1

Compaction Differences in Gels Composed of Collagen and NIH 3T3

Fibroblast Cells with Varying Collagen Concentrations Madeline Grosklos

Abstract

Cells within a matrix is a common and promising approach in tissue engineering.

Fibroblasts dispersed throughout a collagen gel is useful in studying phenomenon that occur in

the body and valuable as a potential solution to tissue engineered skin. Fibroblasts will cause the

collagen gel to compact which in turn determines the physical properties of the gel, but the

extent of compaction is dependent on the amount of both fibroblasts and collagen and the exact

mechanisms of action are not known. We hypothesized that increasing the collagen

concentration in the gels while keeping the number of fibroblasts constant would result in less

compaction. NIH 3T3 fibroblast cells were cultured in Dulbecco’s modified medium (DMEM)

and combined with 5mg/ml bovine collagen and additional DMEM to form the gels, then

allowed to sit and compact in an incubator for 7 days. After this, visual results were observed

and fraction of gel volume compaction was calculated. Visual results showed clear compaction

in the lowest concentration of 1mg/ml collagen, however the compaction in the 2mg/ml and

3mg/ml conditions was not as apparent. Microscopic analysis revealed cells were living and

existed throughout each collagen gel. An ANOVA analysis at significance α=0.05 established

that at least one condition was statistically different with a significance of 0.002 and a Tukey’s

post hoc test revealed that the 1mg/ml condition was statistically different from 2mg/ml and

3mg/ml, however the latter two conditions were not significantly different from each other.

These findings indicate that higher collagen concentrations could cause decreases in gel

compaction. This study helps to gain insight into pathological environments that may have

increased collagen concentrations, and into potential tissue engineered skin substitute methods.

Introduction

This study was performed to test and compare the degrees of compaction in matrices

consisting of collagen and fibroblasts with varying amounts of collagen. The interaction between

collagen and fibroblasts is important to study as collagen is a major protein component of

connective tissue in the body and fibroblasts are the primary cells responsibly for its biosynthesis

and remodeling (Rhee and Grinnell, 2007). Thus, compaction of collagen gels by fibroblasts

gives an in vitro model for studying an important relationship in the body. Additionally,

fibroblasts condensing a collagen matrix creates a tissue-like substance that can be used as an

artificial skin substitute. The contraction of these gels by fibroblasts impacts important physical

properties such as strength and elasticity that determine how the synthetic material will perform

in a biological environment (Bell, et al., 1979). Aside from modeling and replacing connective

tissues, studying compaction and stiffness of collagen gels by cells such as fibroblasts is an

important aspect of research in pathological environments such as breast tumors (Barcus, et al.,

2013). A stiff collagen microenvironment is a characteristic element of tumorigenesis in many

different cancers and compacted gels can be used to mimic this occurrence in studying diseases

(Gkretsi, Vasiliki, and Stylianopoulos, 2018).

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Compaction is what occurs when a cell-populated gel is not in mechanical equilibrium. In

the state of non-equilibrium, the traction forces applied by the cells is greater than the resistance

by elastic forces in the extracellular matrix (ECM) of the mesh. The cells pull the gel inwards,

decreasing the overall volume. As this occurs, the elastic modulus, or stiffness, of the gel

increases. This compaction ceases when there is a balance between the forces exerted by the cells

and the resisting elastic force in the ECM (Stevenson, et al., 2010).

The expression used for fraction of gel volume compaction is calculated in Stevenson et

al. and is used to analyze the samples in this experiment (Stevenson, et al., 2010). This

expression can be seen in Equation 1 below. This equation relates the initial and final volumes of

the gels, assuming the three-dimensional shape to be cylindrical. Isometric compaction is also

assumed, therefore the percent change in the radial plane is the same as the percent change in

height. Thus, the ratio of final radius to initial radius is equal to the ratio of final height to initial

height, allowing the ratio of heights to be replaced by an additional ratio of radii.

