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Imaging of Collagen Gels for Tissue Engineering . ALLISON THROM and REBECCA KUNTZ WILLITS . Department of Biomedical Engineering, Saint Louis University, 3507 Lindell ...

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Imaging of Collagen Gels for Tissue Engineering - BE@SLU

Imaging of Collagen Gels for Tissue Engineering . ALLISON THROM and REBECCA KUNTZ WILLITS . Department of Biomedical Engineering, Saint Louis University, 3507 Lindell ...

Imaging of Collagen Gels for Tissue Engineering

ALLISON THROM and REBECCA KUNTZ WILLITS

Department of Biomedical Engineering, Saint Louis University, 3507 Lindell Boulevard, St. Louis, MO 63103, USA

Collagen I is commonly tested for use as a scaffold for nerve regeneration. To
understand how collagen promotes nerve regeneration, the properties of the gels themselves must
be determined. This study focused on the differences between collagen fiber diameter and
density for gels of varying collagen concentrations and with different extracellular matrix
additives. Gels of 0.4mg/ml, 1mg/ml, and 2mg/ml collagen concentrations with added laminin,
collagen IV, or chondroitin sulfate were formed, dehydrated in graded ethanol, critically point
dried, and then imaged with scanning electron microscopy. Gels with added laminin showed no
significant difference in fiber diameter from the plain gels. However, these images had fibers that
were often fuzzier and difficult to focus on, indicating that the fiber was somehow influenced.
Collagen IV and chondroitin sulfate significantly decreased the fiber diameter by up to 31.6%
and 29.6%, respectively. These additives also gave the gels a matted appearance, in comparison
with the distinguishable fibers of the plain gels. Overall, the physical characteristics of the gels
agreed with previous reports and will ultimately be supplemented with mechanical properties to
further investigate the materials.

INTRODUCTION

Injured neurons do not consistently regenerate in the proper manner in vivo, but scaffolds
composed of the proper materials have the potential to allow necessary nerve extension1. Both
natural and synthetic polymers are currently being looked as scaffolds. Of particular interest in
this study are collagen gels. Type I collagen is the most prevalent form in the ECM2. When used
as a scaffold, it provides favorable conditions for cell adhesion and growth.5 It is capable of
forming fibrils in vitro similar to those seen in vivo. Collagen gels have various properties that
may affect neurite growth. One such property is mechanical stiffness. Mechanical stiffness may
be responsible for the special arrangement of the fibers3. Also important are the physical
characteristics of the collagen fibers, such as fiber diameter and spacing.

However, collagen I is not alone in the body. It interacts with other components of the
ECM, including other types of collagen, glycosaminoglycans (GAGs), proteoglycans, and
noncollagenous proteins.2 One example of another ECM component is collagen IV. Collagen IV
is found only in basement membranes.6 It does not form fibrils.9 Fibronectin is another
component of the ECM. It is important for cell growth, migration, differentiation, and adhesion. 7
Chondroitin sulfate, a GAG, has been shown to support significantly less nerve growth than
fibronectin.8 GAGs consist of a protein core with polysaccharide branches.10 Their function is to
link collagen together to maintain the shape of tissue and organs, especially connective tissue.10
Laminin is often found in conjunction with collagen IV; it serves to attach certain types of cells,
such as epithelial cells, to a substrate in a manner that is analogous to that of fibronectin.9

MATERIALS AND METHODS

Materials

PBS was purchased from MP Biomedicals, Inc of Solon, OH. Ethanol was purchased
from Acros, NJ. Collagen I was obtained from rat tails as defined in a previous paper.4 Sodium
hydroxide was purchased from X. Kaughn’s F12 media, HEPES, chondroitin sulfate, lamanin,
sodium bicarbonate collagen IV, fibronectin, and 70% glutaraldehyde were purchased from
Sigma Aldrich St. Louis, MO. Sodium hydroxide was purchased from Fischer Scientific of Fair
Lawn, NJ.

