Izvestiya of Saratov University.


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ISSN 2542-193X (Online)

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Shvachkina M. E. On the Possibility of Stabilization of a Contracted State after Riboflavin/UV Cross-Linking of Collagenous Tissue in a Partially Dehydrated State. Izvestiya of Sarat. Univ. Physics. , 2019, vol. 19, iss. 3, pp. 210-222. DOI: 10.18500/1817-3020-2019-19-3-210-222

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On the Possibility of Stabilization of a Contracted State after Riboflavin/UV Cross-Linking of Collagenous Tissue in a Partially Dehydrated State

Shvachkina Marina Evgen'evna, Saratov State University

Background and Objectives: The method of riboflavin/UV collagen cross-linking is widely used to strengthen the corneal stroma in the treatment of keratoconus and is of considerable interest as a possible method to improve the biomechanical property of the sclera in the treatment of myopia. Regarding the application of this method to the sclera, one of the important problems is the rapid decrease in the intensity of UV radiation due to scattering as it propagates into the tissue. The depth of penetration of optical radiation into the sclera can be significantly increased using immersion optical clearing of the tissue. Under the action of immersion liquids used for optical clearing, partial dehydration of the tissue occurs. It is known that chemical cross-linking of tissue in a dehydrated state can lead to a significant decrease in the volume of tissue after its rehydration to a saturated state compared to the initial state, that is, it can lead to stabilization of a contracted state. In this case, the cross-linked tissue in the saturated rehydrated state contains less water than in the initial state. In this paper, we investigate the possibility of stabilizing a contracted state of the tissue after its riboflavin/UV cross-linking in a partially dehydrated state. Materials and Methods: Experiments were performed in vitro on samples of rat tail tendon fascicles. Before riboflavin/UV cross-linking, the sample was incubated in a 0.1% solution of riboflavin in normal saline solution for 20 minutes. Then the sample was dehydrated in a riboflavin-doped immersion liquid (37%, 58.5%, or 87% aqueous solution of polyethylene glycol PEG-300 containing 0.1% riboflavin) for 10 minutes (58.5% and 87% PEG solutions) or 15 minutes (37% PEG solution). After, a 4.5 mm-long section of the sample was exposed to UV radiation with a wavelength of 365 nm for 10 minutes. Finally, the sample was rehydrated in normal saline solution for 2 hours. At each stage of the treatment, the average group refractive index and geometry of the sample in UV-irradiated and non-irradiated sections of the sample were monitored using optical coherence tomography (OCT). The water content in the tissue was calculated from the measured values of its average group refractive index. Results: It was experimentally established that the retention of the contracted state of the tissue can occur, provided that the cross-linking is carried out at a volume hydration of the tissue less than 0.8. When the volume hydration of samples was less than 0.5 during UV exposure, the resulting contraction of the fascicle was found to be 8–15%. Conclusion: In this work, it was experimentally shown that riboflavin/UV cross-linking of collagenous tissue in a dehydrated state can lead to a decrease in tissue volume after its rehydration compared to the native one. The top level of hydration of the tissue during UV exposure at which the contracted state can be stabilized was found and the degree of resulting tissue contraction as a function of the degree of tissue hydration during UV exposure was estimated.


1. Bikbov M. M., Khalimov A. R., Usubov E. L. Ultraviolet Corneal Crosslinking. Vestnik Rossiiskoi akademii meditsinskikh nauk, 2016, vol. 71, no. 3, pp. 224–232 (in Russian). DOI: https://doi.org/10.15690/vramn562

2. Iomdina E. N. Biomechanical aspects of keratorefractive surgery and corneal crosslinking. Rossijiskaja pediatricheskaja oftalmologija [Russian Pediatric Ophthalmology], 2015, vol. 10, no. 4, pp. 32–37 (in Russian).

