Corey P Neu

Garrett Shannon

Sherry L Voytik-Harbin

Tyler Novak

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Copyright © 2013 by ASME 1 
INTRODUCTION 
 Osteoarthritis (OA), commonly known as ‘wear and tear’ in 
human joints, affects over 27 million people in the United States [1]. 
There is currently no encompassing solution for the regeneration of 
damaged articular cartilage. One potential solution involves the close 
emulation of the native structure of articular cartilage, with special 
consideration given to maintaining the distinct organized zonal 
ultrastructure, characterized by both random and highly aligned zones 
of collagen fibrils, in order to preserve mechanical and cell signaling 
properties of the extracellular matrix [2]. Techniques such as 
electrospinning achieve high degrees of alignment, but do so at the 
cost of denaturing the collagen molecule [3] that may lead to inferior 
cell recognition and mechanical strength. 
 Collagen fibrils have been shown to align perpendicular to an 
applied magnetic field [4]. The magnetic field induces a torque in the 
diamagnetic planar peptide bonds of the collagen during fibril 
assembly, and results in increased forces as the collagen molecules 
aggregate. However, current methods report inconsistent alignment in 
large scaffolds suitable for cartilage regeneration, perhaps due to 
physical properties of the collagen solutions [5]. We hypothesized that 
the time required for fibril alignment during fibrillogenesis (fibril 
assembly) could be extended using lower temperatures to create 
controlled alignment profiles through the interior of collagen scaffolds. 
Our objective was to manipulate the collagen fibrillogenesis time 
through temperature control to achieve high degrees of alignment 
while maintaining the physiological pH. 
 
METHODS 
Collagen Extraction, Fibrillogenesis, and Turbidity 
 Type I oligomeric collagen was extracted from porcine dermis to 
a concentration of 5.6 mg/mL as previously described [6]. Collagen 
was neutralized to a final concentration of 3.2 mg/mL using 10X PBS 
and 0.1N NaOH [5]. Control (random) scaffold collagen solutions 
were polymerized at various temperatures between 15°C and 37°C in 
an incubator (MIR-154, Sanyo). Aligned scaffolds were polymerized 
within a 9.4 Tesla vertical-bore superconducting magnet (CMX 400, 
Chemagnetics) at either 18°C or 37°C for fibril orientation studies. 
Scaffolds were formed inside (10×10×5 mm3) plastic chambers, bound 
by No. 1 confocal microscopy coverslips (VWR), to facilitate imaging. 
Fibrillogenesis time was assessed using turbidity data. 
 For turbidity of control scaffolds, spectrophotometric data was 
acquired at 30 second intervals using a 313 nm wavelength light 
source (n=3 for each group) [7]. Temperature control was achieved 
through the use of a refrigerated water-pump (Isotemp 3006D, Fisher 
Scientific) and verified by an external thermocouple. 
 
Collagen Alignment Assays 
 Collagen scaffolds were analyzed using polarized light (bulk) and 
confocal reflection (microscale) microscopy. For polarized light 
microscopy (PLM), a full wavelength retardation plate was used to 
distinguish a red-blue color shift in aligned scaffolds. Scaffolds (n=12) 
were placed on a rotating stage and imaged at 5° intervals through a 
180° arc using a Leica DFC480 CCD camera. A micrometer was used 
to shift the field of view and acquire alignment data through the depth 
of the scaffold. The parallelism index (PI) was determined as 
previously described [8]. 
 For confocal reflection microscopy, microscale fibril alignment 
was assessed by an Olympus Fluoview FV1000 microscope system 
(488 nm) with a 40× water immersion objective. An image volume (z-
stack; 5 images with a 50 µm interslice distance) was performed at up 
to nine separate locations within each scaffold (n=12). Fiber alignment 
was assessed using a custom two-dimensional Fast Fourier Transform 
 
TEMPERATURE CONTROL DURING FIBRILLOGENESIS ALLOWS FOR IMPROVED 
MAGNETIC ALIGNMENT OF COLLAGEN 
Garrett Shannon
1 
 
Tyler Novak
1
       Sherry L. Voytik-Harbin
1
   Corey P. Neu
1 
Weldon School of Biomedical Engineering 
Purdue University 
Lafayette, IN, 47907 
United States of America 
Proceedings of the ASME 2013 Summer Bioengineering Conference 
SBC2013 
June 26-29, 2013, Sunriver, Oregon, USA 
SBC2013-14314
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Copyright © 2013 by ASME 2 
(FFT) based algorithm (MATLAB) [9]. Image intensity of the FFT 
results was radially summed to determine preferred alignment angles. 
The aspect ratio of a best-fit (linear) ellipse was determined from long 
and short axes, representing the degree of alignment. 
 
Statistical Analysis 
 Confocal and turbidity data were analyzed using SAS/STAT® 
software. Aspect ratios were compared using a mixed-model ANOVA 
with samples as a random effect. Magnetic field, temperature, z-stack, 
and depth into scaffolds were treated as fixed main effects. All fixed 
effect interactions were included. Fibrillogenesis times were compared 
using a one-way ANOVA with temperature as a fixed main effect. 
 
