Peptide‐modified alginate surfaces as a growth permissive substrate for neurite outgrowth

Abstract

Different strategies are being investigated for treatment of spinal cord injuries, one of the most promising being application of neurotrophic factors, which have been shown to prevent neuronal death and stimulate regeneration of injured axons. Ex vivo gene therapy has emerged as the leading delivery method at the site of the injury, and we have shown previously that encapsulating genetically engineered fibroblasts in an immunoprotective alginate capsule can permit implantation of the factor-secreting cells without need for immunosuppression. This strategy could be greatly enhanced by providing the sprouting neurons with a permissive substrate upon which to attach and grow. We report here studies on the modification of an alginate gel surface by either coating it with laminin or by covalent attachment of YIGSR peptide. Using NB2a neuroblastoma cells, we found that native alginate elicited minimal cell attachment ( approximately 1.5%); however, YIGSR-alginate conjugate elicited a fivefold increase in numbers of cells attached using peptide ratios of 0.5 and 1 mg/g alginate, ranging from 9.5% of the cells at the lower ratio, to about 44% at the higher. Only a further 19% increase was obtained at an increased peptide density of 2 mg/g alginate ( approximately 63% over control). Laminin-coated gels showed approximately 60% cell attachment. However, laminin coating did not stimulate differentiation and neurite growth, whereas both numbers and lengths of outgrowths increased with increasing peptide density on peptide-modified alginate. We demonstrate here the ability of the peptide-modified alginate gels to allow adhesion of NB2a neuroblastoma cells and to promote neurite outgrowth from these cells when attached to the peptide-modified alginate surface. Also, we show that the adhesion of NB2a neuroblastoma cells and neurite outgrowth from the attached cells is a function of the peptide density on the gel surface.

Swelling and Mechanical Properties of Alginate Hydrogels with Respect to Promotion of Neural Growth

“…32,33 Mesenchymal stem cells and primary neural stem cells differentiate into neurons on soft hydrogels with stiffnesses < 1 kPa. [34][35][36][37] Stiff alginate hydrogels were demonstrated to be nonadhesive for neurons; to provide an adhesive surface, these hydrogels required functionalizations with signaling molecules such as laminin and fibronectin [38][39][40] and integration of polyglycolic acid, heparin, or basic fibroblast growth factor (bFGF). [41][42][43][44] Anisotropic, stiff alginate scaffolds, which were covered with collagen or polylysine or incorporated gelatin, were shown to support neurite elongation along capillary channels, but prohibited neural ingrowth into the hydrogel core.…”

Production and in vitro evaluation of macroporous, cell-encapsulating alginate fibres for nerve repair

“…Attempts to resolve this shortcoming have involved modifying alginate or the finished scaffold with extra cellular matrix (ECM) proteins, such as fibronectin, vitronectin, laminin, and collagen for binding with cell adhesive receptors. Laminin-coated and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide-conjugated alginate hydrogel (peptide/alginate ratios of 1 mg/g) improved NB2a neuroblastoma cell attachment by 44 and 60%, respectively, compared with the control [20].…”

“…Alginate hydrogels are known to be non-adhesive for cells inculture and have thus been modified with molecules, such as peptides (eg. Tyr-Ile-Gly-Ser-Arg) [20] or fibronectin [33] to enhance A C C E P T E D M A N U S C R I P T neuron growth. The extensive neurite outgrowth observed in macroporous alginate fibres produced by wet spinning alginate solution containing DRGs and gelatin particles indicates that the gelatin particles function not only as porogens, but also provide cell adhesion molecules (CAMs) for modification of the pore surfaces to promote cell attachment and growth.…”

Neural ECM mimetics

“…Bioengineering scaffold materials via incorporation of proteins or peptides on its surface can be used to design scaffolds with specific cell-scaffold interactions to influence the cellular behavior. Incorporation of specific peptide sequences, which promote cell adhesion, is a common way of designing functionalized ECM mimetics with the potential for enhanced cellular adhesion or better integration of the respective material (Choi et al, 2013;Dhoot et al, 2004;Plant et al, 1997).…”