Characterisation of the stability and bio-functionality of tethered proteins on bioengineered scaffolds: Implications for stem cell biology and tissue repair

In the context of biological applications, the timely delivery of molecules can be critical for cellular and organ function. As such, previous studies have demonstrated the superior long-term protein delivery, by way of protein tethering onto bioengineered scaffolds, compared to conventional delivery of soluble protein in vitro and in vivo. Despite such benefits little knowledge exists regarding the stability, release kinetics, longevity, activation of intracellular pathway and functionality of these proteins over time. By way of example, here we examined the stability, degradation and functionality of a protein, glial derived neurotrophic factor (GDNF), which is known to influence neuronal survival, differentiation and neurite morphogenesis. Enzyme-linked immuno-sorbent assays revealed that GDNF, covalently tethered onto polycaprolactone electrospun nanofibrous scaffolds, remained present on the scaffold surface for 120 days, with no evidence of protein leaching or degradation. The tethered GDNF protein remained functional and capable of activating downstream signalling cascades, as revealed by its capacity to phosphorylate intracellular Erk in a neural cell line. Furthermore, immobilisation of GDNF protein promoted cell survival and differentiation in culture at both 3 and 7 days. This study provides important evidence of the stability and functionality kinetics of tethered molecules.