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Characterizing Matrix Remodeling through Collagen Contraction

Tiffany Zhao, Michael Zhao, Erin Kizer, Nicolas Etcheverry
Bioengineering Department, University of California San Diego

The objectives of this experiment were to examine the relationship between
ECM and cells in vitro using statistical analyses, and to measure and compare
the morphologies of cells grown in different ECM conditions.

Prepare Cell-Seeded Collagen Gels
3T3 Media, an aliquot of 3X DMEM, and trypsin were warmed in a water
bath, and the biosafety cabinet was washed with 70% ethanol. The 3 mg/mL
collagen solution was diluted with 3X DMEM to make 12 mL of 2 mg/ml
collagen in DMEM. The 3T3 cells were removed from the incubator, analyzed
under the microscope, and trypsinized. The cells were spun down at 300g and
resuspended in 2 mL of 3T3 media. The cells were counted using the scepter
and diluted to a final concentration of 1.6E6 cells/mL.
Four tubes were filled with the appropriate cell suspension, collagen
solution, and 3T3 media volumes with respect to Table 12.1 in the lab guide.
The final cell concentrations were: .4x106, .2x106, .1x106, and 0 cells/mL. .8
mL of the mixtures described above were plated onto twelve well plates in
triplicate, and two more gels were plated on a separate twelve plate to form
the constrained configuration using the .1x106 cells/mL concentration. The
mixtures polymerized in the incubator for 15 minutes, and the gels were
detached on the first plate to form the unconstrained configuration. Each gel
received .5 mL of 3T3 media and was left in the incubator for two days.
Measure Gel Contraction and Cell Morphology
The plates were removed from the incubator, and pictures were taken in
order to quantify the different diameters of the gels with respect to cell
concentration. Two microliters of calcein AM were diluted in 4 mL of PBS, and
this solution replaced the media of the two constrained constructs and two of
the .1E6 cells/mL unconstrained gels. The gels were incubated for 10 minutes,
and fluorescence microscopy was performed.
Data Processing
The average gel diameters were determined for the different conditions
and samples, and ANOVA based statistical testing was applied in order to
determine the F value to show that there were differences in the data. The
Tukey test was used to compare between the different groups, and ImageJ
was applied to measure the areas and perimeters of the cells and determine
the shape index.




Average Gel Diameter (mm)

Skin is the largest organ in the human body. Products such as Apligraf®
which are composed of actual human cells are used to aid wound healing
within this organ [4]. In this technology, cells are seeded on a scaffold
composed of collagen and grown to appropriate size and maturity. In natural
healing and maintenance processes in the body, these living skin cells
constantly produce, degrade, and mold their surrounding extracellular matrix
(ECM) [2]. To engineer artificial tissues, one must understand what conditions
in vitro allow cells to develop in collagen as they would in vivo, particularly in
response to different growth conditions.
In this experiment, two main types of matrix models were used to study
cell morphology and matrix reorganization: free floating, and constrained [2].
Cells in polymerized collagen remodel the randomly oriented collagen fibrils
into specific matrices[2]. Generally, a more organized collagen matrix would
lead to a more constrained gel and a smaller gel diameter. Cell morphology can
be measured via a shape index (SI), which ranges from 1 for a perfectly circular
cell to 0 for a very branched cell [2]. SI can be calculated from: ?? = 4?? ?2 .




Fig 2. Five selected
cells from two free
floating gels.







Cell Density (x10^6 cells/mL

Fig 1. Graph of average gel diameters at different
cell concentrations.

Fig 3. Five selected
cells are from the
constrained gels.

