title: In vivo assessment of guided neural stem cell differentiation in growth factor immobilized chitosan-based hydrogel scaffolds.
abstract: In this study, we demonstrate that a unique growth factor-biomaterial system can offer spatial control of growth factors with sustained signaling to guide the specific lineage commitment of neural stem/progenitor cells (NSPCs) in vivo. First, recombinant fusion proteins incorporating an N-terminal biotin tag and interferon-γ (IFN-γ), platelet derived growth factor-AA (PDGF-AA), or bone morphogenic protein-2 (BMP-2) were immobilized to a methacrylamide chitosan (MAC) based biopolymer via a streptavidin linker to specify NSPC differentiation into neurons, oligodendrocytes, or astrocytes, respectively. MAC was mixed with growth factors (immobilized or adsorbed), acrylated laminin, NSPCs, and crosslinked within chitosan conduits. This system mimics regenerative aspects of the central nervous system ECM, which is largely composed of a crosslinked polysaccharide matrix with cell-adhesive regions, and adds the new functionality of protein sequestration. We demonstrated that these growth factors are maintained at functionally significant levels for 28 d in vitro. In the main study, immobilized treatments were compared to absorbed and control treatments after 28 d in vivo (rat subcutaneous). Masson's Trichrome staining revealed that small collagen capsules formed around the chitosan conduits with an average acceptable thickness of 153.07 ± 6.02 μm for all groups. ED-1 staining showed mild macrophage clustering around the outside of chitosan conduits in all treatments with no macrophage invasion into hydrogel portions. Importantly, NSPC differentiation staining demonstrated that immobilized growth factors induced the majority of cells to differentiate into the desired cell types as compared with adsorbed growth factor treatments and controls by day 28. Interestingly, immobilized IFN-γ resulted in neural rosette-like arrangements and even structures resembling neural tubes, suggesting this treatment can lead to guided dedifferentiation and subsequent neurulation.
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, USA.
authors.forename: Hang
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Koenig
authors.affiliation: Akron General Medical Center, Akron, OH, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Sloan
authors.affiliation: Department of Chemical and Biomolecular Engineering, The University of Akron, Akron, OH, USA. Electronic address: nic.leipzig@uakron.edu.
authors.forename: Nic D
authors.initials: ND
authors.lastname: Leipzig
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects; D001672:Biocompatible Materials / Q000737:chemistry; D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry; D002454:Cell Differentiation; D055598:Chemical Phenomena; D048271:Chitosan / Q000737:chemistry*; D005260:Female; D020100:Hydrogels / Q000737:chemistry*; D055767:Immobilized Proteins / Q000737:chemistry; D007371:Interferon-gamma / Q000737:chemistry; D058953:Neural Stem Cells / Q000166:cytology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects; D009836:Oligodendroglia / Q000166:cytology / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000737:chemistry; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D054457:Tissue Scaffolds / Q000737:chemistry*
chemical_list: D000178:Acrylamides; D001672:Biocompatible Materials; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D020100:Hydrogels; D055767:Immobilized Proteins; D010982:Platelet-Derived Growth Factor; D011993:Recombinant Fusion Proteins; C075278:platelet-derived growth factor A; D007371:Interferon-gamma; D048271:Chitosan; C045985:methacrylamide
keywords: Differentiation; Hydrogel scaffold; Neural stem cells; Neurons; Neurulation; Protein immobilization