Osteogenic Surface Modification Based on Functionalized Poly-P-Xylylene Coating

Abstract

The biotechnology to immobilize biomolecules on material surfaces has been developed vigorously due to its high potentials in medical applications. In this study, a simple and effective method was designed to immobilize biomolecules via amine-N-hydroxysuccinimide (NHS) ester conjugation reaction using functionalized poly-p-xylylene coating on material surfaces. The NHS ester functionalized coating is synthesized via chemical vapor deposition, a facile and solvent-less method, creating a surface which is ready to perform a one-step conjugation reaction. Bone morphogenetic protein 2 (BMP-2) is immobilized onto material surfaces by this coating method, forming an osteogenic environment. The immobilization process is controlled at a low temperature which does not damage proteins. This modified surface induces differentiation of preosteoblast into osteoblast, manifested by alkaline phosphatase (ALP) activity assay, Alizarin Red S (ARS) staining and the expression of osteogenic gene markers, Alpl and Bglap3. With this coating technology, immobilization of growth factors onto material surface can be achieved more simply and more effectively.

Fig 1. Schematic figure of (a) synthesis of 4-N-hydroxysuccinimide (NHS) ester-[2.2]paracyclophane and (b) immobilization of BMP-2.

(a) Synthetic route of 4-N-hydroxysuccinimide ester-[2.2]paracyclophane 4. (b) CVD polymerization of 4 to poly(4-N-hydroxy-succinimide ester-p-xylylene-co-p-xylylene) (coating 5) and immobilization of protein by forming an amide bond between protein and coating 5.

Fig 2. IRRAS characterization of (a) coating 5 modified surface and (b) coating 5/BMP-2 modified surface.

Both of the modifications were on a gold-coated silicon substrate. Three significant peaks, which are characters of asymmetric stretching bands of NHS ester C = O were detected as 1770, 1739, and 1716 cm^-1 in S1(a). Peaks at 1217 cm^-1 and 1072 cm^-1 were attributed to N-O and C-O stretch, respectively. In S1 (b), peak of N-O (1207 cm^-1) and C-O (1067 cm^-1) stretch were reduced and one of the C = O peak (1716 cm^-1 in (a) and 1709 cm^-1 in (b)) strongly reduced after immobilization of BMP-2. The characterization peaks of BMP-2 appeared on the bands around 3247 cm^-1 for N-H and 3490 cm^-1 for O-H.

Fig 3. XPS survey spectra of (a) coating 5 modified surface and (b) coating 5/BMP-2 modified surface.

The atomic concentration of C, N and O are 77.3%, 4.55% and 18.22% on the coating 5 modified surface and 65.0%, 9.5% and 25.5% on the coating 5/BMP-2 modified surface. The N composition significantly increased due to immobilization of BMP-2.

Fig 4. QCM analysis of coating 5 modified surface.

Curve (a) is the result of BMP-2 solution injected and flowing through coating 5 modified surface, and at the end of the measurement, the amount of BMP-2 adsorption on coating 5 modified surface is 6.04·10⁻¹² mol cm^-2. Curve (b) is the result of BMP-2 solution injected and flowing through parylene C-modified surface, and at the end of the measurement, the amount of BMP-2 adsorption on parylene C surface is 3.81·10⁻¹² mol cm^-2. Curve (c) is the result of BMP-2 solution injected and flowing through PEG5000 modified surface, and at the end of the measurement, only 1.62·10⁻¹⁴ mol cm^-2 of BMP-2 adsorbed on the surface. Curve (d) is the result of BMP-2 primary antibody solution injected and flowing through coating 5/BMP-2 modified surface, and at the end of the measurement, the amount of BMP-2 primary antibody adsorption on coating 5 modified surface is 2.43·10⁻¹² mol cm^-2. Curve (e) is the result of BMP-2 primary antibody solution injected and flowing through coating 5 modified surface, and at the end of the measurement, the amount of BMP-2 primary antibody adsorption on coating 5 modified surface is 2.35·10⁻¹² mol cm^-2.

Fig 5. ALP activity assay and ARS staining.

ALP activity and calcium deposits of MC3T3-E1 preosteoblasts were evaluated on coating 5/BMP-2 modified, coating 5 modified and unmodified surface. (a) ALP staining assay and (b) quantification results of ALP at day 4, 7, 11. (c) ARS staining and (d) quantification of calcium deposits on days 7, 14 and 21. The asterisks indicate significant differences (*P<0.05 and **P<0.01) between coating 5/BMP-2 modified, coating 5 modified and unmodified surface at each time point.

Fig 6. Relative gene expression of (a) Alpl (gene marker of ALP) and (b) Bglap3 (gene marker of osteocalcin).

The effect of coating 5/BMP-2 modified, coating 5 modified and unmodified surface on relative mRNA expression was assayed on days 0, 4, 7, 11, and 14. The asterisks indicate significant differences (*P<0.05 and **P<0.01) between coating 5/BMP-2 modified, coating 5 modified and unmodified surface at each time point.