Hyaluronan Migration Assay Results

The HA migration assay used intact FN for the two experimental constructs. One construct was HA crosslinked to FN as well as to itself (HAFN). The other construct was HA crosslinked to itself and then later mixed with free FN (HA+FN). Fibrin crosslinked with FN was used as a positive control, as this construct has been used many times in our lab as a matrix that supports fibroblast migration (Greiling et.al., 1997). Finally, pure crosslinked HA was used as a baseline. The results of the first two experiments are summarized in Figure 12.

Figure 12. Migration of fibroblasts seeded onto Cytodex-3 beads into various three-dimensional matrix constructs. The general trend of outmigration from collagen-coated cytodex beads is that there is clear robust migration into HA+FN.

From Figure 12 it is seen that fibroblasts were able to migrate into the Fibrin-FN, HA, and HA+FN matrices from the collagen-coated beads. However there was practically no outmigration into HAFN. Unfortunately, due the fairly high variation in number of cells outmigrating between the randomly selected 10 beads in each well, it is difficult to state significance between outmigration in Fibrin, HA, and HA+FN. Photomicrographs of various matrix conditions are displayed in Figure 13.

HAFN

HA+FN

HA

Fibrin (+FN)

Figure 13. Photomicrographs of fibroblast outmigration from collagen-coated cytodex-3 beads into various HA constructs. Cells were able to migrate into all matrices except for HAFN. Note the “rounded” morphology of the cells in HAFN. Bars are 0.4 mm.

In these photomicrographs, there is clear outmigration visible into the fibrin +FN gel, the HA+FN construct, and even into the pure crosslinked HA. There was no clear outmigration into HAFN. However, the cells that have remained adhered to the collagen-coated beads in the HAFN construct have become rounded, which suggested cytotoxicity might have occurred. To test if there was indeed cytotoxicity, we performed an indirect contact cytotoxicity test.

The outcome of the toxicity test clearly showed leachable cytotoxic material in the HAFN construct that was not present in the pure HA construct. The cytotoxic species affected the proliferation and/or the adhesion of the fibroblasts indirectly exposed to HAFN. The results of the cytotoxicity study are displayed in Figures 14,15.

Figure 14. Graph displaying the quantity of fibroblasts attached to the well after exposed to media conditioned with various constructs over time. Fibroblast proliferation and adhesion while exposed to pure crosslinked HA is not significantly different than when exposed to the two control baselines. However, fibroblasts show dramatic decrease in proliferation/adhesion relative to the two baselines when exposed to HAFN.

Text Box: HA Day 3 Text Box: HA Day 2

Text Box: HAFN Day 3 Text Box: HAFN Day 2

Figure 15. Photomicrographs of fibroblasts attached to the well after exposed to media conditioned with various constructs over time. These four images were captured using phase contrast microscopy. Image A is CF-31 exposed to HA at day 2. Image B is CF-31 exposed to HA at day 3. Image C is CF-31 exposed to HAFN at day 2. Image D is CF-31 exposed to HAFN at day 3. The cells exposed to HA look healthy and are proliferating normally. The cells exposed to HAFN look to have taken on altered morphology and have not proliferated. Bars are 0.4 mm.

The HAFN construct was then redyalized according to the protocol for dialysis of pure HA to remove the leachable cytotoxic chemicals. A second cell toxicity test was performed and the removal of these chemicals was confirmed (Figure 16).

Figure 16. Graph displaying the quantity of fibroblasts attached to the well after exposed to media conditioned with various constructs over time. Fibroblast proliferation and adhesion while exposed to redialized HAFN or pure crosslinked HA is not significantly different than when exposed to the two control baselines. However, fibroblasts show dramatic decrease in proliferation/adhesion relative to the two baselines when exposed to the HAFN that has not been redialyzed.

The HA migration assay was repeated to determine whether the cells would now outmigrate onto the redialyzed HAFN construct. The cells did not move out onto the redialyed HAFN, but once again rounded up on the Cytodex-3 beads. This indicates that soluble chemicals were not the only toxic species in the HAFN construct. There may be contact cytotoxicity from non-soluble chemicals as well.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture