedic porous implant that decreases bacterial adhesion and viability while promoting bone cell growth and differentiation.

Keywords: ALP activity; Porous Ti-6Al-4V implants; additive manufacturing; multifunctional coating; orthopedic implants; osteocalcin release; quercitrin.

Figure 1

Schematic representation of quercitrin coating on Ti-6Al-4V porous implants, and experimental groups used in the study, control and quercitrin-coated implants.

Figure 2

(n = 2). Scanning electron microscope images of 3D uncoated implants. Images show the 3D implants used for the study at different magnification. Scale bars are showed in each image.

Figure 3

Confocal micrographs of DPBA (2-aminoethyl diphenylborinate) staining of 3D implants (n = 2). A: Control uncoated 3D implant. B: Quercitrin-coated 3D implants. C: Image showing a depth projection micrograph of a Quercitrin-coated implant, images are 2D reconstructions of sections acquired repeatedly in sequential steps along the z-axis. The color code corresponds to the z-axis depth of DPBA fluorescence, coded from blue at 0 µm and pink at 500 µm depth.

Figure 4

Biocompatibility of the porous implants. A) Confocal micrographs of MC3T3-E1 cells cultured for 48 h on 3D implants. Cells were stained with Phalloidin-FITC (stains actin fibers) and DAPI (stains nucleus) (n = 2). B) Lactate dehydrogenase (LDH) activity measured in culture media 48 h after seeding onto the 3D implants. Low control (-, 0% toxicity) was obtained from cells seeded on plastic. High control (+, 100% toxicity) was obtained from culture media of cells seeded on plastic and treated with 1% Triton X-100 (n = 12). C) Number of cells growing on 3D implants 48 h after seeding. Cells were stained with DAPI and counted with Image J software (n = 2 replicates, 4 images of each sample). Values represent the mean (SD). Results were statistically compared by Student’s t-test: * p < 0.05 vs. Control.

Figure 5

Bioactivity of the porous implants. A) Alkaline phosphatase activity of MC3T3-E1 cells seeded onto the 3D implants after 14 days of culture (n = 12). B) Osteocalcin levels released to culture media of MC3T3-E1 cells cultured onto the 3D implants at 14 days of culture (n = 12). Values represent mean (SD). Results were statistically compared by Student’s t-test: * p < 0.05 vs. Control.

Figure 6

Implant bioactivity under bacterial challenging conditions. Alkaline phosphatase activity in MC3T3-E1 cells treated with 1µg/mL E. coli LPS (lipopolysaccharide) for 72 h seeded onto coin and 3D implants at 14 of cell culture (n = 6). Values represent mean (SD). Results were statistically compared by Student’s t-test: * p < 0.05 vs. Control.

Figure 7

Effect of the implants on S. epidermidis survival and adhesion. (A) S. epidermidis Live/Dead Ratio cultured for 4 h on the implants (n = 6). (B) S. epidermidis adhesion on the implants after 4 h of incubation (n = 6). Values represent mean (SD). Differences between groups were assessed by Student t-test: * p < 0.05 vs. control.