Different Combined With State Of Chondroitin Sulfate B On The Three-dimensional Culture Of Chick Embryo Dorsal Root Ganglion Neurite Growth Of Mechanistic Studies

ObjectiveIn order to assess effects of chondroitin sulfate type B (CS-B) to the elongation of chicken dorsal root ganglion neurites incubated in agarose hydrogel, we established an in vitro model of neurites out growth by building a three dimensional culture medium to evaluate the difference of chicken DRG neuritis growth between the 3D and 2D medium. Furthermore, we have measured the ordinary agarose gel and the agarose gel covalent with CS-B in aperture area and contour of molecular skeleton by atomic force microscope (AFM). All these effort is to explore how the CS-B polysaccharide chain affects the axon of the DRG neurites extension in three-dimensional culture medium.MethodDay 9 (E9) chicken DRG embedded in the SeaPrep^? agarose gel, a range of agarose gel with differ concentrations (0.5%, 0.75%, 1%, 1.25%, 1.5%) were used. the gel was combined with CS-B by covalent binding. Snap the DRG at the 24h, 48h, 72h, and 96h. And the corresponding length of E9 DRG neurite was measured. The atomic force microscopy was used to observe the ordinary agarose gel and covalent CS-B's agarose gel in differ concentration.ResultsThe chicken dorsal root ganglions can sprout the neurite by three-dimensional way in more than 1.25% concentrations agarose conditioned substrate. But under the concentrations of 1% agarose conditioned substrate, the DRG neurite only can extension by two-dimensional manner. Results From ANOVA demonstrated that statistically lower average neurites lengths in CS-B covalent binding gels and free CS-B gel compared with normal gel in three-dimensional culture(p＜0.05). But have no difference in two-dimensional culture (P＞0.05). Under the high concentrations (1.5% w/v), the free CS-B have no effect to the extension of axons, but the CS-B covalent gel still can inhibit the elongation of neurite. Some neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation. The atomic force microscopy showed us that covalent CS-B can modify the molecular shape of agarose gel. And under some concentration (1%, 1.25%, 1.5%) the covalent CS-B can change the pore area of the hydrogel. The agarose gel pore area Exponential decline when the agarose gel concentration increased. We fitting the exponential curve by formula y=7.209e^-0.91x and y=7.1203e^-0.91x.ConclusionThe biological characteristics of 3D growth of dorsal root ganglion neurite are distinctly differ form 2D growth. CS-B can inhibit neurite growth in vitro, but its specific inhibitory effect depends on the state of CS-B and matrix surrounding the nerve cells. Although covalent CS-B can reduce the pore size of hydrogel, however, this shrinkage does not conduce the inhibiting of the neurite growth. It can also change the agarose-polymer's molecular shape and Aggregation, but whether this change can inhibit the neurite growth still need for further exploration.