Gene-Activated Titanium Surfaces Promote In Vitro Osteogenesis

Abstract

Purpose: Commercially pure titanium (CpTi) and its alloys possess favorable mechanical and biologic properties for use as implants in orthopedics and dentistry. However, failures in osseointegration still exist and are common in select individuals with risk factors such as smoking. Therefore, in this study, a proposal was made to enhance the potential for osseointegration of CpTi discs by coating their surfaces with nanoplexes comprising polyethylenimine (PEI) and plasmid DNA (pDNA) encoding bone morphogenetic protein-2 (pBMP-2).

Materials and methods: The nanoplexes were characterized for size and surface charge with a range of N/P ratios (the molar ratio of amine groups of PEI to phosphate groups in pDNA backbone). CpTi discs were surface characterized for morphology and composition before and after nanoplex coating using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and X-ray powder diffraction (XRD). The cytotoxicity and transfection ability of CpTi discs coated with nanoplexes of varying N/P ratios in human bone marrow-derived mesenchymal stem cells (BMSCs) was measured via MTS assays and flow cytometry, respectively.

Results: The CpTi discs coated with nanoplexes prepared at an N/P ratio of 10 (N/P-10) were considered optimal, resulting in 75% cell viability and 14% transfection efficiency. Enzyme-linked immunosorbent assay results demonstrated a significant enhancement in BMP-2 protein secretion by BMSCs 7 days posttreatment with PEI/pBMP-2 nanoplexes (N/P-10) compared to the controls, and real-time PCR data demonstrated that the BMSCs treated with PEI/pBMP-2 nanoplex-coated CpTi discs resulted in an enhancement of Runx-2, alkaline phosphatase, and osteocalcin gene expressions on day 7 posttreatment. In addition, these BMSCs demonstrated enhanced calcium deposition on day 30 posttreatment as determined by qualitative (alizarin red staining) and quantitative (atomic absorption spectroscopy) assays.

Conclusion: It can be concluded that PEI/pBMP-2 nanoplex (N/P-10)-coated CpTi discs have the potential to induce osteogenesis and enhance osseointegration.

Fig 1

I) Chart showing the particle size and zeta potential of PEI/pBMP-2 nanoplexes formed at N/P ratios of 1, 5, 10, 15 & 20. Values are expressed as mean ± SEM with n = 3. II) SEM images showing the surface topography of (a) Uncoated and (b) PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs.

Fig 2

AFM images showing the surface topography of (I) Uncoated and (II) PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs. a) Amplitude retrace and b) Histograms. III) Graph representing the root mean square roughness (RMS-R_q) of uncoated (red) and PEI/pBMP-2 nanoplex (N/P-10) coated (blue) CpTi discs. Values are expressed as mean ± SEM with n = 3.

Fig 3

Plots representing (I) XPS survey level scan spectra of: A) Uncoated B) pBMP-2 coated C) PEI coated and D) PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs. (II) XPS core level spectra of A) pBMP-2 coated B) PEI coated and C) PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs. III) X-ray diffraction patterns of uncoated (red) and PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs (blue).

Fig 4

I) Chart demonstrating the MTS cell viability analysis of BMSCs treated with PEI/pEGFP nanoplex coated CpTi discs at various N/P ratios (indicated). II) Histogram demonstrating the percent EGFP expression by BMSCs treated with PEI/pEGFP nanoplex coated CpTi discs at various N/P ratios (indicated) obtained by flow cytometry. BMSCs treated with uncoated, pEGFP and PEI coated CpTi discs served as controls. One-way ANOVA was employed to assess the significant differences between treatments and controls followed by Tukey’s post-test (*p < 0.05). Values are expressed as mean ± SEM with n = 3.

Fig 5

I) Gel electrophoresis image demonstrating the effect of heparin sodium salt on separating pBMP-2 from PEI. In the figure, N+H represents PEI/pBMP-2 nanoplex (N/P-10) with heparin, N-H indicates PEI/pBMP-2 nanoplex (N/P-10) without heparin or nanoplex alone, P+H represents pBMP-2 with heparin and P-H represents pBMP-2 without heparin or pBMP-2 alone. pBMP-2 with (P+H) and without (P-H) heparin. II) Graph demonstrating the cumulative release profile of PEI/pBMP-2 nanoplexes (N/P-10) from the CpTi discs with respect to time obtained using PicoGreen assay. pBMP-2 coated CpTi discs served as controls. Values are expressed as mean ± SEM with n = 3.

Fig 6

I) ELISA data demonstrating the amount of BMP-2 produced by BMSCs on day 7 post-treatment. II) Osteogenic gene expression by BMSCs on day 7 post-treatment obtained using real time PCR analysis (a) Runx-2 expression (b) Alkaline phosphatase (ALP) expression and (c) Osteocalcin expression. Here PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs served as treatments and uncoated CpTi discs, pBMP-2 and PEI coated CpTi discs served as controls. One-way ANOVA was employed to assess the significant differences between treatments and controls followed by Tukey’s post-test (*p < 0.05; **p < 0.01; ***p < 0.001). Values are expressed as mean ± SEM with n = 3.

Fig 7

I) Light microscopic images of alizarin red stained BMSCs on day 30 post-treatment as an indication of mineralization. a) BMSCs treated with uncoated; b) pBMP-2 coated; c) PEI coated; and d) PEI/pBMP-2 nanoplex (N/P-10) coated CpTi discs; BMSCs treated with uncoated, pBMP-2 and PEI coated CpTi discs served as controls. II) Chart representing the alizarin red stain quantification data of light microscopic images for indicated treatments obtained using ImageJ software. III) Atomic absorption spectrophotometric results of calcium levels produced by BMSCs on day 30 post-treatment. One-way ANOVA was employed to assess the significant differences between treatments and controls followed by Tukey’s post-test (*p < 0.05; **p < 0.01). Values are expressed as mean ± SEM with n = 3.