𝜃 = 1 − (𝑣𝑓

𝑣𝑖) = 1 − (

4

3𝜋𝑟𝑓

2ℎ𝑓

4

3𝜋𝑟𝑖

2ℎ𝑖) = 1 − (

𝑟𝑓

𝑟𝑖)

2(

ℎ𝑓

ℎ𝑖) = 1 − (

𝑟𝑓

𝑟𝑖)

3 (1)

𝜃 = 𝑐𝑜𝑚𝑝𝑎𝑐𝑡𝑖𝑜𝑛 𝑣 = 𝑣𝑜𝑙𝑢𝑚𝑒 ℎ = ℎ𝑒𝑖𝑔ℎ𝑡 𝑟 = 𝑟𝑎𝑑𝑖𝑢𝑠 𝑓 = 𝑓𝑖𝑛𝑎𝑙 𝑖 = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙

With this equation, the fraction of the volume compaction is between 0 and 1. A compaction of 0

signifies no compaction and the same initial and final volumes while a compaction of 1 would

correlate to compaction of the gel so much that the final volume is zero. The larger the difference

between initial and final volume, the closer to 1 the compaction value will be.

In this study, gels composed of bovine collagen and NIH 3T3 fibroblast cells were

incubated in Dulbecco’s modified medium (DMEM) and allowed to compact. Collagen

concentrations of 1mg/ml, 2mg/ml, and 3mg/ml were studied to determine the impact of collagen

concentration on gel volume compaction. Based on the increased stiffness that is introduced by

additional collagen in the gel, we hypothesize that increasing the concentration of collagen while

keeping the number of cells in the gel constant will decrease the amount of compaction caused

by the fibroblasts.

Materials and Methods

To begin the experiment, NIH 3T3 Fibroblasts were cultured in a T-25 culture flask with

DMEM and placed in an incubator at 37 degrees Celsius and 5% carbon dioxide. The cells were

grown to 50% confluency before being released with 0.25% trypsin in

ethylenediaminetetraacetic acid. The cells were then counted using a c-chip disposable

hemocytometer.

The collagen gels were prepared with the cultured fibroblasts, 5mg/ml bovine collagen,

and DMEM. For the duration of the collagen gel preparation, all contents were kept on ice in

order to prevent the collagen from prematurely forming a gel. Specific volumes of collagen, cell

solution, and extra media that were added for each condition are listed in Table 1 below. Three

total replicates were created for each condition.

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Table 1: Schematic of experimental conditions used to create the collagen gels.

Condition #1 Condition #2 Condition #3 (optional)

Desired Collagen

Concentration in each gel

(mg/ml)

1 2 3

Desired total number of cells in each gel

105 105 105

Ca

lcu

late

d V

olu

me

s

Collagen Volume

(µl)

180 360 540

Cell

Solution (µl)

50 50 50

Extra Media

(µl)

670 490 310

Total Volume (µl) 900 900

900

Number of replicates you will

make per each condition

3 3 3

An hour after being placed in the wells, the edges of the collagen gels were released from

the plate so that they were free to compact and 1mL of DMDM was added to each. The gels were

left to compact in the incubator for 7 days before being removed for compaction analysis.

The first step in gel analysis was microscopic examination of the wells with a dissecting

microscope. This occurred before any other investigation because it was imperative to confirm

whether living cells were present in each well and that the cells were throughout the collagen

matrix rather than persisting on the bottom of the plate or suspended in the media. Once this was

completed, a macroscopic visual analysis of each gel was performed to gain general insight on

the behavior of the gels at a macro scale. We then used ImaegeJ image processing and analysis

software from NIH to measure the diameter of the gels. For gels with irregular border shapes,

five diameters were recorded and averaged to find a representative value. Once diameter values

were established and converted into radii, fraction of gel volume compaction was calculated

using Equation 1. All detailed calculations for radii and compaction can be seen in Appendix A.

