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. 2023 May 21;16(10):3869.
doi: 10.3390/ma16103869.

Strategies for the Covalent Anchoring of a BMP-2-Mimetic Peptide to PEEK Surface for Bone Tissue Engineering

Leonardo Cassari  1 , Annj Zamuner  2 , Grazia Maria Lucia Messina  3 , Martina Marsotto  4 , Hao-Chen Chang  5 , Trevor Coward  5 , Chiara Battocchio  4 , Giovanna Iucci  4 , Giovanni Marletta  3 , Lucy Di Silvio  5 , Monica Dettin  1
Affiliations

Strategies for the Covalent Anchoring of a BMP-2-Mimetic Peptide to PEEK Surface for Bone Tissue Engineering

Leonardo Cassari et al. Materials (Basel). .

Abstract

Researchers in the field of tissue engineering are always searching for new scaffolds for bone repair. Polyetheretherketone (PEEK) is a chemically inert polymer that is insoluble in conventional solvents. PEEK's great potential in tissue engineering applications arises from its ability to not induce adverse reactions when in contact with biological tissues and its mechanical properties, which are similar to those of human bone. These exceptional features are limited by the bio-inertness of PEEK, which causes poor osteogenesis on the implant surface. Here, we demonstrated that the covalent grafting of the sequence (48-69) mapped on the BMP-2 growth factor (GBMP1α) significantly enhances the mineralization and gene expression of human osteoblasts. Different chemical methods were employed for covalently grafting the peptide onto 3D-printed PEEK disks: (a) the reaction between PEEK carbonyls and amino-oxy groups inserted in the peptides' N-terminal sites (oxime chemistry) and (b) the photoactivation of azido groups present in the peptides' N-terminal sites, which produces nitrene radicals able to react with PEEK surface. The peptide-induced PEEK surface modification was assessed using X-ray photoelectron measurements, while the superficial properties of the functionalized material were analyzed by means of atomic force microscopy and force spectroscopy. Live and dead assays and SEM measurements showed greater cell cover on functionalized samples than the control, without any cytotoxicity induction. Moreover, functionalization improved the rate of cell proliferation and the amount of calcium deposits, as demonstrated by the AlamarBlue™ and alizarin red results, respectively. The effects of GBMP1α on h-osteoblast gene expression were assayed using quantitative real-time polymerase chain reaction.

Keywords: 3D printing; BMP-2; PEEK; bone tissue engineering; human osteoblasts; peptides; surface functionalization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Patterned surface of 3D-printed PEEK disk.
Figure 2
Figure 2
X-ray photoelectron spectroscopy results. Signals C1s, N1s, and O1s of PEEK functionalized with BMP through aminooxy chemistry are shown in (a–c), respectively.
Figure 3
Figure 3
Spectroscopy analysis on non-functionalized PEEK and PEEK functionalized with GBMP1α through amino-oxy (PEEK-AoaBMP) and azido groups (PEEK-N3BMP). Surface Young’s modulus (a) and adhesion forces (b) are presented. Values are reported as mean ± SD. n = 3. * p-value < 0.05.
Figure 4
Figure 4
Live and dead staining of human osteoblasts cultured for 48 h on non−functionalized (a,d) and functionalized PEEK (through amino-oxy (b,e); through azido groups (c,f)). Very few dead cells (in red) are seen in (f) in contrast to a large number of viable cells (in green) in all the images (a–f). The magnifications used are 10× (a–c) and 20× (d–f).
Figure 5
Figure 5
Scanning electron microscopy images of human osteoblast cells 48 h after seeding. The samples shown are the unfunctionalized PEEK sample (a) and PEEK functionalized with GBMP1α through amino-oxy (b) and azido groups (c). The scale bars in the images are 20 μm.
Figure 6
Figure 6
Quantification of the amount of calcium deposition through alizarin red assay 21 days after the seeding. The mineralization test was performed on non-functionalized PEEK and PEEK functionalized with GBMP1α through amino-oxy (PEEK-AoaBMP) or azido groups (PEEK-N3BMP). * p-value < 0.05 and **** p-value < 0.0001.
Figure 7
Figure 7
Human osteoblast proliferation evaluated on non-functionalized PEEK and PEEK functionalized with GBMP1α through amino-oxy (PEEK-AoaBMP) and azido groups (PEEK-N3BMP). AlamarBlue™ tests were assessed on days 1, 4, 7, 14, and 21 post-human osteoblast seeding. ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001.
Figure 8
Figure 8
Gene expression of SRY−Box transcription factor 9 (SOX9), Runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteocalcin (OCN) in non-functionalized PEEK and PEEK functionalized with GBMP1α through amino-oxy (PEEK-AoaBMP) and azido groups (PEEK-N3BMP). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. Relative gene expression was calculated with the 2−ΔΔCt method. Time points: 1 day (a), 7 days (b), 14 days (c), and 21 days (d) from cell seeding. * p-value < 0.05, ** p-value < 0.01, *** p-value < 0.001, and **** p-value < 0.0001.
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Grants and funding

This research was funded by the University of Padova, PhD program fund 064984, Department of Industrial Engineering, and by the University of Padova, fund: DOR Dettin 2021, Finanziamento Dipartimenti di Eccellenza 2023-2027 (Art. 1, commi 314-337 Legge 11/12/2016, n. 232).

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