Abstract
Damage of enteric neurons and partial or total loss of selective neuronal populations are reported in intestinal disorders including inflammatory bowel diseases and necrotizing enterocolitis. To develop three-dimensional scaffolds for enteric neurons we propose the decoration of ionic-complementary self-assembling peptide (SAP) hydrogels, namely EAK or EAbuK, with bioactive motives. Our results showed the ability of EAK in supporting neuronal cell attachment and neurite development. Therefore, EAK was covalently conjugated to: RGD, (GRGDSP)4K (fibronectin), FRHRNRKGY (h-vitronectin, named HVP), IKVAV (laminin), and type 1 Insulin-like Growth Factor (IGF-1). Chemoselective ligation was applied for the SAP conjugation with IGF-1 and the other longer sequences. Freshly isolated murine enteric neurons attached and grew on all functionalized EAK but IGF-1. Cell-cell contact was evident on hydrogels enriched with (GRGDSP)4K and HVP. Moreover (GRGDSP)4K significantly increased mRNA expression of neurotrophin-3 and nerve growth factor, two trophic factors supporting neuronal survival and differentiation, whereas IKVAV decoration specifically increased mRNA expression of acetylcholinesterase and choline acetyltransferase, genes involved in synaptic communication between cholinergic neurons. Thus, decorated hydrogels are proposed as injectable scaffolds to support in loco survival of enteric neurons, foster synaptic communication, or drive the differentiation of neuronal subtypes.
Highlights
The extracellular matrix (ECM), the network of glycoproteins providing mechanical support and biochemical signals for tissue homeostasis, plays a key role in the economy of biological tissues
In this study we proposed the enrichment of plain self-assembling peptide (SAP) with self-assembling sequences joining bioactive factors
To keep the SAP side chains available for cell receptor interaction, two strategies were adopted in covalent binding: (i) extension in the protocol of solid phase peptide synthesis to condense short bioactive fragments (≤5 amino acids) to the self-assembling sequence; (ii) chemoselective ligation for the conjugation of SAPs with longer bioactive peptides
Summary
Paola Brun 1, Annj Zamuner[2], Alessandro Peretti[2], Jessica Conti[1], Grazia M. The application of SAPs is strongly jeopardized by the lack of adhesive motives able to properly drive attachment, growth, and differentiation of specific neuronal cells on hydrogels To fill this gap, in this study we proposed the enrichment of plain SAPs with self-assembling sequences joining bioactive factors. To support the in vitro growth of enteric neurons, in this study ionic self-assembling peptides of module II (EAK or EAbuK)[2] were conjugated with (i) RGD motif; (ii) linear peptide of 4 GRGDSP motives previously reported to support adhesion in osteoblasts, cardiomyocytes and endothelial cells[15,22,23]; (iii) (351–359) fragment of the human vitronectin involved in proteoglycan-mediated osteoblast adhesion[22,24,25]; (iv) laminin sequence IKVAV26; (v) insulin-like growth factor-1 (IGF-1) promoting survival of cortical neurons[27]. Our data demonstrated that application of injectable hydrogel matrices decorated with different motives could support gut neuronal plasticity possibly fostering in loco migration and differentiation of definite intestinal neuronal subtypes but avoiding inoculation of foreign biological materials
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