Visualization and quantification of the bioactive molecules immobilized at the outmost surface of PLLA-based biomaterials

Abstract

Our ongoing work concerns the modification of poly(L-lactic acid) (PLLA) scaffolds with oligo(lactic acid)-oligo peptide heteroconjugates as a means to improve their bioactivity. Regrettably, the initial cell attachment exhibited by these scaffolds was unsatisfactory, most likely owing to an insufficient density of peptide molecules bound to the outermost surface of the scaffolds. However, to distinguish surface-bound peptides from those embedded in the matrices is difficult by conventional surface analysis techniques such as energy dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy because they detect signals from the bulk surface to a given depth. In the present study, we succeeded in visualizing and quantifying the peptide molecules at the outmost surface only by staining them with an aqueous solution of fluorescein isothiocyanate (FITC). FITC reacts only with the peptides at the surface but not those embedded in the matrices. The uniformity of the peptide coverage can be observed using fluorescence microscopy, and the peptide surface density can be quantified by dissolving the matrices and measuring the intensity of the resultant solution. In addition, by using this quantification method, an effective strategy to increase the peptide density at the PLLA surface was developed. This strategy involves immersing an electrospun nanofibrous sheet composed of PLLA and oligo(D-lactic acid)-IleLysValAlaVal (IKVAV) heteroconjugate (99:1 in weight) in water, warming it to above the glass transition temperature of PLLA (60 °C), and slowly cooling it to room temperature. The surface IKVAV density was highest when treated at 60 °C. During the electrospinning, the IKVAV peptide is mainly embedded in the matrices, but it is exposed to the aqueous phase at the scaffold surface and fixed there by the in-water heating/cooling process.