Calcified Surfaces

G. K. Toworfe, R. J. Composto, C. S. Adams, I. M. Shapiro, P. Ducheyne.  Initial attachment of MC3T3-E1 osteoblast cells  on functionalized surfaces coated with calcium phosphate.  In:  Biomaterials in Regenerative  Medicine - The Advent of Combination Products. 
Soc Biomater Trans 2004, 104.

The use of synthetic calcium phosphate grafts for bone tissue repair therapy has received considerable attention.Tissue engineering techniques which combine synthetic grafts with molecules and cells are considered as viable long term solutions for bone tissue repair and reconstruction procedures.This study models the changing surface characteristics of calcium phosphates.By combining matrices and biological molecules, the synergistic effect on bone cell function is determined.  Materials that are compatible with bone tissue either have a calcium phosphate surface upon implantation, or develop such surfaces (1) with characteristics similar to those of the bone mineral phase.The self assembled monolayer (SAM) technology enables modeling of surface functional groups on biomaterials while the concept of ion-selective precipitation reaction causes formation of calcium phosphate (CaP) layer on these functionalized surfaces (2).Strongly negative surfaces (COOH or OH terminated) have more powerful induction capability for the heterogeneous nucleation of hydroxyapatite-like layer, while nucleation could be prohibited on an NH2-terminated surface.

In this study, we use the SAM technology to create highly controlled surface chemistries, specifically surfaces with hydroxyl, carboxyl and amine-terminated groups.  We hypothesized that: the type of coverage of CaP is controlled by functional groups on the surface and that bone cell adhesion, spreading and function is also dependent on the type of functional group.   Using the SAM technique, 3-aminopropyltriethoxysilane (APTES), triethoxysilylpropyl succinic anhydride (TESPSA) and glycidoxypropyl tri-methoxysilane (GPTMS) were grafted onto treated silicon wafers to yield NH2 and –COOH and –OH functionalized surfaces respectively.The SAM surfaces were characterized by contact angle measurements and ellipsometry to confirm the presence of grafted molecules and surface wettability.A CaP film was formed on the SAMs by immersing specimens in a solution which simulates the electrolyte content of physiological fluids, i.e. tris buffer solution (pH 7.4, at 37oC), complemented with 2.5 mM Ca 2+, 152 mM Na+, 136 mM Cl-, 5.0 mM K+, 1.5 mM Mg+, 27 mM HCO3-, and 0.5 mM SO4 2-.  The phosphate concentration, i.e. 2.5 mM HPO4 2-, was slightly elevated in order to increase solution supersaturation.Prior to cell attachment, Fn molecules were adsorbed on the CaP-coated SAM model surfaces.

The structure and composition of the CaP film formed on the homogeneous SAM surfaces (NH2, COOH, OH-terminated) were characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis (SEM/EDX), and Rutherford backscattering spectrometry (RBS) to determine compound stochiometry, crystal structure, variation of composition and depth profiling.FTIR spectra obtained on the substrates show the dependence of the morphology of the CaP coating on both the type of surface functionality and on the duration of coating.FTIR spectrum of the CaP coating on GPTMS-grafted silicon oxide surfaces exhibited P-O (1089 cm-1, 970 cm-1, 595 cm-1), P-OH (1216 cm-1, 865 cm-1) and C-O doublet (1457 cm-1, 1421 cm-1) absorption bands characteristic of a poorly crystallized calcium hydroxyapatite (c-Ap) (Fig. 1).The phosphate bands were stronger on the hydroxyl-terminated surfaces.

Initial cell attachment and proliferation of MC3T3-E1 cells (with integrins that are responsive to the RGD binding motif) maintained in culture medium (Dulbecco’s modified Eagle’s medium) supplemented with 2 mM L-glutamine, 10% fetal calf serum and antibiotics was studied by plating 2 x105 cells/cm2 on substrates for 30 min, 1 h, 5 h, and 24 h.Figure 2 shows increased initial cell attachment on the CaP coating on OH-terminated SAM, after cells were seeded and allowed to attach for 1h, compared to the COOH and NH2 terminated surfaces.

References

1. Ducheyne et al.Clin. Orthop. Rel. Res. (1992)

2. Marcolongo et al, J. Biomed. Mater. Res. (1998)