Overcoming PEEK bioinertness with peptide functionalization for bone regeneration

The rise in life expectancy in Western countries has led to an increased preva-lence of bone and cartilage pathologies. Consequently, there is a growing demand for the optimization of orthopedic implants. While traditional metal-lic implants have been utilized for many years, their mismatch with the elastic modulus of cortical bone can pose issues. Polymeric materials, though generally unable to withstand repetitive loads without plastic deformations, have exceptions such as polyetheretherketone (PEEK). PEEK, a thermoplastic polymer, has recently been employed in bone tissue engineering due to its biocompatibility and mechanical properties comparable to human bone. How-ever, PEEK is a bio-inert material and, when implanted, fails to interact with host tissues, resulting in poor integration. Our approach to address this limi-tation involves the chemical functionalization of PEEK surface with bioactive motifs. In our grafting strategy, carbonyl groups on the PEEK surface react with an amino-oxy group (Aoa) ad hoc inserted into a peptide sequence. This method allows for the covalent anchoring of bioactive peptides in a single step without the use of condensing agents or organic solvents. In this study, 3D-printed PEEK disks enriched with biochemical factors, specifically peptides derived from human proteins: the (351-359) sequence of human Vitronectin Precursor, its dimeric retroinverted analog D2HVP, and the (48-69) sequence of human Bone Morphogenetic Protein 2 (GBMP1α), are used to enhance interaction with human osteoblasts (HOB). Moreover, the PEEK surface was enriched with self-assembling peptides (SAP) (EAK and EYK). When these peptides self-assemble, they generate an environment that mimics extracel-lular matrices, serving as a physiological scaffold to augment cell activities. The effect of GBMP1α grafted via oxime was compared with the anchoring of the same sequence through the photoactivation of an azido group introduced at its N-terminus, while EAK, grafted via oxime, was compared with EYK self-aggregated onto the PEEK surface. The functionalized PEEK surfaces, characterized through X-ray Photoelectron Spectroscopy, Water Contact An-gle, and Force Spectroscopy assays, exhibit improved properties compared to plain PEEK. Subsequent biological tests, including Live and Dead and SEM images, AlamarBlueTM and AlizarinRed assays, and Quantitative-Real Time Polymerase Chain Reaction (qRT-PCR), reveal enhanced cell activities compared to control. Because surface morphology plays a critical role in cell interactions the optimal roughness was identified. PEEK sanblasted with corundum sizes 110 µm (R110-PEEK) shows increased hydrophobicity and the highest enhancements in cell proliferation. Moreover, R110-PEEK function-alized simultaneously with GBMP1α and EAK via oxime exhibits synergistic effects, as demonstrated by heightened cell proliferation, calcium deposition, and expression of genes related to osteogenic activity compared to plain PEEK.