3. Bikbov M. M., Surkova V. K., Usubov E. L., Astrelin M. N. Scleral crosslinking with ribofl avin and ultraviolet A (UVA). A review. Oftalmologia [Ophthalmology], 2016, vol. 12, no. 4, pp. 4–8 (in Russian). DOI: https://doi.org/10.18008/1816-5095-2015-4-4-8

4. Meek K. M., Hayes S. Corneal cross-linking – a review. Ophthalmic and Physiological Optics, 2013, vol. 33, no. 2, pp. 78–93. DOI: https://doi.org/10.1111/opo.12032

5. Gamidov G. A., Mushkova I. A., Kostenev S. V. Modifi - cations of corneal collagen cross-linking in keratoconus treatment. Literature review. Prakticheskaja medicina [Practical Medicine], 2018, vol. 3, no. 114, pp. 52–56 (in Russian).

6. Hayes S., Kamma-Lorger C. S., Boote C., Young R. D., Quantock A. J., Rost A., Khatib Y., Harris J., Yagi N., Terrill N., Meek K. M. The effect of ribofl avin/UVA collagen cross-linking therapy on the structure and hydrodynamic behaviour of the ungulate and rabbit corneal stroma. PloS One, 2013, vol. 8, no. 1, pp. e52860. DOI: https://doi.org/10.1371/journal.pone.0052860

7. Wollensak G., Spoerl E., Seiler T. Ribofl avin/ultraviolet-A–induced collagen crosslinking for the treatment of keratoconus. American Journal of Ophthalmology, 2003, vol. 135, no. 5, pp. 620–627. DOI: https://doi.org/10.1016/S0002-9394(02)02220-1

8. Wollensak G., Spoerl E., Seiler T. Stress-strain measurements of human and porcine corneas after ribofl avin–ultraviolet- A-induced cross-linking. Journal of Cataract & Refractive Surgery, 2003, vol. 29, no. 9, pp. 1780–1785. DOI: https://doi.org/10.1016/S0886-3350(03)00407-3

9. Wollensak G., Spoerl E. Collagen crosslinking of human and porcine sclera. Journal of Cataract & Refractive Surgery, 2004, vol. 30, no. 3, pp. 689–695. DOI: https://doi.org/10.1016/j.jcrs.2003.11.032

10. Wollensak G., Iomdina E. Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet A (UVA). Acta Ophthalmologica, 2009, vol. 87, no. 2, pp. 193–198. DOI: https://doi.org/10.1111/j.1755-3768.2008.01229.x

11. Zhang Y., Li Z., Liu L., Han X., Zhao X., Mu G. Comparison of ribofl avin/ultraviolet-A cross-linking in porcine, rabbit, and human sclera. BioMed Research International, 2014, vol. 2014, pp. 1–5. DOI: https://doi.org/10.1155/2014/194204

12. Dotan A., Kremer I., Gal-Or O., Livnat T., Zigler A., Bourla D., Bourla D., Weinberger D. Scleral cross-linking using ribofl avin and ultraviolet-A radiation for prevention of axial myopia in a rabbit model. J. Vis. Exp., 2016, no. 110, pp. e53201. DOI: https://doi.org/10.3791/53201

13. Iomdina Е. N., Tarutta Е. P., Semchishen V. А., Korigodskiy А. R., Zakharov I. D., Khoroshilova-Maslova I. P., Ignat’eva N. Yu., Kiseleva Т. N., Sianosyan А. А., Milash S. V. Experimental realization of minimally invasive techniques of scleral collagen cross-linking. Vestnik Oftalmologii [Bulletin of Ophthalmology], 2016, vol. 132, no. 6, pp. 49–58 (in Russian). DOI: https://doi.org/10.17116/oftalma2016132649-56

14. Spaide R. F., Ohno-Matsui K., Yannuzzi L. A. Pathologic Myopia. New York, Springer, 2014. 376 p.

15. Morgan I. G., Ohno-Matsui K., Saw S. M. Myopia. The Lancet, 2012, vol. 379, no. 9827, pp. 1739–1748. DOI: https://doi.org/10.1016/S0140-6736(12)60272-4

16. Chechneva A. V., Sotnikova L. F., Iomdina E. N. Corneal Collagen crosslinking to treatment complications of infectious keratoconjunctivitis in cats. Izvestia mezhdunarodnoji akademii agrarnogo obrazovania [News of the International Academy of Agrarian Education], 2018, vol. 2, iss. 42, pp. 117–121 (in Russian).