Gel Preparation

Collagen gels were made on poly-l-lysine coated cover slips. Gels were made by a
previously defined method (Appendix A); NaHCO3, H2O, collagen I, NaOH, F12K, HEPES, and
an additive were mixed over ice. Gels without additives were poured at concentrations ranging
from .4-2mg/ml. Gels with additives were prepared at collagen concentrations of .4, 1, and
2mg/ml and additive concentrations of 1, 10, and 100ug/ml. Additives included laminin,
fibronectin, collagen IV, and chondroitin sulfate. After mixing, the gels were pipetted on to the
poly-l-lysine coated coverslips in 12-well plates. Gels were allowed to set in a dry incubator.
After gels were set, they were fixed with 2% glutaraldehyde, then washed three times with PBS
and placed on a mixer plate for ten minutes.

Preparation for Scanning Electron Microscopy (SEM)

The gels were dehydrated with an ethanol gradient. In this dehydration, the PBS was
removed and replaced with 10% ethanol. The gels were then placed on a mixer plate for 10
minutes. The 10% ethanol was then replaced with 20% ethanol and the mixer plate process was
repeated. This process was then repeated with each of the gels with increasing concentrations of
ethanol. The following concentrations of ethanol were used: 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 95%, 100%, 100%, and 100%. Specimens were then critical point dried
using CO2 as a transition medium with a BAL-TEC CPD 030. Immediately after being dried, the
samples were sputter coated with a BAL-TEC SCD 005 using an Au/Pd plate with a current of
30mA at a pressure of .5mbar for two 50 second segments.

SEM Imaging and Analysis

Gels were imaged on a JEOL JSM5800 scanning electron microscope with an
accelerating voltage of 10mV and a working distance of 20mm. Images were then analyzed for
fiber diameter using ImageJ by drawing a perpendicular line across the fiber diameter. Four
fibers per image were analyzed. Fiber diameters were then statistically analyzed using a student’s
t-test.

RESULTS

Plain Collagen Gels

For most concentrations of the plain collagen gels, there was no significant difference in
fiber diameter (Table 1). However, the diameters 2mg/ml and 1.5mg/ml gels were significantly
greater than several of the other gels. This is likely due to the aggregation of the fibers in the
higher concentrations. In the images of the plain collagen gels, fibers and aggregations of fibers
were generally easily distinguishable (Figure 1), unlike gels with additives.

Table 1. Average fibril diameter for varying collagen concentrations

Fiber 

Concentration  diameter  Standard 

(mg/ml)  (nm)  Error 

0.4  126.4781  2.219 

0.6  121.3118  1.995 

0.8  121.8483  2.899 

1  122.0997  2.376 

1.25  121.3659  1.988 

1.5  128.9659  2.552 

2  128.5034  2.05 

 

Figure 1. SEM images depicting collagen I gels of varying concentrations without additives.

Collagen Gels with Additives

The different additives to the gels had varying effects. At all concentrations, laminin did
not significantly change the fiber diameter. However, as seen from the images, the fibers coated
with laminin were often blurry and difficult to focus in upon (Figures 2, 3, and 4); this may have
been due to laminin altering the properties of the fibers.

Collagen IV significantly decreased fiber diameter for all concentrations of collagen and
additives (Table 2, 3, and 4). In addition, the appearance of the gels was altered; the plain gels
had clear, distinct fibers while the gels with collagen IV have a matted appearance (Figures 2, 3,
and 4).

Chondroitin Sulfate also significantly decreased fiber diameter for all collagen and
additive concentrations (Tables 2, 3, and 4). Chondroitin sulfate also gave the gels a matted
appearance (Figures 2, 3, and 4).

Experiments with fibronectin were also done, but not enough images were obtained for
analysis.

 

Figure 2. SEM images depicting .4mg/ml collagen gels with various additives
Table 2. Mean fiber diameters in nm of 4mg/ml gels with various additives.

