1. Application of Raman Scattering to the Measurement of Ligament Tension M.W. Winchester 1,2 L.W. Winchester 1 N. Chou 1 1 C.W. Optics, Inc. Seaford, Virginia, U.S.A. 2 University of Mary Washington, Fredericksburg, Virginia, U.S.A. 1 (Patent Pending)

2. Knee Anatomy 2

3. ACL Statistics 275,000 ACL reconstructive surgeries performed each year in U.S. 1 Increase in activity. 8% of patients experience either recurrent instability or graft failure. 2 1. aaos.org July 2008 Newsletter 2. Revision ACL Considerations. Southern California Orthopedic Institute. © 2008. 3

4. ACL Grafts Bone-patellar tendon-bone Hamstring tendon Allograft or autograft 4

5. Ligament Structure ACL is composed of fascicular subunits with larger functional bands. Comprised of mainly collagen Collagen backbone and hydroxy-proline cross- links provide strength Collagen in the core exhibits crimp, a sinusoidal pattern allowing for an increase in resistance or load. 5

6. Experiments Tension ▫Validate force measurement procedure ▫Determine relaxation parameters Spectroscopy ▫Measure Raman spectrum ▫Develop algorithms for peak identification Develop model for tension computation 6

7. Animal Measurements Minks and rabbits Hind limb tendons were harvested Rabbit tendons were larger than mink tendons. 7

8. Sample Holder 8

9. Mechanical Results The tendons of both minks and rabbits display nonlinear and linear elasticity regions and viscoelasticity. 9

10. Raman Scattering Inelastic scattering First-order spectrum is discrete (peaks) Second-order spectrum can be continuous Spectrum is characteristic of the material Changes in structure and force constants can be observed for Raman-active modes 10

11. Energy Level Diagram 11

12. Block Diagram 12

13. Typical Spectra 13

14. Data Processing Remove fluorescence with cubic spline fit Locate Raman peaks ▫Peak detection Algorithm (single) ▫SVD fit (multiple) 14

15. Frequency Shift with Tension 15 Blue and green = collagen backbone Red and black = amino acid residues

16. Determine Dependence of Shift T (∆υ) = C 0 + C 1 ∆υ + C 2 (∆υ) 2 + C 3 (∆υ ) -1 T = tension ∆υ = change in Raman shift C n (n = 0 to 3) = coefficient 16

17. Results 17

18. Bland-Altman 18

19. Conclusion The frequency shift of the spectral lines of the Raman spectrum of collagen depends on applied tension. Raman scattering is a means of determining the tension in ligaments and tendons. 19

20. Future Work Move on to cadavers Move on to humans 20

21. Acknowledgments This work was funded in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, under grant AR053791. M.W. Winchester would like to thank the Virginia Biotechnology Association for the 2008 Summer Internship Grant. 21