3D Collagen Hydrogels Research Articles

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.

Quantification by SIFT-MS of acetaldehyde released by lung cells in a 3D model

Our previous studies have shown that both lung cancer cells and non-malignant lung cells release acetaldehyde in vitro. However, data from other laboratories have produced conflicting results. Furthermore, all these studies have been carried out in 2D models which are less physiological cell growth systems when compared to 3D models. Therefore, we have carried out further work on the release of acetaldehyde by lung cells in 3D collagen hydrogels. Lung cancer cells CALU-1 and non-malignant lung cells NL20 were seeded in these hydrogels at different cell concentrations and the release of acetaldehyde was measured with the Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) technique. The data obtained showed that the amount of acetaldehyde released by both cell types grown in a 3D model is higher when compared to that of the same cells grown in 2D models. More importantly, acetaldehyde from the headspace of lung cancer cells could be measured even at a low cell concentration (10(5) cells per hydrogel). The differential of acetaldehyde release could be, depending on the cell concentration, more than 3 fold higher for cancer cells when compared to non-malignant lung cells. This pilot study is the first to study acetaldehyde emission from albeit only two cell types cultured in 3D scaffolds. Clearly, from such limited data the behaviour of other cell types and of tumour cells in vivo cannot be predicted with confidence. Nevertheless, this work represents another step in the search for volatile biomarkers of tumour cells, the ultimate goal of which is to exploit volatile compounds in exhaled breath and other biological fluids as biomarkers of tumours in vivo.

Engineering stable topography in dense bio-mimetic 3D collagen scaffolds

Topographic features are well known to influence cell behaviour and can provide a powerful tool for engineering complex, functional tissues. This study aimed to investigate the mechanisms of formation of a stable micro-topography on plastic compressed (PC) collagen gels. The uni-directional fluid flow that accompanies PC of collagen gels creates a fluid leaving surface (FLS) and a non-fluid leaving surface (non-FLS). Here we tested the hypothesis that the resulting anisotropy in collagen density and stiffness between FLS and non-FLS would influence the fidelity and stability of micro-grooves patterned on these surfaces. A pattern template of parallel-aligned glass fibres was introduced to the FLS or non-FLS either at the start of the compression or halfway through, when a dense FLS had already formed. Results showed that both early and late patterning of the FLS generated grooves that had depth (25 ±7 µm and 19 ±8 µm, respectively) and width (55 ±11 µm and 50 ±12 µm, respectively) which matched the glass fibre diameter (50 µm). In contrast, early and late patterning of the non-FLS gave much wider (151 ±50 µm and 89 ±14 µm, respectively) and shallower (10 ±2.7 µm and 13 ±3.5 µm, respectively) grooves than expected. The depth to width ratio of the grooves generated on the FLS remained unaltered under static culture conditions over 2 weeks, indicating that grooves were stable under long term active cell-mediated matrix remodelling. These results indicate that the FLS, characterised by a higher matrix collagen density and stiffness than the non-FLS, provides the most favourable mechanical surface for precise engineering of a stable micro-topography in 3D collagen hydrogel scaffolds.

Collagen Three-Dimensional Hydrogel Matrix Carrying Basic Fibroblast Growth Factor for the Cultivation of Mesenchymal Stem Cells and Osteogenic Differentiation

Three-dimensional (3D) collagen hydrogels have been extensively used for cell culture experiments and are more closely representative of in vivo conditions than monolayer (2D) culture. Here we cultured rat bone marrow-derived mesenchymal stem cells (MSCs) in collagen hydrogels containing varying concentrations of basic fibroblast growth factor (bFGF) to examine the effect of bFGF on MSC proliferation and osteogenic differentiation in 3D culture. The optimal bFGF concentration that promoted the greatest degree of cell proliferation and expression of the early osteogenic induction marker alkaline phosphatase was also determined. Subsequent quantitative real-time polymerase chain reaction analysis of gene expression demonstrated that bFGF promoted significant upregulation of the bone-related genes: collagen type I, osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) for periods of up to 21 days. Immunofluorescence staining and fluorescence-activated cell sorting analysis further supported the enhanced osteogenic differentiation of cells as a greater proportion of cells were found to express OPN. Matrix mineralization within the collagen hydrogels was enhanced in the presence of bFGF, as assessed by calcium detection using von Kossa staining. These results clearly demonstrate a positive effect of bFGF on proliferation and osteogenic induction of MSCs in 3D collagen hydrogels when applied at the appropriate concentration. Moreover, collagen hydrogel constructs containing MSCs and appropriate growth factor stimulus might be a potentially useful biological tool for 3D bone tissue engineering.

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.

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.

Electrophysiological characterization of embryonic hippocampal neurons cultured in a 3D collagen hydrogel

Rat embryonic hippocampal neurons were cultured in (1) 3D collagen hydrogels as ‘entrapped’ evenly distributed cells, (2) at the interface of two collagen layers (sandwich model), and (3) on the surface of collagen coated coverslips (2D model). In the ‘entrapment’ model the neuronal processes grew out of the plane of the cell body and extended into the collagen matrix, in contrast to the sandwich model where the cells and their processes rarely left the plane in which they were seeded. Hippocampal neurons ‘entrapped’ in the 3D collagen gel grew the same number, but shorter, processes and exhibited improved survival compared to neurons cultured in the 2D model. There was no difference in the electrophysiological properties of the neurons cultured in the 3D compared to the 2D model except in the resting membrane potential and in the duration of the after-hyperpolarization. Spontaneous postsynaptic currents were recorded in 14- and 21-day-old 3D cultures evidencing functional synapse formation. Our results indicate that the physiological characteristics of 3D neuronal cultures are similar to traditional 2D cultures. However, functional 3D networks of hippocampal neurons will be necessary for multi-level circuit formation, which could be essential for understanding the basis of physiological learning and memory.

Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three-dimensional collagen gels.

We describe the application of three-dimensional collagen matrices to the study of nerve cord repair in the leech. Our experiments show that ganglia and connectives of the leech ventral nerve cord can be maintained for up to four weeks embedded in 3D gels constructed from mammalian type I collagen. Severed nerve cords embedded in the collagen gel reliably repaired within a few days of culture. The gel was penetrable by cells emigrating from the cut ends of nerves and connectives, and we consistently saw regenerative outgrowth of severed peripheral and central axons into the gel matrix. Thus, 3D gels provide an in vitro system in which we can reliably obtain repair of severed nerve cords in the dish, and visualize cell behaviour underlying regenerative growth at the damage site: and which offers the possibility of manipulating the regenerating cells and their extracellular environment in various ways at stages during repair. Using this system it should be possible to test the effect on the repair process of altering expression of selected genes in identified nerve cells.