The formation of protein concentration gradients mediated by density differences of poly(ethylene glycol) microspheres

Abstract

A critical element in the formation of scaffolds for tissue engineering is the introduction of concentration gradients of bioactive molecules. We explored the use of poly(ethylene glycol) (PEG) microspheres fabricated via a thermally induced phase separation to facilitate the creation of gradients in scaffolds. PEG microspheres were produced with different densities (buoyancies) and centrifuged to develop microsphere gradients. We previously found that the time to gelation following phase separation controlled the size of microspheres in the de-swollen state, while crosslink density affected swelling following buffer exchange into PBS. The principle factors used here to control microsphere densities were the temperature at which the PEG solutions were reacted following phase separation in aqueous sodium sulfate solutions and the length of the incubation period above the ‘cloud point’. Using different temperatures and incubation times, microspheres were formed that self-assembled into gradients upon centrifugation. The gradients were produced with sharp interfaces or gradual transitions, with up to five tiers of different microsphere types. For proof-of-concept, concentration gradients of covalently immobilized proteins were also assembled. PEG microspheres containing heparin were also fabricated. PEG-heparin microspheres were incubated with fluorescently labeled protamine and used to form gradient scaffolds. The ability to form gradients in microspheres may prove to be useful to achieve better control over the kinetics of protein release from scaffolds or to generate gradients of immobilized growth factors.

Comparison of Gelation Time and Polyalcohol Effect on Hydrogels from Domestic and Wild Silk Fibroins

“…The presence of abundant oxethyl groups in the PEG molecule that are capable of forming hydrogen bonds with water makes PEG a highly water-soluble material. PEG is also a nontoxic, nonimmunogenic, and amphipathic polymer, and it possesses a variety of properties pertinent to biomedical and biotechnical applications [18][19][20] . Thus, adding PEG to A. pernyi SF solutions was selected as a method for reducing gelation time in this study.…”

A modular, plasmin-sensitive, clickable poly(ethylene glycol)-heparin-laminin microsphere system for establishing growth factor gradients in nerve guidance conduits

“…For microspheres to form, a pre-incubation step had to be added, in which the undiluted PEG-(VRN) 8 and PEG 8 -VS (200 mg/mL total PEG) were incubated for > 30 min (1 h in final protocol) at 37°C before dilution in 0.6 M sodium sulfate and subsequent incubation at 70°C. The incubation time at 70°C in the phase separated state is known to determine the extent of crosslinking within the microsphere, affecting its swelling, buoyancy and the rate of diffusion of proteins through the material [19, 60]. Because the time in phase separated state ultimately controls the rate of release of growth factor, this time length was carefully monitored during microsphere formation.…”

“…Our laboratory has created nearly linear gradients in reversibly-bound GDNF within heparinated poly(ethylene glycol) (PEG) microsphere scaffolds [18, 19]. These GDNF gradients persist for more than a week and potentially might enhance nerve regeneration within an NGC.…”

Synthetic PEG Hydrogels as Extracellular Matrix Mimics for Tissue Engineering Applications

“…In addition to the above mentioned techniques, gradients in PEG hydrogels have also been formed by microsphere packing (Roam et al, 2010). This technique relies upon the formation of PEG microspheres of varying density by thermally induced phase separation.…”