Injectable and biomimetic poly (ethylene glycol) hydrogel systems for in situ therapeutic cell delivery
Date of Issue2017-05-02
School of Materials Science and Engineering
Biomimetic poly (ethylene glycol) (PEG)-based hydrogels have been widely explored as extracellular matrix (ECM) mimicking hydrogels as they conserve the advantages of both ECM (cell-responsive molecules) and synthetic hydrogels (controllable structure and mechanical properties), but did not mimic natural ECM well enough due to limited instructive cues. The aim of this work is to synthesize and characterize a novel and facile prepared multi ECM proteins incorporated PEG hydrogel system that can better mimic natural ECM and innately guide cell behavior. Gelatin, elastin and PEG diacrylate (PEGDA) were designed to synthesize biomimetic hydrogels in this study for developing ECM-mimicking synthetic hydrogels. In this thesis, the thiolation of ECM proteins were finished first; thiolated ECM protein was then conjugated to one end of PEGDA by Michael type addition reaction. Two kinds of elastin-PEG hydrogels were fabricated and encapsulated with smooth muscle cells (SMCs), but SMCs were not able to attach in 3-dimensional (3-D), presumably due to lack of strong cell adhesion peptides or sequences. Therefore, gelatin was considered that combining other biological cues with elastin to support ADCs attachment and growth. Two kinds of gelatin-PEG precursors, GP30 and GP60, were synthesized by adjusting the amount of Traut's reagent. FRP (NHDFs) were encapsulated into the gelatin-PEG hydrogel by crosslinking the remaining double bonds of precursor under UV light in situ with high cell viability. In particular, this study proved that a minimum amount of cell-binding motifs (gelatin > 2.3 wt/v %) are required for attachment; and appropriate initial mechanical properties (storage modulus <~100 Pa or mesh size >~150 nm) can accelerate the attachment of cells and improve cell viability. Therefore, this gelatin-PEG hydrogel system with tunable mechanical properties (storage modulus: 40~2000Pa) can support cell attachment, growth and help to rebuild a new ECM in 3-D microenvironments. Further studies of gelatin-PEG hydrogels on better mimicking natural ECM were done by covalently conjugating soluble elastin. Elastin has been conjugated into gelatin-PEG hydrogel to innately guide cell behavior and help the remolding of new ECM in 3-D microenvironment for better mimicking natural ECM. To evaluate the benefits of covalently-bound elastin, NHDFs were encapsulated into the hybrid hydrogel systems in situ and demonstrated high viability (>95%). This work showed that covalently conjugating elastin to gelatin-PEG hydrogel is more effective in guiding fibroblast behavior by promoting the spreading and proliferation of NHDFs and finally to secret their-own ECM protein in 3-D. Results from NHDFs studies showed that proliferation rate of NHDFs in GEP45 (13.5%) and GEP30 (11.4%) were higher than that of control (7.3%) on day 9. Live/dead staining showed that NHDFs in GEP45 and GEP30 could form extensive intercellular networks, while NHDFs in GPE control showed limited spreading and networks. Besides, F-actin and ECM proteins (collagen type I and elastin) staining revealed that cells in GPE45 and GPE30 showed significant cytoplasmic spreading and F-actin bundling compared with GPE control hydrogels. Taken together, the ability of elastin to alter the biological response of gelatin-PEG hydrogel in 3-D has led to a better ECM-mimicking construct highly suitable for soft tissues (especially dermal substitutes). To the best of our knowledge, this is the first injectable and multiple ECM-proteins incorporated ECM-mimicking PEG hydrogel with tunable mechanical properties that can effectively help to rebuild new ECM for the specific application of soft tissue replacement.