Research Papers

Analysis of Collagen Organization in Mouse Achilles Tendon Using High-Frequency Ultrasound Imaging

[+] Author and Article Information
Corinne N. Riggin, Benjamin R. Freedman

McKay Orthopaedic Research Laboratory,
University of Pennsylvania,
424 Stemmler Hall,
36th and Hamilton Walk,
Philadelphia, PA 19104

Joseph J. Sarver

McKay Orthopaedic Research Laboratory,
University of Pennsylvania,
424 Stemmler Hall,
36th and Hamilton Walk,
Philadelphia, PA 19104;
School of Biomedical Engineering,
Science and Health Systems,
Drexel University,
3141 Chestnut Street,
Philadelphia PA 19104

Stephen J. Thomas

McKay Orthopaedic Research Laboratory,
University of Pennsylvania,
424 Stemmler Hall,
36th and Hamilton Walk,
Philadelphia, PA 19104;

Division of Nursing and Health Sciences
Athletic Training,
Neumann University,
1 Neumann Rd.
Aston, PA 19014

Louis J. Soslowsky

McKay Orthopaedic Research Laboratory,
University of Pennsylvania,
424 Stemmler Hall,
36th and Hamilton Walk,
Philadelphia, PA 19104
e-mail: soslowsk@upenn.edu

Contributed by the Bioengineering Division of ASME for publication in the Journal of Biomechanical Engineering. Manuscript received September 3, 2013; final manuscript received December 12, 2013; accepted manuscript posted December 19, 2013; published online February 5, 2014. Editor: Victor H. Barocas.

J Biomech Eng 136(2), 021029 (Feb 05, 2014) (6 pages) Paper No: BIO-13-1407; doi: 10.1115/1.4026285 History: Received September 03, 2013; Revised December 12, 2013; Accepted December 19, 2013

Achilles tendon ruptures are traumatic injuries, and techniques for assessing repair outcomes rely on patient-based measures of pain and function, which do not directly assess tendon healing. Consequently, there is a need for a quantitative, in vivo measure of tendon properties. Therefore, the purpose of this study was to validate ultrasound imaging for evaluating collagen organization in tendons. In this study, we compared our novel, high-frequency ultrasound (HFUS) imaging and analysis method to a standard measure of collagen organization, crossed polarizer (CP) imaging. Eighteen mouse Achilles tendons were harvested and placed into a testing fixture where HFUS and CP imaging could be performed simultaneously in a controlled loading environment. Two experiments were conducted: (1) effect of loading on collagen alignment and (2) effect of an excisional injury on collagen alignment. As expected, it was found that both the HFUS and CP methods could reliably detect an increase in alignment with increasing load, as well as a decrease in alignment with injury. This HFUS method demonstrates that structural measures of collagen organization in tendon can be determined through ultrasound imaging. This experiment also provides a mechanistic evaluation of tissue structure that could potentially be used to develop a targeted approach to aid in rehabilitation or monitor return to activity after tendon injury.

Copyright © 2014 by ASME
Your Session has timed out. Please sign back in to continue.


