Fluorescent hydrogels for embryoid body formation and osteogenic differentiation of embryonic stem cells.

Substrate mechanics (e.g., stiffness and topography of the microenvironment) are likely critical for driving normal morphogenesis and tissue development. As such, substrate mechanics imposed by hydrogels have been exploited to guide the lineage differentiation of stem cells and to drive stemness. In this work, we chemically modified gelatin hydrogels through glyceraldehyde cross-linking to render them suitable for cell culture. The modified hydrogels proved to be ideal for embryonic stem cell osteogenesis, initially providing a soft nonadhesive surface for the formation of embryoid bodies. They subsequently degraded in culture to afford a harder surface during osteoblast differentiation. The gels synthesized are highly fluorescent, relatively easy to prepare, and can potentially aid in overcoming the challenge of imaging changes to the microenvironments of cells during three-dimensional cell culture. Exploiting these materials could lead to the development of tissue-engineered products of increased complexity and rational treatment strategies.

The structural analysis of three-dimensional fibrous collagen hydrogels by Raman microspectroscopy.

To investigate molecular effects of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), EDC/N-hydroxysuccinimide (NHS), glyceraldehyde cross-linking as well as polymerization temperature and concentration on the three-dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm(-1) Raman bands corresponding to the C-C stretch of a protein backbone and a shift in the amide III bands from 1241 cm(-1)/1268 cm(-1) in controls to 1247 cm(-1)/1283 cm(-1) in glyceraldehyde-treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm(-1) band and the appearance of a strong peak at 1468 cm(-1) reflected a change in the motion of lysine/arginine CH2 groups. For the EDC-treated collagen hydrogels, the increased intensity of 823 cm(-1) peak corresponding to the C-C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm(-1) to 1271 cm(-1) amide III band intensities in the EDC-modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH2 groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm(-1) band corresponding to a COO(-) stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable.

Effects of zero-length and non-zero-length cross-linking reagents on the optical spectral properties and structures of collagen hydrogels.

We compared the effects of zero-length cross-linkers 1-ethyl-3 (3dimethylaminopropyl) carbodiimide (EDC) and non-zero-length cross-linkers glycolaldehyde and glyceraldehyde on the optical and structural properties of three-dimensional (3D) collagen hydrogels. We evaluated these effects by multiphoton microscopy (MPM) that combined two-photon fluorescence (TPF) and second harmonic generation (SHG) contrasts and transmission electron microscopy (TEM). The collagen hydrogels were incubated separately with the above-mentioned reagents present at the concentration of 0.1 M. The incubation with glycolaldehyde and glyceraldehyde induced strong autofluorescence within the gels. We followed the formation of fluorescence with TPF signals in situ and in real time as well as characterized the micro- and nanostructures within cross-linked hydrogels by examining SHG and TEM images respectively. As detected in the SHG images, glycolaldehyde- and glyceraldehyde-modified 5-10 µm "fiberlike" collagen structures to longer, 20 µm and more, aggregated strands while EDC had minimal effect on the microstructure. TEM revealed that glycolaldehyde and glyceraldehyde either completely eliminated collagen's characteristic native fibrillar striations or generated uncharacteristic fibrils with extensions. EDC preserved the native striation patterns, decreased the fibril diameters and effectively homogenized the fibrils within hydrogels assembled at 1.8-4.68 g/L collagen concentrations and 37 °C. Our findings provide a clear understanding on how different cross-linking reagents have very different effects on the collagen hydrogels. This understanding is critical for advancing tissue engineering and wound healing applications.

Effect of genipin crosslinking on the optical spectral properties and structures of collagen hydrogels.