  .4mg/ml collagen 
Concentration additive (ug/ml) 
laminin 0  Fiber diameter  Standard Error 

collagen IV 0  126.4781 2.219 

chondroitin sulfate 0  1 134.4774 2.526 

10 127.1057 2.537 

100 119.8543 4.128 

126.4781 2.219 

1 90.2649 2.736 

10 89.32866 1.874 

100 88.15418 2.649 

126.4781 2.219 

1 86.20376 1.87 

10 92.37093 2.841 

100 88.7001 2.487 

 

 

Figure 3. SEM images depicting 1mg/ml collagen gels with various additives
Table 3. Mean fiber diameter in nm for 1mg/ml collagen gels with different additives.

  1mg/ml   
Standard 
Concentration additive  Fiber  Error 

(ug/ml)  diameter  2.376
2.525
Laminin 0  122.0997 2.537
4.127
1  121.1063 2.376
1.631
10  113.8054 3.064
1.749
100  112.3894 2.376
2.252
Collagen IV 0  122.0997 3.148
3.261
1  89.05265

10  91.03883

100  83.60394

Chondroitin sulfate 0  122.0997

1  89.11265

10  85.23118

100  95.02843

 

 

Figure 4. SEM images depicting 2mg/ml collagen gels with various additives.

Table 4. Mean fiber diameter in nm of 2mg/ml gels.

Concentration  Fiber  Standard 

additive (ug/ml)  diameter  Error 

Laminin 0  128.5034  2.05

1  125.4952  2.869

10  129.357  3.174

100  128.6474  5.931

Collagen IV 0  128.5034  2.05

1  88.09646  2.479

10  87.29205  3.664

100  88.49707  2.631

Chondroitin 

sulfate 0  128.5034  2.05

1  89.53108  2.62

10  91.46846  1.758

100  100.225  3.494

DISCUSSION

Our results confirm that chondroitin sulfate and collagen IV do influence the collagen
fiber diameter and that laminin has no significant effect. Chondroitin sulfate decreased the fiber

diameters as expected. A previous study also found that chondroitin sulfate decreased the
collagen I fiber diameter at all concentrations.2 This is consistent with the function of a GAG in
maintaining structure in connective tissue by linking with collagen to form interpenetrating
fibers.10 This interweaving of collagen I and chondroitin sulfate may have been responsible for
the matted appearance of the gels. Additionally, chondroitin sulfate increased the rate of
fibrillogenesis by either increasing the nucleation sites or by changing their shapes.11 This may
have led to decreased fibril diameter by promoting aggregation of the ends of collagen
molecules.2

Laminin did not significantly change the fiber diameter. Since it functions in attaching
cells to the basement membrane, it was expected to increased fiber diameter. Images were
blurrier than others and more difficult to focus. This may have been a result of the laminin
altering the fiber characteristics. More work is needed to determine if laminin does affect the gel.

Type IV collagen significantly decreased fiber diameter. Collagen IV is found only in the
basement membrane.6 The triple helices of the molecule form a scaffold for the ECM to act
upon.6 To the author’s knowledge, no other work has measured fibril diameter of collagen I gels
with collagen IV to allow for comparison.

REFERENCES

1Blewitt, M. J. and R. K. Willits. The effect of soluble peptide sequences on neurite extension on
2D collagen substrates and within 3D collagen gels. Annals of Biomedical Engineering 35(12):
2159-2167, 2007.

2Panitch, A. and K. Stuart. Characterization of gels composed of blends of collagen I, collagen
III, and chondroitin sulfate.

3Skornia, S. L. and R. K. Willits. Effect of collagen gel stiffness on neurite extension.

4Silver, F. H. and R. L. Trelstad. Type I collagen in solution: structure and properties of fibril
fragments. The Journal of Biological Chemistry. 255(19): 9247-9433, 1980.

5Iversen, P. L., F. Moatamed, L. M. Partlow, and L. J. Stensaas. Characterization of a variety of
standard collagen substrates: ultrastucture, uniformity, and capacity to bind to and promote
growth of neurons. In Vitro. 17(5): 541-552, 1980.

6Hudson, B. G., J. Khoshnoodi, V. Pedchenko. Mammalian collagen IV. Microscopy Research
and Technique. 71: 357-370, 2008.

