2 Dimensional Assay

Various recombinant fragments of FN were coated onto the plates to test their ability to sustain fibroblast migration in two dimensions. The relevant fragments are as follows:

FN C-His 7-11, or C, is a length of fibronectin type III repeats, stretching from repeat 7-11. This fragment contains the cell-binding domain.
FN C-His 12-15, or H, is a length of fibronectin type III repeats, stretching from repeat 12-15. This fragment contains the hep II domain.
FN C-His 7-15, or CH, represents the recombinant FN fragment stretching from repeat 7-15.
FN C-His 7-15-V, or CHV, represents the recombinant FN fragment stretching from repeat 7-15, including the IIICS variable region located in between type III repeats 14 and 15.
FN C-His 12-15-V, or HV, represents the recombinant FN fragment stretching from repeat 12-15, including the IIICS variable region located in between type III repeats 14 and 15.
FN C-His 7-11 with FN C-His 12-15, or C+H, represents the concurrent coating of the C and H fragments
FN C-His 7-11 with FN C-His 12-15-V, or C+HV, represents the concurrent coating of the C and HV fragments
FN 2-11, or 110K, is the commercially isolated FN fragment of type III repeats 2-11 through digestion. This fragment contains the cell binding domain, but no strong matrix-binding domains.
FN C-His 1-6, or 1-6, represents the recombinant FN fragment stretching from type III repeat 1-6. This fragment does not contain any of the three critical domains and is used as a negative control.

The tested fragments were coated at molar equivalents as compared to the positive control of intact fibronectin. The reason for this was to provide the fibroblasts with domain equivalency between the control experimental groups, as there is only one of each domain per intact FN subunit. Relative sizes and subsequent coating concentrations are exhibited in Table 1.

Fragment

Intact FN

1-6

110K

CHV

C+H

C+HV

Molecular Size (A.A.)

2000

564

1000

458

342

462

800

920

458+

342

458+

462

Coating Concentration (mg/mL)

100

C:25

H:19

C:25

HV:25

Table 1. Relative molecular sizes and coating concentrations of FN and FN fragments.

The results of the 2-D Agarose droplet migration assay are summarized in the following graph (Figure 7). The cells were able to migrate on CHV and on C+HV as well as they could migrate on the FN positive control. The cells were able to migrate on C, H, CH, C+H, and HV as well, although not to the same extent as on intact FN. Cells were not able to migrate on the 1-6 fragment or on the BSA control to a significant extent. There were a few cells that exited the gel perimeter on those two mediums, however, the cells displayed different morphology than those cells that migrated on FN. They seemed to not be as elongated nor oriented as radially as were the cells that migrated on FN.

Figure 7. Migration of fibroblasts on FN fragments relative to migration on intact FN in the agarose-droplet migration assay.

Performing ANOVA statistical analysis with a 95% confidence level on the data provided five important results. There is statistical significance between the BSA negative control group and each of the FN constructs, with the exception of 1-6. There is statistical significance between the intact FN group and each of the C, CH, H, HV, C+H, 110K, 1-6, and BSA groups. There is statistical significance between the CH and the CHV groups, as well as between the H and the HV groups. There is no statistical significance between the two negative controls of BSA and 1-6. There is no statistical significance between the intact FN group and the CHV and C+HV groups.

A sample of the relative extents of migration onto the coated surfaces is displayed in Figure 8.

CHV

FN

BSA

Figure 8. Photomicrographs of fibroblast outmigration across various coated media in the agarose-droplet migration assay. CF-31 fibroblasts migrate as robustly on 715V as they do on intact FN. Relative to migration on FN functional domains, CF-31 fibroblasts do not migrate significantly on BSA. Ruler length is 2.0 mm.

Introduction

Product Design

Materials

Methods

Recombinant FN Fragment Purification

Cell Culture