Suchak, A. A., Bostick, G., Reid, D., Blitz, S., and Jomha, N., 2005, “The Incidence of Achilles Tendon Ruptures in Edmonton, Canada,” Foot Ankle Int., 26(11), pp. 932–936. [PubMed]
Houshian, S., Tscherning, T., and Riegels-Nielsen, P., 1998, “The Epidemiology of Achilles Tendon Rupture in a Danish County,” Injury, 29(9), pp. 651–654. [CrossRef] [PubMed]
Moller, A., Astron, M., and Westlin, N., 1996, “Increasing Incidence of Achilles Tendon Rupture,” Acta Orthop. Scand., 67(5), pp. 479–481. [CrossRef] [PubMed]
Lin, T. W., Cardenas, L., and Soslowsky, L. J., 2004, “Biomechanics of Tendon Injury and Repair,” J. Biomech., 37(6), pp. 865–877. [CrossRef] [PubMed]
Saxena, A., Ewen, B., and Maffulli, N., 2011, “Rehabilitation of the Operated Achilles Tendon: Parameters for Predicting Return to Activity,” J. Foot Ankle Surg., 50(1), pp. 37–40. [CrossRef] [PubMed]
Mullaney, M. J., McHugh, M. P., Tyler, T. F., Nicholas, S. J., and Lee, S. J., 2006, “Weakness in End-Range Plantar Flexion After Achilles Tendon Repair,” Am. J. Sports Med., 34(7), pp. 1120–1125. [CrossRef] [PubMed]
Robinson, J. M., Cook, J. L., Purdam, C., Visentini, P. J., Ross, J., Maffulli, N., Taunton, J. E., and Khan, K. M., 2001, “The VISA-A Questionnaire: A Valid and Reliable Index of the Clinical Severity of Achilles Tendinopathy,” Br. J. Sports Med., 35(5), pp. 335–341. [CrossRef] [PubMed]
Silbernagel, K. G., Nilsson-Helander, K., Thomee, R., Eriksson, B. I., and Karlsson, J., 2010, “A New Measurement of Heel-Rise Endurance With the Ability to Detect Functional Deficits in Patients With Achilles Tendon Rupture,” Knee Surg. Sports Traumatol. Arthrosc., 18(2), pp. 258–264. [CrossRef] [PubMed]
Chinn, L., and Hertel, J., 2010, “Rehabilitation of Ankle and Foot Injuries in Athletes,” Clin. Sports Med., 29(1), pp. 157–167. [CrossRef] [PubMed]
Miller, K. S., Connizzo, B. K., Feeney, E., and Soslowsky, L. J., 2012, “Characterizing Local Collagen Fiber Re-Alignment and Crimp Behavior Throughout Mechanical Testing in a Mature Mouse Supraspinatus Tendon Model,” J. Biomech., 45(12), pp. 2061–2065. [CrossRef] [PubMed]
Miller, K. S., Edelstein, L., Connizzo, B. K., and Soslowsky, L. J., 2012, “Effect of Preconditioning and Stress Relaxation on Local Collagen Fiber Re-alignment: Inhomogeneous Properties of Rat Supraspinatus Tendon,” ASME J. Biomech. Eng., 134(3), p. 031007. [CrossRef]
Gimbel, J. A., Van Kleunen, J. P., Mehta, S., Perry, S. M., Williams, G. R., and Soslowsky, L. J., 2004, “Supraspinatus Tendon Organizational and Mechanical Properties in a Chronic Rotator Cuff Tear Animal Model,” J. Biomech., 37(5), pp. 739–749. [CrossRef] [PubMed]
Dourte, L. M., Perry, S. M., Getz, C. L., and Soslowsky, L. J., 2010, “Tendon Properties Remain Altered in a Chronic Rat Rotator Cuff Model,” Clin. Orthop. Relat. Res., 468(6), pp. 1485–1492. [CrossRef] [PubMed]
Lake, S. P., Miller, K. S., Elliott, D. M., and Soslowsky, L. J., 2009, “Effect of Fiber Distribution and Realignment on the Nonlinear and Inhomogeneous Mechanical Properties of Human Supraspinatus Tendon Under Longitudinal Tensile Loading,” J. Orthop. Res., 27(12), pp. 1596–1602. [CrossRef] [PubMed]
Quinn, K. P., and Winkelstein, B. A., 2008, “Altered Collagen Fiber Kinematics Define the Onset of Localized Ligament Damage During Loading,” J. Appl. Physiol., 105(6), pp. 1881–1888. [CrossRef] [PubMed]
Quinn, K. P., Bauman, J. A., Crosby, N. D., and Winkelstein, B. A., 2010, “Anomalous Fiber Realignment During Tensile Loading of the Rat Facet Capsular Ligament Identifies Mechanically Induced Damage and Physiological Dysfunction,” J. Biomech., 43(10), pp. 1870–1875. [CrossRef] [PubMed]
Thomopoulos, S., Williams, G. R., Gimbel, J. A., Favata, M., and Soslowsky, L. J., 2003, “Variation of Biomechanical, Structural, and Compositional Properties Along the Tendon to Bone Insertion Site,” J. Orthop. Res., 21(3), pp. 413–419. [CrossRef] [PubMed]
Franchi, M., Torricelli, P., Giavaresi, G., and Fini, M., 2013, “Role of Moderate Exercising on Achilles Tendon Collagen Crimping Patterns and Proteoglycans,” Connect. Tissue Res., 54, pp. 267–274. [CrossRef] [PubMed]
Lau, T. Y., Ambekar, R., and Toussaint, K. C., 2012, “Quantification of Collagen Fiber Organization Using Three-Dimensional Fourier Transform-Second-Harmonic Generation Imaging,” Opt. Express, 20(19), pp. 21821–21832. [CrossRef] [PubMed]