Once the compaction values were determined, a statistical analysis in SPSS software was

performed on the data. Because there were three conditions, a one-way ANOVA test with

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significance level of α=0.05 was performed to determine if any condition was significantly

different from the others. Then, a post hoc Tukey’s test was completed to further analyze

specifically which of the three conditions were significantly different from the others. This

statistical information provides insight as to whether the increase in collagen concentration

decreased the gel compaction as hypothesized. The data was analyzed with the theory that if each

condition is significantly different from the others with decreasing compaction values as the

collagen concentration increases, our hypothesis will have strong support.

Results

Microscopic examination revealed that living cells existed in each of the wells. By

focusing on the edges of the gel, we established that fibroblasts were in fact dispersed within the

collagen in each well. Figure 1 below shows the microscopic images from the edge of the gel

within each well. Fibroblast cells can be seen living within the edges of the gels. The research

team continued with visual and numerical analysis once it was established that the fibroblasts

were both alive and dispersed throughout the gel.

Figure 1: Microscopic images of the outer edge of all 9 gels; fibroblasts can be seen in the gels

and contrasted with the surrounding media. From left to right the columns have repetitions of the

1mg/ml condition, 2mg/ml condition, and 3mg/ml condition respectively.

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It is important to note that additional findings from the microscopic analysis revealed

that there was a population of fibroblasts that remained suspended in the media as well as a

population adhered to the bottom surface of the wells. The implications of this will be detailed in

the discussion.

After one week, the gels in column one with 1mg/mL collagen concentration had

visibly compacted while the other two columns did not have as much of a visible change. Figure

2 below shows all twelve wells after 7 days in incubation. Column two with 2mg/ml collagen

concentration and column three with 3 mg/ml collagen concentration each had wells with

nonuniform bubbles around the edges. These appear to be localized regions of fibroblasts

compacting the collagen. This can be seen highlighted in Figure 3.

As stated previously, the extent of compaction of the gels is reliant on mechanical

equilibrium and ceases when there is a balance between the forces exerted by the cells and the

resisting elastic force in the ECM (Stevenson, et al., 2010). The gel compaction measurements

can be seen in Table 2 below. At 1mg/ml, 2mg/ml, and 3mg/ml of collagen, the average fraction

of gel volume compaction was 0.804±0.100, 0.368±0.106, and 0.273±.113 respectively. These

results can also be seen visually in Figure 4.

Table 2: Fraction of gel volume compaction for each sample under each condition along with averages

and standard deviations for each condition.

Sample 1 Sample 2 Sample 3 Average Stdv

1mg/ml 0.855 0.688 0.868 0.804 0.100

2mg/ml 0.485 0.340 0.279 0.368 0.106

3mg/ml 0.15 0.372 0.298 0.273 0.113

Figure 2: Macroscopic results of gel compaction after 7

days. Columns from left to right have 1mg/ml, 2mg/ml, and

3mg/ml collagen concentration.

Figure 3: Arrows pointing to “bubble-like”

localized regions of fibroblast activity

causing irregular shapes of the gels.

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Figure 4: Boxplot showing the variance of compaction of 1, 2, and 3 mg/ml collagen concentration. It is

clear that 1mg/ml collagen concentration has significantly higher compaction result compared to 2mg/ml

and 3mg/ml.

The AVOVA analysis of the three conditions resulted in a significance of 0.002. This

is less than the significance level of α=0.05, therefore at least one condition is significantly

different than the others. The Tukey’s post hoc test revealed that the 1mg/ml condition was

significantly different from the 2mg/ml and 3mg/ml conditions, however the 2mg/ml and

3mg/ml conditions did not vary from each other. The detailed results of the ANOVA analysis

and Tukey’s test can be seen in Appendix B.

Discussion

As noted in the results, microscopic analysis of the wells revealed that while some

fibroblasts were dispersed in the collagen gels, there were also cells that remained suspended in

the media and cells adhered to the bottom surface of the wells. Due to this, it is improbable that

there were 105 fibroblasts exerting force on the collagen gel in any of the wells. This would have

the largest impact on the gels of higher collagen concentration because they are inherently stiffer.

