The Study of the Properties of Collagen Hydrogel and 3T3 Fibroblast Cells’ Behavior Within Collagen Hydrogel by Multiphoton Microscopy (MPM)

by Xuye Lang

Institution: University of California – Riverside
Year: 2016
Keywords: Biochemistry; Biomedical engineering; Biophysics; Collagen hydrogel; cross-linking; Fibroblast; MPM; Scaffold; Tissue engineering
Posted: 02/05/2017
Record ID: 2134508
Full text PDF: http://www.escholarship.org/uc/item/6fz8622q


The goal of tissue engineering is to create the functional engineered tissue to replace or repair the damaged one in human body. The complex multi-phase collagen hydrogel systems are more and more popular in the tissue engineering field where they are used as scaffolds because of biodegradability and biocompatibility. The properties of collagen hydrogel are very important because they can lead to different cellular behavior. My study employed the non-invasive technologies to probe the properties of collagen-based materials (optical, microstructural and mechanical properties) and fibroblast cells’ behavior within collagen hydrogels. We identified that the ion types and ionic strength, initial pH and self-assembly temperature all affect the microstructure of collagen hydrogels, which was detected with the Second Harmonic Generation (SHG) images. Besides that, initial collagen concentration can affect the mechanical properties of collagen hydrogels, which was determined with rheology (storage modulus of 4 g/l collagen hydrogels is five times higher compared to 2 g/l collagen hydrogels).Collagen hydrogel needs to be crosslinked with cross-linkers to improve its mechanical strength since it is generally too soft to support cellular behavior, such as, cell proliferation and migration. According to our experience, the 3D collagen hydrogel collapsed into film after 2 days cell culture. Zero-length cross-linker (EDC) and non-zero-length cross-linker (Genipin) were employed to crosslink collagen hydrogel. Genipin can introduce very strong fluorescence into the collagen hydrogels and weaken their SHG signal. Genipin crosslinked collagen hydrogel has higher cross-linking degree (~85%) compared to EDC cross-linked collagen hydrogel (~10%). The storage modulus of genipin cross-linked collagen hydrogel is more than two times of EDC cross-linked collagen hydrogel. Different “aged” genipin solution can result in different properties of collagen hydrogel. For the optical properties, older genipin solution can increase the fluorescence signal in the green channel (470 nm – 550 nm) and weaken fluorescence signal in the red channel (570 nm – 610 nm). For the microstructural properties, older genipin can result in a thinner collagen fiber within a hydrogel. For the mechanical properties, the storage modulus of genipin cross-linked collagen hydrogel dropped down along with the age increasing of genipin solutions. The embryonic BALB/3T3 (clone A31) fibroblasts cultured on collagen hydrogels cross-linked with stored genipin reagent are disproportionately more round compared to cells cultured on unmodified collagen hydrogels and collagen hydrogels stabilized with fresh genipin solutions. The best correlation of cellular extension is with morphological properties of synthesized hydrogels and no correlation is identified with mechanical properties of materials.