Hydrofluoric acid and nitric acid cotreatment for biofunctionalization of polyetheretherketone in M2 macrophage polarization and osteogenesis

Abstract

Due to its excellent mechanical and low‐friction properties, polyetheretherketone (PEEK) has been widely investigated for use in orthopedic applications over the past decade. However, the bioinertness and poor osteogenic properties of PEEK have hampered its clinical application. In this study, the surface of PEEK was modified by co‐treatment with hydrofluoric acid and nitric acid (AFN). The microstructures of the modified PEEK surfaces were investigated using scanning electron microscopy. The water contact angles of the surfaces were also measured. To evaluate their cytocompatibility, PEEK samples were used as substrates to culture rat bone mesenchymal stem cells, and cell adhesion, viability, and expression of specific marker genes were measured. Treatment of PEEK with AFN (PEEK‐AFN) was found to enable better osteoblast adhesion, spreading, and proliferation; the activity of alkaline phosphatase (an early osteogenic differentiation marker) was also found to be enhanced post‐treatment. Furthermore, PEEK‐AFN was able to modulate macrophage polarization and down regulated the expression of proinflammatory factors via inhibiting the NF‐κB pathway. Thus, treatment of PEEK with AFN could promote M2 polarization of the macrophages and stimulate the differentiation of osteoblasts. These results provide valuable information that could facilitate the use of PEEK‐based composites as bone implant materials.

Translating Material Science into Bone Regenerative Medicine Applications: State-of-The Art Methods and Protocols

“…The morphology of the biomaterial-seeded cells can also be examined by scanning electron microscopy (SEM) [ 14 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ], as well as the intracellular structures by transmission electron microscopy (TEM) [ 65 , 68 ]. Cell morphology and distribution can also be assessed using immunofluorescence staining of actin filaments, by means of anti-phalloidin antibodies among others, which allows for the analysis of the cytoskeleton conformation of cell growth in contact with the biomaterials [ 14 , 52 , 54 , 55 , 57 , 60 , 62 , 64 , 65 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 ].…”

“…Fluorescent live/dead assays are able to discriminate between live and dead cells by evaluating plasma membrane integrity and the activity of the esterase enzyme, both maintained only in viable cells [ 54 , 55 , 60 , 62 , 70 , 74 , 75 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 ]. Other commercial colorimetric assays for the evaluation of cell viability are available on the market and, among the most widely used are the cell counting kit-8 (CCK-8) assay that allows a sensitive colorimetric measure of viable cells, using a water-soluble tetrazolium salt that produces, in presence of active dehydrogenases in living cells, an orange formazan product, and the amount of formazan produced is directly proportional to the number of viable cells [ 55 , 57 , 58 , 62 , 73 , 76 , 80 , 81 , 85 , 86 , 87 , 88 , 89 ]. Moreover, the Alamar Blue assay method is frequently used to assess the metabolic activity of proliferating cells: the active resazurin compound, upon entering living cells, is reduced to resorufin, a red fluorescent molecule that can be quantified [ 14 , 59 , 64 , 72 , 75 , 90 , 91 ].…”

“…They can proliferate and differentiate into osteoblasts upon appropriate stimuli. MSCs isolated from bone marrow are regarded as the gold standard [ 14 , 53 , 55 , 57 , 67 , 68 , 73 , 76 , 77 , 79 , 83 , 91 , 94 , 98 , 107 , 108 , 109 ]. MSC-like cells isolated from the stromal vascular fraction of the adipose tissue, namely, adipose-derived stem cells, ASC, are seldom used in selected applications [ 2 , 65 , 72 , 82 , 89 , 104 , 109 , 110 , 111 , 112 ].…”

“…In the vast majority of papers in the literature, the expression levels of one or more genes associated with osteoblast differentiation are usually evaluated by quantitative real-time PCR (qRT-PCR) and/or Western blot and/or immunofluorescence analysis and/or ELISA assay [ 14 , 55 , 57 , 58 , 60 , 62 , 64 , 65 , 68 , 69 , 70 , 72 , 73 , 76 , 77 , 79 , 80 , 82 , 83 , 84 , 86 , 87 , 89 , 90 , 91 , 94 , 96 , 98 , 100 , 108 , 109 , 113 ]. Table 3 reports the most representative osteogenic genes usually analyzed.…”

Bioinspired Modifications of PEEK Implants for Bone Tissue Engineering

“…Fukuda et al found that the plasma treatment and subsequent phosphorylation on the PEEK surface attenuated the phenotypic polarization of RAW264.7 macrophages to an inflammatory phenotype (Fukuda et al, 2019). A recent study revealed that PEEK modified with hydrofluoric acid and nitric acid could promote the polarization of macrophages (M2 phenotype) and inhibit the expression of proinflammatory factors via the NF-κB pathway (Huo et al, 2020).…”