Decreased number of cells in the gel could have been a factor in why they did not show

compaction. The number of cells within the gel was likely inconsistent from one gel to the next,

introducing a source of discrepancy amongst the wells. Cell number was intended to be

consistent throughout each gel so that the impact of collagen concentration could be studied

without any additional factors at play. This was not fully achieved and the variance in cell

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population amongst the wells should be considered as a source of error while analyzing the gel

compaction results.

The initial visual results (Fig. 2) suggest that changing the collagen concentration

could impact the ability of the fibroblasts to compact the gel. The gels with a collagen

concentration of 1mg/ml were visually smaller whereas this result was not present at

concentrations of 2mg/ml and 3mg/ml. Unlike the results for 1mg/ml, there was no visually

noticeable difference between the gels at 2mg/ml and 3mg/ml collagen concentration. The gels

under both of these conditions did not exhibit any evidence of isometric compaction, rather had

localized regions of possible compaction around the outer edges of the gels.

The statistical analysis supports the visual observation that the gel with the lowest

collagen concentration compacted significantly more than the two gels with higher collagen

concentration. When analyzing the higher collagen concentrations, the Tukey’s test revealed that

these conditions were not significantly different from each other and only deviated when

compared to condition one. Because only one condition showed significant results, the statistics

do not fully support the hypothesis that increasing the concentration of collagen while keeping

the number of cells in the gel constant will decrease the amount of compaction caused by the

fibroblasts. However, the results still show promise and do not disprove the hypothesis.

There are multiple sources of experimental error that must be accounted for when

discussing these results. First, the hypothesis relied on the assumption that each gel had a

constant number of cells which was proven incorrect by the microscopic analysis. Additionally,

the compaction measurements for the 2mg/ml and 3mg/ml conditions could be a

misrepresentation due to our attempt to account for the irregular shape of the gels. To eliminate

these errors in the future, the twelve-well plate should be surface treated to prevent cell adhesion

and the gels with irregular shapes should be treated with a different compaction equation that

does not assume they are cylindrical. More replicates of each collagen concentration would also

increase the chances for statistically significant results. The results of the gel with 1mg/ml

collagen concentration are promising and further, more refined experimentation should be done

to better address the hypothesis.

Conclusions

This study was conducted to determine if increased collagen concentration in gels

seeded with a constant number of fibroblasts will decrease the overall compaction of the gel. The

compaction of a collagen gel due to the action of fibroblasts is important to study because of its

implications in regenerative medicine as a tissue substitute and in studying pathological disease

states such as cancers. The experiment resulted in average compactions of 0.804±0.100 for gels

with 1mg/ml collagen concentration, 0.368±0.106 for gels with 2mg/ml collagen concentration,

and 0.273±.113 for gels with 3mg/ml collagen concentration. An ANOVA analysis followed by

Tukey’s post hoc test revealed that the 1mg/ml condition was significantly different from the

2mg/ml and 3mg/ml conditions. This overall decrease in compaction, although only significant

in one condition of this study, is seen throughout literature. Feng, Zhonggang, et al. found that

gels with initial collagen concentration of 0.5mg/ml had a significantly smaller final volume ratio

than gels with initial collagen concentration of 1.0mg/ml or 1.5mg/ml (Feng, Zhonggang, et al.,

2014). This trend was also observed in Bell, E., et al. as they observed that hydrated protein

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lattices with 220μg of collagen protein compacted more over 10 days than gels with 360μg or

570μg of protein (Bell, E., et al., 1979).

These results aid in the understanding of collagen-fibroblast interactions and cell and

tissue engineering in general. On a broader scale, the variance in compaction of the gels revealed

how cell behavior adapts in different microenvironments. Additionally, it can be reasoned that

because the gels responded differently due to varying concentrations of collagen, complex

physiological environments may also respond differently based on this factor. The preliminary

findings in this experiment suggest that the properties of a material consisting of collagen and

cells can drastically change with different collagen concentrations, which is an important

consideration in tissue engineering. Further experiments could follow up on these results to study

other applications.