Gusachenko, I., Tran, V., Houssen, Y. G., Allain, J. M., and Schanne-Klein, M. C., 2012, “Polarization-Resolved Second-Harmonic Generation in Tendon Upon Mechanical Stretching,” Biophys. J., 102(9), pp. 2220–2229. [CrossRef] [PubMed]
Abraham, T., Fong, G., and Scott, A., 2011, “Second Harmonic Generation Analysis of Early Achilles Tendinosis in Response to in vivo Mechanical Loading,” BMC Musculoskelet. Disord., 12, p. 26.
Vidal Bde, C., and Mello, M. L., 2011, “Collagen Type I Amide I Band Infrared Spectroscopy,” Micron, 42(3), pp. 283–289. [CrossRef] [PubMed]
Alfredson, H., Masci, L., and Ohberg, L., 2011, “Partial Mid-portion Achilles Tendon Ruptures: New Sonographic Findings Helpful for Diagnosis,” Br. J. Sports Med., 45(5), pp. 429–432. [CrossRef] [PubMed]
Maranho, D. A., Nogueira-Barbosa, M. H., Simao, M. N., and Volpon, J. B., 2009, “Ultrasonographic Evaluation of Achilles Tendon Repair After Percutaneous Sectioning for the Correction of Congenital Clubfoot Residual Equinus,” J. Pediatr. Orthop., 29(7), pp. 804–810. [CrossRef] [PubMed]
Mangat, K. S., Kanwar, R., Johnson, K., Korah, G., and Prem, H., 2010, “Ultrasonographic Phases in Gap Healing Following Ponseti-Type Achilles Tenotomy,” J. Bone Joint Surg. Am., 92(6), pp. 1462–1467. [CrossRef] [PubMed]
Vadala, A., De Carli, A., Vulpiani, M. C., Iorio, R., Vetrano, M., Scapellato, S., Suarez, T., DiSalvo, F., and Ferretti, A., 2012, “Clinical, Functional and Radiological Results of Achilles Tenorraphy Surgically Treated With Mini-open Technique,” J. Sports Med. Phys. Fitness, 52(6), pp. 616–621. [PubMed]
Poposka, A., Georgieva, D., and Dzoleva-Tolevska, R., 2012, “Significance of Ultrasound in the Diagnosis and Treatment of Achilles Tendon Rupture,” Prilozi, 33(1), pp. 209–216. [PubMed]
Chamberlain, C. S., Duenwald-Kuehl, S. E., Okotie, G., Brounts, S. H., Baer, G. S., and Vanderby, R., 2013, “Temporal Healing in Rat Achilles Tendon: Ultrasound Correlations,” Ann. Biomed. Eng., 41(3), pp. 477–487. [CrossRef] [PubMed]
Duenwald-Kuehl, S., Lakes, R., and Vanderby, R., Jr., 2012, “Strain-Induced Damage Reduces Echo Intensity Changes in Tendon During Loading,” J. Biomech., 45(9), pp. 1607–1611. [CrossRef] [PubMed]
Bashford, G. R., Tomsen, N., Arya, S., Burnfield, J. M., and Kulig, K., 2008, “Tendinopathy Discrimination by Use of Spatial Frequency Parameters in Ultrasound B-Mode Images,” IEEE Trans. Med. Imaging, 27(5), pp. 608–615. [CrossRef] [PubMed]
Kulig, K., Landel, R., Chang, Y. J., Hannanvash, N., Reischl, S. F., Song, P., and Bashford, G. R., 2013, “Patellar Tendon Morphology in Volleyball Athletes With and Without Patellar Tendinopathy,” Scand. J. Med. Sci. Sports, 23(2), pp. e81–e88. [CrossRef] [PubMed]
Chen, X. M., Cui, L. G., He, P., Shen, W. W., Qian, Y. J., and Wang, J. R., 2013, “Shear Wave Elastographic Characterization of Normal and Torn Achilles Tendons: A Pilot Study,” J. Ultrasound Med., 32(3), pp. 449–455. [PubMed]
Shah, S. A., Santago, P., and Rubin, B. K., 2005, “Quantification of Biopolymer Filament Structure,” Ultramicroscopy, 104(3–4), pp. 244–254. [CrossRef] [PubMed]
Gimbel, J. A., Mehta, S., Van Kleunen, J. P., Williams, G. R., and Soslowsky, L. J., 2004, “The Tension Required at Repair to Reappose the Supraspinatus Tendon to Bone Rapidly Increases After Injury,” Clin. Orthop. Rela.t Res., 4(26), pp. 258–265. [CrossRef]
Garcia, T., Hornof, W. J., and Insana, M. F., 2003, “On the Ultrasonic Properties of Tendon,” Ultrasound Med. Biol., 29(12), pp. 1787–1797. [CrossRef] [PubMed]
Khoury, V., Guillin, R., Dhanju, J., and Cardinal, E., 2007, “Ultrasound of Ankle and Foot: Overuse and Sports Injuries,” Semin. Musculoskelet. Radiol., 11(2), pp. 149–161. [CrossRef] [PubMed]
VisualSonics, 2007, VisualSonics 700-Series RMV Scanhead Selector, VisualSonics, Toronto.
Hansen, P., Haraldsson, B. T., Aagaard, P., Kovanen, V., Avery, N. C., Qvortrup, K., Larsen, J. O., Krogsgaard, M., Kjaer, M., and Peter Magnusson, S., 2010, “Lower Strength of the Human Posterior Patellar Tendon Seems Unrelated to Mature Collagen Cross-Linking and Fibril Morphology,” J. Appl. Physiol., 108(1), pp. 47–52. [CrossRef] [PubMed]
Ros, S. J., Andarawis-Puri, N., and Flatow, E. L., 2013, “Tendon extracellular matrix damage detection and quantification using automated edge detection analysis,” J. Biomech., 46(16), pp. 2844–2847. [CrossRef]


