Introduction: Precursor and mature osteoblasts are involved in bone formation, but mechanisms that control this differentiation are poorly understood. The objectives of our research program are the development of a smart and bioactive material mimicking the native bone extracellular matrix (ECM) and their impact on the differentiation of human mesenchymal stem cell (hMSC).
For this purpose, peptides were grafted onto the surface of polyethylene terephthalate (PET) in order to induce an osteogenic differentiation of MSC. Our objective is to mimic ECM by controlling the spatial organization of these peptides on the surface at the micro and/or nanoscale[1]. Four grafted peptides were used: a RGD peptide and the heparin-binding peptide FHRRIKA were selected to promote cell adhesion and BMP-2[2] and the OGP (osteogenic growth protein)[3] were chosen due to their ability to promote bone differentiation.
Experimental Methods: In order to design the above-mentioned material, PET surfaces were modified according to the method described by Chollet et al[4]. First, materials were modified by hydrolysis and oxidation to generate COOH functionalities onto PET. Then, PET was immersed in 0.2 M of 1-Éthyl-3-(3-diméthylaminopropyl)carbodiimide + 0.1 M N-Hydroxysuccinimide in 0.1 M 4-Morpholineethanesulfonic acid buffer in order to activate the surface for further peptide grafting. COOH densities were determined by the toluidine blue-O method. All samples were analysed with XPS, AFM, contact angle and fluorescence microscopy.
Results: Figure 1 shows a 6-fold increase of COOH density after material hydrolysis and oxidation. As expected the decrease in COOH density observed after conversion of acidic groups into activated esters and peptide immobilization confirme successful grafting.
These results were further confirmed by XPS (not shown). In addition the surface grafting of fluorophore-labeled peptides allowed measuring a steady fluorescent signal throughout the PET and confirm peptide homogeneous grafting at a density close to 1 pmol.mm-2 after one week of rinsing (fig. 2).
In order to evaluate the eventual impact of roughness and hydrophilicity on cell behavior, we used AFM and contact angle measurement to characterize surfaces at each step of the functionalization. Although as slight increase of the roughness and a hydrophobicity decrease over the modification steps, both features are similar on the peptide grafted surfaces.
Finally a 4-week cell culture has been performed on the homogenous surfaces, and several markers (STRO-1, OPN, Runx2 and ALP) are currently under investigation to evaluate the differentiation into the osteoblastic lineage.
Conclusion: All the characterisations have been performed on homogenous surfaces, and cell culture preliminary results exhibit different hMSC behaviors. Further work will consist in the production, characterisation and biological assessment of the micro and nano-structured surfaces in comparison of homogenous surfaces.
Pascale Chevallier; Christine Labrugère
References:
[1] L Hench, & J. Polak, Science, 2002; 295(5557): 1014-1017
[2] OF Zouani, C Chollet, B Guillotin, MC Durrieu, Biomaterials, 2010, 31(32), 8245-8253
[3] S Panseri, L Russo, M Montesi, F Taraballi, C Cunha, M Marcacci, & L Cipolla, MedChemComm, 2014, 5(7), 899-903.
[4] C Chollet, C Chanseau, B Brouillaud, & MC Durrieu, Biomolecular engineering, 2007, 24, 5:477-482