In future studies, a method should be established to ensure that less cells end up

suspended in the media or adhered to the bottom surface and that the number of cells within the

gels is consistent. Surface treatment of the plates before introducing the collagen and cells would

be a viable approach. Additionally, it may be beneficial to study smaller increases in collagen

concentration. It is possible that the higher collagen concentrations used in this experiment were

too stiff for the number of fibroblasts and resulted in a lack of significant compaction results. A

basic yet important consideration is that more replicates of each condition would lead to stronger

statistics and more convincing outcomes. Lastly, measuring the amount of compaction over time

rather than only at the end of the experiment would provide insight towards the dynamic

environment and aid in supporting a hypothesis.

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REFERENCES

Barcus, Craig E., et al. “Stiff Collagen Matrices Increase Tumorigenic Prolactin Signaling in

Breast Cancer Cells.” Journal of Biological Chemistry, vol. 288, no. 18, 2013, pp. 12722–

12732., doi:10.1074/jbc.m112.447631.

Bell, E., et al. “Production of a Tissue-like Structure by Contraction of Collagen Lattices by

Human Fibroblasts of Different Proliferative Potential in Vitro.” Proceedings of the

National Academy of Sciences, vol. 76, no. 3, 1979, pp. 1274–1278.,

doi:10.1073/pnas.76.3.1274.

Feng, Zhonggang, et al. “The Mechanisms of Fibroblast-Mediated Compaction of Collagen Gels

and the Mechanical Niche around Individual Fibroblasts.” Biomaterials, vol. 35, no. 28,

2014, pp. 8078–8091., doi:10.1016/j.biomaterials.2014.05.072.

Gkretsi, Vasiliki, and Triantafyllos Stylianopoulos. “Cell Adhesion and Matrix Stiffness:

Coordinating Cancer Cell Invasion and Metastasis.” Frontiers in Oncology, vol. 8, 2018,

doi:10.3389/fonc.2018.00145.

Rhee, S, and F Grinnell. “Fibroblast Mechanics in 3D Collagen Matrices☆.” Advanced Drug

Delivery Reviews, vol. 59, no. 13, 2007, pp. 1299–1305., doi:10.1016/j.addr.2007.08.006.

Stevenson, Mark D., et al. “Pericellular Conditions Regulate Extent of Cell-Mediated

Compaction of Collagen Gels.” Biophysical Journal, vol. 99, no. 1, 2010, pp. 19–28.,

doi:10.1016/j.bpj.2010.03.041.

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Appendix A Calculations for compaction and average radius (when applicable)

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Compaction in column 1 (1mg/ml collagen concentration condition):

𝐶𝑜𝑙. 1 𝑊𝑒𝑙𝑙 1: 𝜃 = 1 − (0.611𝑐𝑚

1.163𝑐𝑚)

3

= 0.855

𝐶𝑜𝑙. 1 𝑊𝑒𝑙𝑙 2: 𝜃 = 1 − (0.789𝑐𝑚

1.163𝑐𝑚)

3

= 0.688

𝐶𝑜𝑙. 1 𝑊𝑒𝑙𝑙 3: 𝜃 = 1 − (0.592𝑐𝑚

1.163𝑐𝑚)

3

= 0.868

𝐴𝑣𝑔 𝐶𝑜𝑙. 1: 𝜃 =[0.855 + 0.688 + 0.868]

3= 0.804

Average radius values for column 2 (2mg/ml collagen concentration condition):