Grahic Jump Location
Fig. 5

(a) Changes in CSD in response to load for the CP images. There is a significant decrease in CSD (increase in organization) with increased loading. Statistics for each load compared to 0 N (*p < 0.05) and to 0.5 N (∼p < 0.05). (b) CSD measures for CP images before and after a biopsy punch excisional injury. There is an increase in CSD (decrease in organization) with injury (*p < 0.05).

Grahic Jump Location
Fig. 4

(a) Changes in CSD in response to load for the HFUS images. There is a significant decrease in CSD (increase in organization) with increased loading. Statistics for each load compared to 0 N (*p < 0.05) and to 0.5 N (∼p < 0.05). (b) CSD measures for HFUS images before and after a biopsy punch excisional injury. There is a significant increase in CSD (decrease in organization) with injury (*p < 0.05).

Grahic Jump Location
Fig. 1

Schematic of setup for capturing CP and HFUS imaging simultaneously. The tendon is secured between two grips in a PBS tank, where the ultrasound probe can be centered over top of the tendon, submerged in the PBS. Two polarizing sheets are located on either side of the tank, with a linear backlight on one side and a camera on the other (not depicted in this image). A custom-built tensiometer is attached to a sliding shaft to allow for load control. Inset figure (below) shows representative images of a tendon as captured by the CP camera through one rotation cycle.

Grahic Jump Location
Fig. 3

Alignment maps and histograms depicting the distribution of localized fiber directions throughout the tendon that were produced through CP analysis for (a) injured and (b) uninjured specimens

Grahic Jump Location
Fig. 2

(a) HFUS image of Achilles tendon with excisional injury, (b) filtered HFUS image, (c) hyper- and hypoechoic regions that create the banded pattern that is quantified as the CSD of the fiber orientations



Some tools below are only available to our subscribers or users with an online account.

Related Content

Customize your page view by dragging and repositioning the boxes below.

Related Journal Articles
Related eBook Content
Topic Collections

Sorry! You do not have access to this content. For assistance or to subscribe, please contact us:

  • TELEPHONE: 1-800-843-2763 (Toll-free in the USA)
  • EMAIL: asmedigitalcollection@asme.org
Sign In