Table 1. Tukey test q values for p = 3,
νd = 8, qcrit = 4.529, p < 0.05.
Tukey Test Results
4 to 1


4 to 2


4 to 3


3 to 1


3 to 2


2 to 1


Fig 4. Free floating collagen
gels. Left to right cell density:
0.4x106, 0.2x106, 0.1x106, 0

After two days of ECM remodeling, the first column of gels shows
the greatest amount of contraction, and the gels in the last column did
not contract at all (Fig 4). The bar graph shows the relationship
between cell density and average gel diameter. The average gel
diameter is measured in each of the four groups of cell density, along
with the standard deviations of the individual samples (n = 3) (Fig 1). D
In order to test the relationship between cell contraction and cell
density, we performed a one-way ANOVA test. The null hypothesis of
the test states that cell density does not affect the extent of gel
contraction. With a p<0.05, we have calculated the F-value to be 352,
for numerator d.f. of 3 and a denominator d.f. of 8. A Tukey post-hoc
test was used to further examine the significant differences by
comparing two means at a time, shown in Table 1.
The shape index is calculated in order to examine the cell
morphology of the fibroblasts. Five representative cells are selected
from each of the unconstrained and constrained wells as shown in
Figures 2 and 3. After measuring the area and perimeter of the cells, we
calculated the shape indices to be 0.10 ± 0.03 and 0.23 ± 0.07 for
unconstrained cells and constrained cells, respectively. Constrained cell
pictures were provided by Group 8 because our gel did not adhere.

In this experiment we examined the cell-matrix interaction of 3T3
fibroblasts cell on a collagen matrix, effects of cell concentration on
matrix remodeling and the shape index. Based on our reported F value of
352 (p < 0.05) we can reject the null hypothesis that cell concentration
has no effect on matrix remodeling. A positive correlation between cell
concentration and matrix remodeling was observed across our sample
groups as reported in Figure 1. Our results makes sense with respect to
the mechanism of free floating gel contraction during cell remodeling.
The collagen gels that were constrained the most were in wells with the
highest cellular concentration while the wells without any cells had the
least constrained gels. Matrix contraction requires the interconnection of
the fibril networks within the gel [3]. A higher cellular concentration will
allow more cells to adhere onto and within the gel, giving it a higher
chance to interconnect and increasing gel contraction. Further post hoc
analysis with Tukey’s test revealed that values reported in Table 1 agreed
with our prior rejection of the null hypothesis with all but one of the
pairwise comparisons. The only comparison that did not pass the post
hoc test was between the wells with the lower concentration of cells and
the wells without any cells. This could simply be explained by a low cell
concentration that resulted in a less visible remodeling effect on the
collagen gel since the contractile forces are dependent on cell
interconnectivity. It could also be plausible for there to be a minimum cell
concentration required for matrix remodeling to occur, but further
experiments are required to provide a substantive answer regarding this
possibility. However the trend in our data suggests remodeling still occurs
in the wells with the lowest cell concentration.
We calculated shape indices for the constrained gels to be .23 ±
.07 and the unconstrained cells to be .10 ± .03. Examining figure 3, we
expect to have a low shape index because the cells are elongated in one
direction. We also expected the constrained gel to have a higher shape
index because there is an opposing force by the well to the inward force
the cells exert on the matrix in the radial direction. Our data showed high
variability, indicated by the relatively large standard deviations, and this
may have resulted from our deviation of protocol. Instead of incubating
the calcein dye in 37⁰C, we left the cells at room temperature after
calcein dye treatment to avoid contaminating the incubator. This resulted
in poor quality images from the fluorescent microscope that were difficult
to analyze. Future experiments should focus on obtaining higher quality
images with better dye treatment to ensure improved results.
This experiment can be further expanded in the future by studying a
wider range of cell concentrations to observe effects of extreme cell
concentrations and adding other ECM components such as fibronectin to
better mimic in vivo conditions.
[1] Micou, Melissa. "Appendix 1: Trypsinizing a Cell Monolayer." A Laboratory Course in Tissue
Engineering. 1st ed. Taylor and Francis Group, 2012. 237-39.
[2] Micou, Melissa. "Ch. 12: Characterizing Matrix Remodeling through Collagen Gel
Contraction." A Laboratory Course in Tissue Engineering. 1st ed. Taylor and Francis Group,
2012. 135-44.
[3] Grinnell, Frederick, and W. Matthew Petroll. “Cell Motility and Mechanics in ThreeDimensional Collagen Matrices.” Annual Review of Cell and Developmental Biology 26(2010):
435–61. Accessed November 26, 2010.
[4] "Apligraf." What Is Apligraf? Organogenesis, 2012.