The dependence of MG63 osteoblast responses to (meth)acrylate-based networks on chemical structure and stiffness

Abstract

The cell response to an implant is regulated by the implant's surface properties including topography and chemistry, but less is known about how the mechanical properties affect cell behavior. The objective of this study was to evaluate how the surface stiffness and chemistry of acrylate-based copolymer networks affect the in vitro response of human MG63 pre-osteoblast cells. Networks comprised of poly(ethylene glycol) dimethacrylate (PEGDMA; Mn approximately 750) and diethylene glycol dimethacrylate (DEGDMA) were photopolymerized at different concentrations to produce three compositions with moduli ranging from 850 to 60 MPa. To further decouple chemistry and stiffness, three networks comprised of 2-hydroxyethyl methacrylate (2HEMA) and PEGDMA or DEGDMA were also designed that exhibited a range of moduli similar to the PEGDMA-DEGDMA networks. MG63 cells were cultured on each surface and tissue culture polystyrene (TCPS), and the effect of copolymer composition on cell number, osteogenic markers (alkaline phosphatase specific activity and osteocalcin), and local growth factor production (OPG, TGF-beta1, and VEGF-A) were assessed. Cells exhibited a more differentiated phenotype on the PEGDMA-DEGDMA copolymers compared to the 2HEMA-PEGDMA copolymers. On the PEGDMA-DEGDMA system, cells exhibited a more differentiated phenotype on the stiffest surface indicated by elevated osteocalcin compared with TCPS. Conversely, cells on 2HEMA-PEGDMA copolymers became more differentiated on the less stiff 2HEMA surface. Growth factors were regulated in a differential manner. These results indicate that copolymer chemistry is the primary regulator of osteoblast differentiation, and the effect of stiffness is secondary to the surface chemistry.

Figure 1

Representative load versus displacement curves of bone wafers using nanoindentation. The copolymer networks tested in this study are included for comparative purposes. Calculated elastic moduli for each bone type, region, and degree of mineralization are listed in Table 3

Figure 2

FTIR-ATR spectra of (A) PEGDMA-co-DEGDMA and (B) 2HEMA-PEGDMA/DEGDMA surfaces. Peaks corresponding to key chemical components of the networks are indicated. Dotted lines represent the wavenumber corresponding to the C=O and C-O-C peaks for 90PEG. It can be observed that these peak wavenumbers shift for the other copolymer systems relative to the 90PEG.

Figure 3

Scanning electron micrographs of MG63 cells after 24h in culture on 10PEG, 50PEG, and 90PEG (A–F) and 65HEMA, 80HEMA, and 98HEMA (G–L) at 500× (A–C and G–I) and 2kX (D–F and J–L) magnifications. Scale bar represents 40µm and 10µm for 500× and 2kX images, respectively.

Figure 4

Effect of composition and stiffness on differentiation of MG63 cells. Cells were cultured on tissue culture polystyrene (TCPS) or the copolymer surfaces with various weight ratios of PEGDMA to DEGDMA: 90%PEGDMA (90PEG), 50%PEGDMA (50PEG), and 10%PEGDMA (10PEG). At confluence (A) cell number, (B) alkaline phosphatase specific activity, (C) osteocalcin, (D) OPG, (E) VEGF-A, and (F) total TGF-β1 levels were measured. Data is the ratio of polymer surfaces to TCPS control for at least four independent experiments.*p<0.05, polymer surface vs. TCPS; #p<0.05, polymer surface vs. 10PEG; $p<0.05, 90PEG vs. 50PEG.

Figure 5

Effect of composition and stiffness on differentiation of MG63 cells. Cells were cultured on either tissue culture polystyrene (TCPS) or the copolymer surfaces (65HEMA, 80HEMA, and 98HEMA) with similar stiffness values to PEGDMA-DEGDMA materials. 90PEG was included as a positive control for the PEGDMA-DEGDMA surfaces. At confluence, (A) cell number, (B) alkaline phosphatase specific activity, (C) osteocalcin, (D) OPG, (E) VEGF-A, and (F) total TGF-β1 levels were measured. *p<0.05, HEMA copolymer surface vs. TCPS; #p<0.05, HEMA copolymer surface vs. 90PEG; ‡p<0.05, 80HEMA or 98HEMA vs. 65HEMA.