AVS 60th International Symposium and Exhibition
    In Situ Spectroscopy and Microscopy Focus Topic Friday Sessions
       Session IS+AS+SP-FrM

Paper IS+AS+SP-FrM7
Changes to the Microstructure of Fibrous Collagen Hydrogels formed under Different Physicochemical Parameters and Upon Cross-Linking with Non-Toxic Reagents are Detected with In Situ Multiphoton Microscopy Imaging

Friday, November 1, 2013, 10:20 am, Room 203 B

Session: Evolving In Situ Microscopic and Spectroscopic Techniques and Applications
Presenter: J.G. Lyubovitsky, University of California, Riverside
Authors: Y.J. Hwang, University of California, Riverside
X. Lang, University of California, Riverside
J.G. Lyubovitsky, University of California, Riverside
Correspondent: Click to Email
This presentation will highlight the knowledge developed by our laboratory regarding the microstructure of 3D collagen hydrogels detected in situ with multi-photon imaging while employing second harmonic generation (SHG) and two-photon fluorescence (TPF) contrasts. The materials were prepared under different physicochemical parameters and independently stabilized with non toxic cross-linkers. The effects of collagen solid content, incubation temperature and ionic strength as well as cross-linking with genipin and carbodiimide (EDC) on 3D collagen hydrogel microstructure will be addressed. Second Harmonic generation (SHG) contrast was employed to follow modifications and/or evolutions of the hydrogels’ microstructure in real time and two-photon fluorescence (TPF) was beneficial in monitoring the extent of the chemical reaction between collagen and genipin as well as spatial locations of the newly induced fluorescent fibers. The induced microstructures differ dramatically upon changing collagen solid content, incubation temperature or ionic strength. For example, short lag time, fast assembly rates and short, tightly connected fibers were detected upon assembly from 30 mM phosphate buffer. In 0.9 M NaCl adjusted 30 mM phosphate buffer only unconnected one micron fiber nuclei with low second harmonic generation contrast formed. Fiber width and length was somewhat similar upon assembly of collagen fibers from 30 mM phosphate buffer adjusted with 0.3 M or 0.6 M NaCl while pore structure depended on the polymerization temperature. Non-zero-length cross-linker genipin induced formation of long aggregated fluorescent strands throughout hydrogels. The SHG imaging suggested that modification with genipin partially disaggregated initial collagen microstructure within hydrogels at the expense of forming these new fluorescent fibers. On the other hand, zero-length cross-linker EDC, even with an addition of N-Hydroxysuccinimide (NHS), does not affect micro-structures. Imaging of the interactions of these important materials with embryonic stem cells induced to differentiate into a neural lineage and implication for tissue engineering will be discussed as well.