Genipin, a natural cross-linking reagent extracted from the fruits of Gardenia jasminoides, can be effectively employed in tissue engineering applications due to its low cytotoxicity and high biocompatibility. The cross-linking of collagen hydrogels with genipin was followed with one-photon fluorescence spectroscopy, second harmonic generation, fluorescence and transmission electron microscopy. The incubation with genipin induced strong auto-fluorescence within the collagen hydrogels. The fluorescence emission maximum of the fluorescent adducts formed by genipin exhibit a strong dependence on the excitation wavelength. The emission maximum is at 630 nm when we excite the cross-linked samples with 590 nm light and shifts to 462 nm when we use 400 nm light instead. The fluorescence imaging studies show that genipin induces formation of long aggregated fluorescent strands throughout the depth of samples. The second harmonic generation (SHG) imaging studies suggest that genipin partially disaggregates 10 µm "fiberlike" collagen structures because of the formation of these fluorescent cross-links. Transmission electron microscopy (TEM) studies reveal that genipin largely eliminates collagen's characteristic native fibrillar striations. Our study is the first one to nondestructively follow and identify the structure within collagen hydrogels in situ and to sample structures formed on both micro- and nanoscales. Our findings suggest that genipin cross-linking of collagen follows a complex mechanism and this compound modifies the structure within the collagen hydrogels in both micro- and nanoscale.

Collagen hydrogel characterization: multi-scale and multi-modality approach.

The complex supramolecular architecture of collagen biopolymer plays an important role in tissue development and integrity. Developing methods to report on collagen structures assembled in vitro would accelerate the pace of utilizing it in biomedical applications. Employing imaging techniques and turbidity measurements, we mapped the light scattering properties of 3D collagen hydrogels formed at initial concentrations of 1 mg ml-1 to about 5 mg ml-1 and several incubation temperatures. The transmission electron microscopy (TEM) images show that collagen scattering features consist of both native-like fibrils and filamentous structures that do not have the characteristic fibrillar striation observed in this protein. Spindle-shaped fibrils appear at the concentrations of 1, 2, 2.5 and 4 mg ml-1 and the spiral-shaped fibrils are formed at the concentrations of 2 and 2.5 mg ml-1. The multiphoton microscopy (MPM) images reveal that in the 3D collagen hydrogels a unified relationship between second harmonic generation (SHG) signal directionality and fibril morphology and/or sizes is not likely. The MPM images, however, showed important micro-structural details. These details lead us to conclude that the dependence of SHG signals on the number of interfaces created upon assembly of 3D collagen hydrogels can account for the strength of the detected backscattered signals.

Multiphoton imaging of actin filament formation and mitochondrial energetics of human ACBT gliomas.

We studied the three-dimensional (3D) distribution of actin filaments and mitochondria in relation to ACBT glioblastoma cells migration. We embedded the cells in the spheroid form within collagen hydrogels and imaged them by in situ multiphoton microscopy (MPM). The static 3D overlay of the distribution of actin filaments and mitochondria provided a greater understanding of cell-to-cell and cell-to-substrate interactions and morphology. While imaging mitochondria to obtain ratiometric redox index based on cellular fluorescence from reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide we observed differential sensitivity of the migrating ACBT glioblastoma cells to femtosecond laser irradiation employed in MPM. We imaged actin-green fluorescent protein fluorescence in live ACBT glioma cells and for the first time observed dynamic modulation of the pools of actin during migration in 3D. The MPM imaging, which probes cells directly within the 3D cancer models, could potentially aid in working out a link between the functional performance of mitochondria, actin distribution and cancer invasiveness.

Multiphoton optical image guided spectroscopy method for characterization of collagen-based materials modified by glycation.

The cross-linking with reducing sugars, known as glycation, is used to increase stiffness and strength of tissues and artificial collagen-based scaffolds. Nondestructive characterization methods that report on the structures within these materials could clarify the effects of glycation. For doing this nondestructive evaluation, we employed an in situ one-photon fluorescence as well as multiphoton microscopy method that combined two-photon fluorescence and second harmonic generation signals. We incubated collagen hydrogels with glyceraldehyde, ribose, and glucose and observed an increase in the in situ fluorescence and structural alterations within the materials during the course of glycation. The two-photon fluorescence emission maximum was observed at about 460 nm. The fluorescence emission in the one-photon excitation experiment (λ(ex) = 360 nm) was broad with peaks centered at 445 and 460 nm. The 460 nm emission component subsequently became dominant during the course of glycation with glyceraldehyde. For the ribose, in addition to the 460 nm peak, the 445 nm component persisted. The glucose glycated hydrogels exhibited broad fluorescence that did not increase significantly even after 6 weeks. As determined from measuring the fluorescence intensity at the 460 nm maximum, glycation with glyceraldehyde was faster compared to ribose and generated stronger fluorescence signals. Upon excitation of glycated samples with 330 nm light, different emission peaks were observed.