7Pankov, R. and K. M. Yamada. Fibronectin at a glance. Journal of Cell Science. 115: 3861-
3863, 2002

8Carbonetto, S., M.M. Gruver, and D.C. Turner. Nerve fiber growth in culture on fibronectin,
collagen, and glycosaminoglycan substrates. The Journal of Neuroscience. 3(11): 2324-2335,
1983.

9Kleinman, H.K., R.J. Klebe, and G.R. Martin. Role of collagenous matrices in the adhesion and
growth of cells. The Journal of Cell Biology. 88: 473-485, 1981.

10Beuermen, R., S. Ramakrishna, W.E. Teo, L.Y.L. Yung, S. Zhong, and X. Zhu. Formation of
collagen-glycosaminoglycan blended nanofibrous scaffolds and their biological properties.
Biomacromolecules. 6: 2998-3004, 2005.

11Wood, G.C. The formation of fibrils from collagen solutions: effect of chondroitin sulfate and
some other naturally occurring polyanions on the rate of formation. Biochem J. 75: 605-612,
1960.

Appendix A

Collagen Gel Recipes

Collagen Gels ***New Recipe with F12K pH
3.5 mg/ml Collagen 8.0

Control (no 0.4 0.6 Collagen Concentrations 1.5 2
additives) 1.2 1.2 0.8 1 1.25 1.2 1.2
Ingredients (uL) 146.94 135.51 81.09 51.51
1.2 1.2 1.2
NaHCO3 22.86 34.29 124.09 112.66 96.37 85.71 114.29
1 1 4 5
H2O 20 20 45.71 56.64 71.43 20 20
Collagen (3.5 8 8 1 1.5 3 8 8
mg/mL) 20 20 20
0.1 M NaOH 0 0 8 8 8 0 0
10x F12K media 200 200 200 200
HEPES 000
Additive (100 200 200 200
ug/mL)
Total (uL)

1 ug/mL Additive 0.4 0.6 Collagen Concentrations 1.5 2
Ingredients (uL) 1.2 1.2 0.8 1 1.25 1.2 1.2
126.91 114.55 1.2 1.2 1.2 55.32 19.84
NaHCO3 101.92 89.47 72.37
22.86 34.29 85.71 114.29
H2O 1.03 1.96 45.71 56.64 71.43 9.77 16.67
Collagen (3.5 20 20 3.17 4.69 7 20
mg/mL) 20 20 20 20
0.1 M NaOH 8 8 8 8 8
10x F12K media 8 8
HEPES 20 20 20 20
Additive (100 200 200 20 20 20 200 200
ug/mL) 200 200 200
Total (uL)

10 ug/mL Additive 0.4 0.6 Collagen Concentrations 1.5 2
Ingredients (uL) 1.2 1.2 0.8 1 1.25 1.2 1.2
126.91 114.55 1.2 1.2 1.2 55.32 19.84
NaHCO3 101.92 89.47 72.37
H2O

Collagen (3.5 22.86 34.29 45.71 56.64 71.43 85.71 114.29
mg/mL) 1.03 1.96 3.17 4.69 7 9.77 16.67
0.1 M NaOH 20 20 20 20 20 20
10x F12K media 8 20
HEPES 8 8 8 8 8 8
Additive (100 20
ug/mL) 20 20 20 20 200 20 20
Total (uL) 200 200 200 200 200 200

100 ug/mL Additive 0.4 0.6 Collagen Concentrations 1.5 2
Ingredients (uL) 1.2 1.2 0.8 1 1.25
126.91 114.55 1.2 1.2 1.2 1.2 1.2
NaHCO3 101.92 89.47 72.37 55.32 19.84
22.86 34.29
H2O 1.03 1.96 45.71 56.64 71.43 85.71 114.29
Collagen (3.5 20 20 3.17 4.69 7 9.77 16.67
mg/mL) 20 20 20 20
0.1 M NaOH 8 8 8 20
10x F12K media 20 20 8 8 20 8 8
HEPES 200 200 20 20 20 20
Additive (1 mg/mL) 200 200 200 200 200
Total (uL)


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