RGD-Functionalized Nanofibers Increase Early GFAP Expression during Neural Differentiation of Mouse Embryonic Stem Cells

Abstract

Stem cell differentiation towards a specific lineage is controlled by its microenvironment. Polymer scaffolds have long been investigated to provide microenvironment cues; however synthetic polymers lack the specific signaling motifs necessary to direct cellular responses on their own. In this study, we fabricated random and aligned poly(ε-caprolactone) nanofiber substrates, surfacefunctionalized with RGD via strain-promoted azide-alkyne cycloaddition, that were used to investigate the role of a covalently tethered bioactive peptide (RGD) and nanofiber orientation on neural differentiation of mouse embryonic stem cells. Gene and protein expression showed neural differentiation progression over 14 days, with similar expression on RGD random and aligned nanofibers for neurons and glia over time. The high levels of glial fibrillary acidic protein expression at early time points were indicative of neural progenitors, and occurred earlier than on controls or in previous reports. These results highlight the influence of RGD binding versus topography in differentiation.

RGD-Modified Nanofibers Enhance Outcomes in Rats after Sciatic Nerve Injury

“…In our study, the use of SPAAC resulted in efficient tethering of peptide to the surface of PCL nanofibers. While nanofibers functionalized with peptides via SPAAC have been previously used for directing stem cells [22,23,24] as well as Schwann cells [16] in vitro , the current study is the first effort to evaluate those substrates in vivo in a peripheral nerve injury model. With the ability to tether peptides onto nanofibers [16,22,24], we hypothesized that a longitudinally aligned scaffold functionalized with bioactive molecules would enhance nerve regeneration [31] and analyzed this assumption through functional tests and histological evaluations.…”

“…While nanofibers functionalized with peptides via SPAAC have been previously used for directing stem cells [22,23,24] as well as Schwann cells [16] in vitro , the current study is the first effort to evaluate those substrates in vivo in a peripheral nerve injury model. With the ability to tether peptides onto nanofibers [16,22,24], we hypothesized that a longitudinally aligned scaffold functionalized with bioactive molecules would enhance nerve regeneration [31] and analyzed this assumption through functional tests and histological evaluations. As RGD had been previously shown to enhance SC adhesion and proliferation compared to other peptides [32], and SC are critical to nerve regeneration [9], we investigated if RGD functionalization would improve the neuroregenerative response compared to non-functionalized fibers.…”

“…DIBO-end-functionalized poly( ɛ -caprolactone) was synthesized (Scheme 1), electrospun, and modified with GRGDS peptide using methods described previously [24]. Briefly, using standard drying techniques, an ampoule was filled with ɛ -CL (0.3340 mol, 37.01 mL), DIBO (0.5148 mmol, 113.25 mg), toluene (120.93 mL), and Mg(BHT) 2 (THF) 2 (0.285 mmol, 173.00 mg).…”

“…An efficient tool to chemically modify a polymeric fiber with peptides is strain-promoted azide–alkyne cycloaddition (SPAAC), a facile and quantitative post-fabrication modification chemistry requiring no catalyst or chemical activation. SPAAC has been used to fabricate YIGSR- [22,23] and RGD- [24] functionalized nanofiber substrates for neural differentiation in vitro . While RGD peptides have been shown to positively affect nerve regeneration [25,26], the method of integration of the peptide can alter the overall response, requiring further investigation of SPAAC integrated peptides.…”

In Situ functionalization of Poly(hydroxyethyl methacrylate) Cryogels with Oligopeptides via β-Cyclodextrin–Adamantane Complexation for Studying Cell-Instructive Peptide Environment

“…Picomolar surface concentrations (per cm 2 ) of RGD-containing sequences immobilized on synthetic hydrogel and nanofiber biomaterials were reported. 52,53 Photopolymerization of acryloyl-PEG-RGDS peptide derivative on preformed PEG hydrogel film provided spatially controlled surface immobilization of the peptide in microgram amounts. 54 In addition, the simultaneous introduction of two adamantylated peptides 3 and 4 into the materials was achieved as a result of coincubation (Figure 6).…”