𝐴𝑣𝑔 𝑟 𝐶𝑜𝑙. 2 𝑊𝑒𝑙𝑙 1 =[1.802 + 2.118 + 1.915 + 2.089 + 2.116]𝑐𝑚

5=

1.864𝑐𝑚

2= 0.932𝑐𝑚

𝐴𝑣𝑔 𝑟 𝐶𝑜𝑙. 2 𝑊𝑒𝑙𝑙 2 =[1.769 + 2.137 + 1.913 + 2.158 + 2.150]𝑐𝑚

5=

2.025𝑐𝑚

2= 1.103𝑐𝑚

𝐴𝑣𝑔 𝑟 𝐶𝑜𝑙. 2 𝑊𝑒𝑙𝑙 3 =[2.223 + 2.155 + 1.997 + 2.001 + 2.053]𝑐𝑚

5=

2.086𝑐𝑚

2= 1.043𝑐𝑚

Compaction in column 2 (2mg/ml collagen concentration condition):

𝐶𝑜𝑙. 2 𝑊𝑒𝑙𝑙 1: 𝜃 = 1 − (0.932𝑐𝑚

1.163𝑐𝑚)

3

= 0.485

𝐶𝑜𝑙. 2 𝑊𝑒𝑙𝑙 2: 𝜃 = 1 − (1.103𝑐𝑚

1.163𝑐𝑚)

3

= 0.340

𝐶𝑜𝑙. 2 𝑊𝑒𝑙𝑙 3: 𝜃 = 1 − (1.043𝑐𝑚

1.163𝑐𝑚)

3

= 0.279

𝐴𝑣𝑔 𝐶𝑜𝑙. 2: 𝜃 =[0.485 + 0.340 + 0.279]

3= 0.368

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Average radius values for column 3 (3mg/ml collagen concentration condition):

𝐴𝑣𝑔 𝑟 𝐶𝑜𝑙. 3 𝑊𝑒𝑙𝑙 2 =[1.707 + 2.135 + 1.998 + 2.076 + 2.045]𝑐𝑚

5=

1.992𝑐𝑚

2= 0.996𝑐𝑚

𝐴𝑣𝑔 𝑟 𝐶𝑜𝑙. 3 𝑊𝑒𝑙𝑙 3 =[1.902 + 2.132 + 1.917 + 2.201 + 2.182]𝑐𝑚

5=

2.067𝑐𝑚

2= 1.034𝑐𝑚

Compaction in column 3 (3mg/ml collagen concentration condition):

𝐶𝑜𝑙. 3 𝑊𝑒𝑙𝑙 1: 𝜃 = 1 − (1.102𝑐𝑚

1.163𝑐𝑚)

3

= 0.150

𝐶𝑜𝑙. 3 𝑊𝑒𝑙𝑙 2: 𝜃 = 1 − (0.996𝑐𝑚

1.163𝑐𝑚)

3

= 0.372

𝐶𝑜𝑙. 3 𝑊𝑒𝑙𝑙 3: 𝜃 = 1 − (1.034𝑐𝑚

1.163𝑐𝑚)

3

= 0.298

𝐴𝑣𝑔 𝐶𝑜𝑙. 3: 𝜃 =[0.150 + 0.372 + 0.298]

3= 0.273

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Appendix B SPSS Statistics Tables

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Table B1: Results of the ANOVA test performed on 1mg/ml, 2mg/ml, and 3mg/ml collagen concentration

conditions.

Table B2: Multiple comparisons results from SPSS with compaction as the dependent variable for a

Tukey’s Post Hoc assessment.

(I) cond_num (J) cond_num

Mean

Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

1.00 2.00 .43567* .08699 .006 .1688 .7026

3.00 .53033* .08699 .002 .2634 .7972

2.00 1.00 -.43567* .08699 .006 -.7026 -.1688

3.00 .09467 .08699 .555 -.1722 .3616

3.00 1.00 -.53033* .08699 .002 -.7972 -.2634

2.00 -.09467 .08699 .555 -.3616 .1722

*. The mean difference is significant at the 0.05 level.

Sum of Squares df Mean Square F Sig.

Between Groups .480 2 .240 21.146 .002

Within Groups .068 6 .011

Total .548 8

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Table B3: Tukey’s test results from SPSS. The cond_num corresponds to the collagen concentration

present. This shows cond_num 1.00 (1mg/ml collagen concentration) as significantly different from the

other two.