Isotopic tracing for calculating the surface density of arginine-glycine-aspartic acid-containing peptide on allogeneic bone

Abstract

Objective: To investigate the feasibility of determining the surface density of arginine-glycine-aspartic acid (RGD) peptides grafted onto allogeneic bone by an isotopic tracing method involving labeling these peptides with (125) I, evaluating the impact of the input concentration of RGD peptides on surface density and establishing the correlation between surface density and their input concentration.

Methods: A synthetic RGD-containing polypeptide (EPRGDNYR) was labeled with (125) I and its specific radioactivity calculated. Reactive solutions of RGD peptide with radioactive (125) I-RGD as probe with input concentrations of 0.01 mg/mL, 0.10 mg/mL, 0.50 mg/mL, 1.00 mg/mL, 2.00 mg/mL and 4.00 mg/mL were prepared. Using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linking agent, reactions were induced by placing allogeneic bone fragments into reactive solutions of RGD peptide of different input concentrations. On completion of the reactions, the surface densities of RGD peptides grafted onto the allogeneic bone fragments were calculated by evaluating the radioactivity and surface areas of the bone fragments. The impact of input concentration of RGD peptides on surface density was measured and a curve constructed.

Results: Measurements by a radiodensity γ-counter showed that the RGD peptides had been labeled successfully with (125) I. The allogeneic bone fragments were radioactive after the reaction, demonstrating that the RGD peptides had been successfully grafted onto their surfaces. It was also found that with increasing input concentration, the surface density increased.

Conclusion: It was concluded that the surface density of RGD peptides is quantitatively related to their input concentration. With increasing input concentration, the surface density gradually increases to saturation value.

Figure 1

The spectrum of ¹²⁵I‐RGD sephadex‐G75.

Figure 2

Curve showing the relationship between surface density and input concentration of RGD peptides.

Figure 3

The complex stereo surface micromorphology of allogeneic bone as shown by scanning electron microscopy (×2000).