Aspartic and Glutamic Acid Templated Peptides Conjugation on Plasma Modified Nanofibers for Osteogenic Differentiation of Human Mesenchymal Stem Cells: A Comparative Study

Abstract

Optimization of nanofiber (NF) surface properties is critical to achieve an adequate cellular response. Here, the impact of conjugation of biomimetic aspartic acid (ASP) and glutamic acid (GLU) templated peptides with poly(lactic-co-glycolic acid) (PLGA) electrospun NF on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) was evaluated. Cold atmospheric plasma (CAP) was used to functionalize the NF surface and thus to mediate the conjugation. The influence of the CAP treatment following with peptide conjugation to the NF surface was assessed using water contact angle measurements, Fourier-Transform Infrared Spectroscopy (FTIR) and X-ray Photoelectron Spectroscopy (XPS). The effect of CAP treatment on morphology of NF was also checked using Scanning Electron Microscopy (SEM). Both the hydrophilicity of NF and the number of the carboxyl (-COOH) groups on the surface increased with respect to CAP treatment. Results demonstrated that CAP treatment significantly enhanced peptide conjugation on the surface of NF. Osteogenic differentiation results indicated that conjugating of biomimetic ASP templated peptides sharply increased alkaline phosphatase (ALP) activity, calcium content, and expression of key osteogenic markers of collagen type I (Col-I), osteocalcin (OC), and osteopontin (OP) compared to GLU conjugated (GLU-pNF) and CAP treated NF (pNF). It was further depicted that ASP sequences are the major fragments that influence the mineralization and osteogenic differentiation in non-collagenous proteins of bone extracellular matrix.

Enhanced osteogenesis of human mesenchymal stem cells by self‐assembled peptide hydrogel functionalized with glutamic acid templated peptides

“…This result suggests that increase in glutamic acid repeat units induces cell proliferation. Our results are consistent with an early report that showed enhanced hMSCs proliferation when seeded on glutamic acid conjugated synthetic electrospun nanofibers (Karaman et al, 2016;Onak et al, 2018). The potential of glutamic acid in cell proliferation can be related to the presence and more influential role of glutamic acid in integrin binding motif.…”

“…All chemicals used for peptide synthesis were purchased from AAPPTEC (Louisville, KY, USA). KLD (the peptide with sequence of Ac-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-NH 2 ), KLD-EEGGC (the peptide with sequence of Ac-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Glu-Glu-Gly-Gly-Cys-NH 2 ), and KLD-EEEEE (the peptide with sequence of Ac-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Lys-Leu-Asp-Leu-Glu-Glu-Glu-Glu-Glu-NH 2 ) were synthesized on 4-methylbenzhydrylamine (MBHA) resin (0.67 mmol/g loading capacity) by using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry as previously described by using the automated peptide synthesis device (AAPPTEC Focus Xi, Louisville, KY, USA) (Onak et al, 2018 (4.2 mM), KCl (3.0 mM), K 2 HPO 4 (1.0 mM), MgCl 2 .6H 2 O (1.5 mM), CaCl 2 (2.5 mM), and NaSO 4 (0.5 mM) in deionized water (Kokubo & Takadama, 2006). Then 60-mM NaHCO 3 solution was added until pH 7.4.…”

“…As an alternate approach, incorporation of small peptides within a scaffold that can guide biomineralization has been proposed due to these peptides' capability of high resistance to pH or temperature changes and precise control over the chemical composition. A glutamic acid-rich peptide (a sequences of glutamic acids) derived from non-collagenous proteins such as osteonectin, osteocalcin (OC), OP, and bone sialoprotein mimics the terminal region of the glycoprotein of bone and acts as a calcium ion nucleation points (Onak et al, 2018;Sarvestani, He, & Jabbari, 2008).…”

“…Furthermore, glutamic acid-rich peptide-conjugated electrospun nanofibers significantly enhanced the osteogenic differentiation of human bone marrow derived mesenchymal stem cells (hMSCs) (Karaman et al, 2016;Onak et al, 2018). Because relatively twodimensional (2D) structure of nanofibers has been reported to remain insufficient environment for bone tissue regeneration, researchers have been focused to develop three-dimensional (3D) scaffolds that enhance precise formation and mineralization of functional bone tissues.…”

“…In this study, first, the functional glutamic acid peptides (EEGGC and EEEEE) was applied to modify KLD peptide for designing and fabricating KLD-EEGGC and KLD-EEEEE and self-assembled in different concentrations (0.5%, 1%, and 2%). The performance of functionalization of KLD self-assembled peptide with different glutamic acid templated peptides in different concentrations was investigated and then the influence of the EEGGC and EEEEE peptide sequences, which are previously reported as osteogenesis and biomineralization inducing epitopes (Onak et al, 2018).…”