RESULTS 
 Full thickness collagen alignment was successfully demonstrated 
using temperature-controlled fibrillogenesis. Collagen scaffolds were 
successfully created and imaged at varying temperatures (18°C and 
37°C). Turbidity data showed an inverse relationship between 
temperature and fibrillogenesis time (p < 0.0001) (Figure 1). Under 
crossed polarizers, control collagen scaffolds (formed absent of a 
magnetic field) appeared similar at differing temperatures, whereas 
scaffolds formed within a 9.4T magnetic field showed a depth-
dependent PI influenced by temperature (Figure 2a). Although the 
maximum magnitude of the PI was consistent between temperatures, 
confocal data demonstrated that lower temperatures promoted a more 
aligned microstructure in a 9.4T magnetic field (p = 0.010) (Figure 2b-
c). Spatial image data revealed non-homogenous alignment throughout 
the scaffold thickness (p=0.0029).  
 
 
Figure 1. Collagen fibrillogenesis time depends on temperature. 
(a) A representative (sigmoidal) turbidity profile generated at 18°C. 
(b) Fibrillogenesis times, expressed as time to reach 95% maximum 
absorbance, follow a two-parameter exponential decay with increasing 
temperatures (R2 = 0.9969). 
 
DISCUSSION 
 This work introduces temperature as a tunable parameter for 
obtaining targeted degrees of fibril alignment for collagen scaffolds 
formed within a magnetic field. Superior alignment achieved through 
other methods such as electrospinning is a primary deterrent to 
magnetic alignment techniques; however, scaffold formation at lower 
temperatures offers increased alignment potentially making this a 
more attractive option for biomimetic scaffold development for the 
treatment of OA. 
 Temperature control also influenced the volume of the scaffold 
that appeared aligned. While scaffolds formed at 37°C showed 
alignment only near the center, with decreasing PI throughout the 
scaffold thickness, collagen formed at 18°C showed alignment 
throughout the full width and thickness of the scaffold. Images 
acquired under PLM using a retardation plate confirmed the 
temperature-dependent spatial variation in alignment throughout full 
thickness scaffolds. These data indicate that temperature control 
during fibrillogenesis could serve to modulate the spatial variation in 
alignment; however, further work is needed to establish and define this 
relationship to exploit temperature as a tunable parameter. 
 
 
Figure 2. Improved alignment of collagen is observed using low 
temperature fibrillogenesis at 9.4T. The parallelism index (PI) 
indicates control (random; 0T) (a) and bulk (9.4T) alignment (b) in 
collagen following 18°C (blue) and 37°C (red) exposure. Samples 
formed at lower temperatures maintain alignment throughout the depth 
of the scaffold, indicating more consistent alignment throughout the 
scaffold volume. The gap (dashed line) represents the acquisition of 
two adjacent fields of view. (b) Qualitative differences between 18°C 
and 37°C magnetic alignment can be seen by confocal microscopy. (c) 
Polar plots of FFT magnitude reveal preferred fibril distributions. 
Collagen gels formed at 18°C at 9.4T exhibit much higher alignment, 
as shown by FFT-derived elliptical aspect ratios. 
 
ACKNOWLEDGEMENTS 
 Funding from the Trask Fund (PRF) is gratefully acknowledged. 
We also thank Tyler Smith and Catherine Whittington for their help 
with collagen extractions, and the Purdue Interdepartmental NMR 
Facility for magnet access. 
 
REFERENCES 
[1] C. G. Helmick, et al., Arthritis and Rheumatism, vol. 58, pp. 15-
25, Jan 2008. 
[2] Z. G. Ge, et al., Journal of Biomedical Materials Research Part 
A, vol. 100A, pp. 2526-2536, Sep 2012. 
[3] D. I. Zeugolis, et al., Biomaterials, vol. 29, pp. 2293-2305, May 
2008. 
[4] J. Torbet and M. C. Ronziere, Biochemical Journal, vol. 219, pp. 
1057-1059, 1984. 
[5] Y. Yang, et al., Conference on Optics in Tissue Engineering and 
Regenerative Medicine III, San Jose, CA, 2009. 
[6] S. T. Kreger, et al., Biopolymers, vol. 93, pp. 690-707, Aug 2010. 
[7] B. R. Williams, et al., Journal of Biological Chemistry, vol. 253, 
pp. 6578-6585, 1978. 
[8] J. Rieppo, et al., Microscopy Research and Technique, vol. 71, 
pp. 279-287, Apr 2008. 
[9] E. A. Sander and V. H. Barocas, Journal of Biomedical Materials 
Research Part A, vol. 88A, Feb 2009. 
 
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Temperature Control During Fibrillogenesis Allows for Improved Magnetic Alignment of Collagen

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Temperature Control During Fibrillogenesis Allows for Improved Magnetic Alignment of Collagen

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