Collagen hydrogels are natural biomaterials that comprise 3D networks of high water content and have viscoelastic properties and biocompatibility similar to native tissues. Consequently, these materials play an important role in tissue engineering and regenerative medicine for quite some time. Second harmonic generation (SHG) and two-photon fluorescence (TPF) contrasts transpire as valuable label-free spectroscopic probes for analysis of these biomaterials and this presentation will report the structural, mechanical and physicochemical parameters leading to the observed optical SHG and TPF effects in synthesized 3D collagen hydrogels. We will present results regarding understanding the dependency of collagen fiber formation on ion types, new results regarding strengthening of these biomaterials with a nontoxic chemical cross-linker genipin and polarization selection of collagen fibers’ orientations.
Collagen is widely used in tissue engineering applications because of its biocompatibility and biodegradability. Detecting collagen microstructure can help to accelerate its applications in tissue engineering. In this study, we followed the changes in microstructure of collagen hydrogels that were digested with collagenase by MPM using Second Harmonic Generation (SHG) and Two photon Fluorescence (TPF) signals. The collagen hydrogels were modified by cross-linkers genipin, EDC or EDC+NHS. For unmodified collagen hydrogels, SHG images showed degradation was underway by about 20 min. For collagen hydrogels modified with EDC or EDC+NHS, preliminary data did not indicate obvious degradation after 22 hours. Modification with genipin induced new fibers that had TPF centered at about 490 nm and 600 nm. The SHG signals were weaker in genipin modified collagen hydrogels. TPF images illustrated that the degradation of the newly induced fluorescent fibers at the surface of the materials was underway after about 2 hours.
The architecture of collagen is important in maintenance and regeneration of higher vertebrates’ tissues. We had been studying the changes to this architecture with in situ multi-photon optical microscopy that combines nonlinear optical phenomena of second harmonic generation (SHG) and two-photon fluorescence (TPF) signals from collagen hydrogels prepared from different collagen solid content, polymerized at different temperatures, with different ions as well as modified with reducing sugars. We incubated 2 g/l collagen hydrogels with 0.1 M fructose at 37 °C and after about 20 days observed a significant induction of in situ fluorescence. The twophoton fluorescence emission was centered at about 460 nm for 730 nm excitation wavelength and shifted to 480 nm when we changed the excitation wavelength to 790 nm. The one-photon fluorescence emission was centered at about 416 nm when excitation was 330 nm. It red shifted and split into two peaks centered at about 430 nm and 460 nm for 370 nm excitation; 460 nm peak became predominant for 385 nm excitation and further shifted to 470 nm for 390 nm excitation. SHG and TPF imaging showed restructuring of hydrogels upon this modification. We will discuss these findings within the context of our ongoing dermal wound repair research.
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