This study focuses on the development of a novel method of non-enzymatic glycation of fibrillar collagen gels. In contrast to previous studies in which type I collagen gels were glycated in the solid state, this study presents a method for glycation in solution. Type I collagen in solution or gels was exposed to a range of ribose concentrations from 0–250mM. The binding of ribose to collagen was documented using Fourier Transform Infrared (FTIR) Spectroscopy. Formation of advanced glycation endproducts (AGEs) was quantified by fluorescence measurement. The bulk compressive modulus and viscoelastic time constant of processed gels were determined in stress relaxation studies. Both methods of glycation enhanced ribose addition and AGE formation in a dose dependent manner, with glycation in the gel state being more efficient. Both methods enhanced mechanical properties similarly, with 250mM ribose treatment resulting in a 10-fold increase in bulk modulus.
1. Introduction 4716 1.1. Cells and Tissues of Bones and Teeth 4716 1.1.1. The Mineral and Extracellular Matrix of Bones and Teeth 4716 1.1.2. Cells in Mineralized Tissues 4717 2. Is the Mineral Formed in Culture Similar to "Physiologic Mineral"? 4717 2.1. X-ray Diffraction 4718 2.2. SEM and TEM and Related Techniques 4718 2.3. Atomic Force Microscopy 4718 2.4. Light Microscopy 4719 2.5. Vibrational Spectroscopy and Vibrational Spectroscopic Imaging 4719 2.6. Radiographic and Related Methods 4720 2.6.1. Magnetic Resonance Methods 4720 2.6.2. Microcomputed Tomography (microCT) or X-ray Microtomography (XMT) 4720 2.7. Chemical Analyses 4721 3. Systems for Studying Mineralization in Culture 4721 3.1. Cell Sources 4721 3.1.1. Organ Culture 4721 3.
Using a custom galvanotaxis chamber and time-lapse digital video microscopy, we report the novel observation that cultured chondrocytes exhibit cathodal migration when subjected to applied direct current (DC) electric fields as low as 0.8 V/cm. The response was dose-dependent for field strengths greater than 4 V/cm. Cell migration appeared to be an active process with extension of cytoplasmic processes in the direction of movement. In some cells, field application for greater than an hour induced elongation of initially round cells accompanied by perpendicular alignment of the long axis with respect to the applied field. Antagonists of the inositol phospholipid pathway, U-73122 and neomycin, were able to inhibit cathodal migration. Cell migration toward the cathode did not require the presence of serum during field application. However, the directed velocity was nearly threefold greater in studies performed with serum. Studies performed at physiologic temperatures (approximately 37 degrees C) revealed a twofold enhancement in migration speed compared to similar studies at room temperature (approximately 25 degrees C). Findings from the present study may help to elucidate basic mechanisms that mediate chondrocyte migration and substrate attachment. Since chondrocyte migration has been implicated in cartilage healing, the ability to direct chondrocyte movement has the potential to impact strategies for addressing cartilage healing/repair and for development of cartilage substitutes.
Collagen glycated with ribose (250 mM) in solution (pre-glycation) and as a gel (post-glycation) was seeded with chondrocytes and the effects of glycation on chondrocyte matrix assembly in culture were determined. Pre-glycation enhanced GAG accumulation significantly over controls at both 2 and 4 weeks (p < 0.05), although at both time points there were no statistical differences in cell number between pre-glycated and control gels. The increased proteoglycan accumulation was shown to be in part due to significantly increased GAG retention by the pre-glycated constructs (p < 0.05). Total collagen content in these pre-glycated gels was also significantly higher than unglycated gels at 4 weeks (p < 0.05). With post-glycation of collagen gels, chondrocyte number and GAG accumulation were all significantly lower than controls (p < 0.05). Post-glycation also inhibited GAG retention by the constructs (p < 0.05). Given these results, pre-glycation may be an improved processing method for collagen gels for tissue engineering techniques. ß
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