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.
The bioinspired approaches to tissue strengthening and preservation rely on non-toxic
cross-linking agents one of which is glyceraldehyde. In this study we used multiphoton
microscopy that employs second harmonic generation (SHG) contrast to evaluate
collagen microstructures and two-photon fluorescence (TPF) contrast to monitor
progression of cross-linking upon treatment of tissues with glyceraldehyde. We examined
collagen hydrogels assembled at 37 °C and 27 °C, bovine scleral and corneal tissues, skin
as well as rat tail tendons. The results show a different effect of glyceraldehyde on
collagen microstructures within the above tissues. This effect depends on the original
microstructural assembly of collagen within a specific tissue. Our data suggests that
epidermis (in skin and cornea) will protect collagen from cross-linking with
glyceraldehyde. The work highlights benefits of monitoring progression of collagen
cross-linking and effects of cross-linking on fiber microstructures as imaged with SHG
and TPF signals.
In spite of the adverse ageing effects of glycation in vivo, in vitro this process is widely employed to increase
stiffness and strength of tissues' and artificial scaffolds'.
In-situ optical characterization methods that report on the
structures within these materials could clarify the effects of glycation. We employed one-photon fluorescence and
multiphoton microscopy method that combined two-photon fluorescence and second harmonic generation signals to
characterize collagen hydrogels modified with glyceraldehyde, ribose and glucose. We observed an increase in the
in situ fluorescence as well as structural alterations within the materials during the course of glycation.
Employing a reflectance multi-photon microscopy (MPM) technique, we developed novel method to
quantitatively study the three-dimensional assembly of structural proteins within bulk of dermal
ECMs. Using a structurally simplified model of skin with enzymatically dissected epidermis, we find
that low resolution MPM clearly discriminates between normal and pathological dermis. High-resolution
images revealed that the backscattered MPM signals are affected by the assembly of collagen fibrils and fibers within this system. Exposure of tissues to high concentrations of potentially denaturing chemicals also resulted in the reduction of SHG signals from structural
proteins which coincided with the appearance of aggregated fluorescent structures.
We report multiphoton in situ optical sectioning of hair follicles in mice and a preliminary investigation of the pathological hair follicles in a transgenic mouse model. Using this imaging technology, we rapidly obtain detailed three-dimensional (3-D) reconstructions of individual hair follicles. No staining or mechanical sectioning is involved, since multiphoton microscopy coregisters two-photon excited fluorescence (TPF) from cells and second harmonic generation (SHG) signals from the extracellular matrix (ECM). These signals are ideally suited for estimating molecularly encoded hair follicular 3-D geometries, including sizes of the follicular orifices and their angles relative to the skin surface. In the normal hair follicles, spectral separation of SHG signals generated by the ECM of the hair follicle from that of intrinsic cellular fluorescence revealed intricate spatial interaction of the cellular components with the surrounding connective tissue. In the pathological hair follicles, these were clearly modified. In particular, in the transgenic mice, we observed lack of cellular fluorescence and significantly shallower angles of follicular orifices with respect to the skin surface. The combination of TPF with SHG is sensitive to structural changes in cells and extracellular matrix brought on by normal hair follicle physiology and specific gene alterations.
A transgenic mouse model with a Clim [co-factor of LIM (a combination of first letters of Lin-11 (C. elegans), ISL1 (rat), and Mec-3 (C. elegans) gene names) domain proteins] gene partially blocked in the epithelial compartment of its tissues is used to establish the sensitivity of intrinsic reflectance nonlinear optical microscopy (NLOM) to stromal and cellular perturbations in the cornea. Our results indicate dysplasia in the squamous epithelium, irregular collagen arrays in the stroma, and a compromised posterior endothelium in the corneas of these mice. As suggested by biochemical data, the collagen alterations are likely due to collagen III synthesis and deposition during healing and remodeling of transgenic mice corneal stromas. All of the topographic features seen in NLOM images of normal and aberrant corneas are confirmed by coregistration with histological sections. In this work, we also use ratiometric redox fluorometry based on two-photon excited cellular fluorescence from reduced nicotinamide adenine dinucleotide (NAD)(P)H and oxidized flavin adenine dinucleotide (FAD) to study mitocondrial energy metabolism. Employing this method, we detect higher metabolic activity in the endothelial layer of cornea compared to an epithelial layer located further away from the metabolites. The combination of two-photon excited fluorescence (TPF) with second harmonic generation (SHG) signals allows imaging to aid in understanding the relationship between alternation of specific genes and structural changes in cells and extracellular matrix.
An intrinsic cellular emission allied with second harmonic signals are promising in-vivo clinical diagnostic tools for corneal abnormalities, cancer and wound healing. The extent of corneal damage in K14-DN-Clim mice will be addressed.
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