Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants

Abstract

Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.

Keywords: Biomedical applications; Engineered proteins; Functional coatings; Hydrogels; Tissue engineering.

Figure 1

ELP processing to form a covalently crosslinked, thin film on titanium. A) Schematic of proposed ELP conjugation to titanium substrates upon exposure to UV light. B) Contact angle measurements of H₂O on Ti6Al4V discs before and after PBS spin coating and UV exposure (top) and after exposure to 10% benzoyl chloride solution (bottom). C) Long-term passive stability of spin or dip coated ELP films of both scrambled and RGD variants.

Figure 2

Active stability of ELP coatings on implants after clinically relevant implantation procedures. A) SEM of spin coated cp-Ti dental screw pre-implantation and post-extraction from a mandible mimetic polyurethane foam block. The presence of a buildup of ELP is highlighted with a white arrow (top left). A small defect in the ELP film is shown post-implantation (bottom right). B) Mean fluorescence measurements of press fit Ti6Al4V rods dip coated in fluorescently-labeled ELP solution, * p<0.0001, n = 9. C) Transilluminator (top) and fluorescence confocal microscopy (bottom) images of press fit Ti6Al4V rods dip coated with fluorescently-labeled ELP solution.

Figure 3

Spreading, adhesion and mineralization of MG63 cells seeded onto ELP coatings on glass and Ti6Al4V. A) Spreading of MG63 cells onto scrambled ELP- and RGD ELP-coated glass substrates. B) Cell morphology and spreading of MG63 cells on scrambled ELP- and RGD ELP-coated glass. C&D) Adhesion of MG63 cells on spin (C) and dip (D) coated Ti6Al4V over 24 hours. E&F) Total MG63 calcium mineralization deposition on spin (E) and dip (F) coated Ti6Al4V over 14 days. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 4

MG63 and mineralization morphology on uncoated, scrambled ELP, and RGD ELP spin coated Ti6Al4V. SEM images were taken at 1, 7, and 14 days post-seeding of MG63s in mineralization medium (standard growth medium + 8 mM CaCl₂).

Figure 5

Spreading, adhesion and mineralization of hMSCs seeded onto ELP coatings on glass and Ti6Al4V. A) Spreading of hMSCs onto scrambled ELP- and RGD ELP-coated glass substrates. B) Representative phase contrast micrographs of hMSCs on scrambled ELP- and RGD ELP-coated glass at 4 hours. C) Adhesion of hMSCs on ELP-coated and bare Ti6Al4V over 24 hours. D–F) Total DNA concentration (D), ALP activity normalized to DNA concentration (E), and total calcium deposition (F) for hMSCs on ELP-coated and bare Ti6Al4V over 28 days. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Figure 6

In vivo response to spin coated ELP films on cp-Ti dental screws implanted into rat femurs. A) Bone-implant contact (BIC) area quantification for ELP coated and uncoated dental screws over 8 weeks post-surgery. n=8, * p<0.05. B) Histological sections of inserted screws at 1 week (top). Expanded histological images of bone contact with screw threads for BIC area quantification (bottom). Section thickness = 20–30 μm. Toluidine blue staining.

Figure 7

In vivo response to spin coated ELP films on cp-Ti dental screws implanted into rat femurs and tibias was tested using removal torque (RTQ) to measure the interfacial strength between the implant surfaces and peri-implant bone. Quantification extended over 8 weeks post-surgery; n=6 at 1 week and n=8 at weeks 4 and 8; * p<0.05.