17. Wollensak G., Aurich H., Wirbelauer C., Pham D. T. Potential use of ribofl avin/UVA cross-linking in bullous keratopathy. Ophthalmic Research, 2009, vol. 41, no. 2, pp. 114–117. DOI: https://doi.org/10.1159/000187630

18. Wollensak G., Iomdina E. Crosslinking of scleral collagen in the rabbit using glyceraldehydes. Journal of Cataract & Refractive Surgery, 2008, vol. 34, no. 4, pp. 651–656. DOI: https://doi.org/10.1016/j.jcrs.2007.12.030

19. Tanaka Y., Shi D., Kubota A., Takano Y., Fuse N., Yamato M., Okano T., Nishida K. Irreversible optical clearing of rabbit dermis for autogenic corneal stroma transplantation. Biomaterials, 2011, vol. 32, no. 28, pp. 6764–6772. DOI: https://doi.org/10.1016/j.biomaterials.2011.05.081

20. Iomdina E. N., Nazarenko L. A., Kiseleva O. A. Are biomechanical properties of the sclera and eye hydrodynamics related an experimental study. Vestnik Nizhegorodskogo universiteta im. N. I. Lobachevskogo [Vestnik of Lobachevsky University of Nizhni Novgorod], 2011, vol. 2, no. 4, pp. 445–447 (in Russian).

21. Cherfan D., Verter E. E., Melki S., Gisel T. E., Doyle F. J., Scarcelli G., Yun S. H., Redmond R. W., Kochevar I. E. Collagen cross-linking using rose bengal and green light to increase corneal stiffness. Investigative Ophthalmology & Visual Science, 2013, vol. 54, no. 5, pp. 3426–3433. DOI: https://doi.org/10.1167/iovs.12-11509

22. Zhu H., Alt C., Webb R. H., Melki S., Kochevar I. E. Corneal crosslinking with rose bengal and green light: effi cacy and safety evaluation. Cornea, 2016, vol. 35, no. 9, pp. 1234–1241. DOI: https://doi.org/10.1097/ICO.0000000000000916

23. Iseli H. P., Körber N., Koch C., Karl A., Penk A., Huster D., Reichenbach A., Wiedemann P., Francke M. Scleral cross-linking by ribofl avin and blue light application in young rabbits: damage threshold and eye growth inhibition. Graefes Archive for Clinical and Experimental Ophthalmology, 2016, vol. 254, no. 1, pp. 109–122. DOI: https://doi.org/10.1007/s00417-015-3213-x

24. Karl A., Makarov F. N., Koch C., Körber N., Schuldt C., Krüger M., Reichenbach A., Wiedemann P., Bringmann A., Iseli H. P., Francke M. The ultrastructure of rabbit sclera after scleral crosslinking with ribofl avin and blue light of different intensities. Graefes Archive for Clinical and Experimental Ophthalmology, 2016, vol. 254, no. 8, pp. 1567–1577. DOI: https://doi.org/10.1007/s00417-016-3393-z

25. Schilde T., Kohlhaas M., Spoerl E., Pillunat L. E. Enzymatic evidence of the depth dependence of stiffening on ribofl avin/UVA treated corneas. Der Ophthalmologe: Zeitschrift der Deutschen Ophthalmologischen Gesellschaft, 2008, Bd. 105, no. 2, S. 165–169. DOI: https://doi.org/10.1007/s00347-007-1587-9

26. Kamaev P., Friedman M. D., Sherr E., Muller D. Photochemical kinetics of corneal cross-linking with ribofl avin. Investigative Ophthalmology & Visual Science, 2012, vol. 53, no. 4, pp. 2360–2367. DOI: https://doi.org/10.1167/iovs.11-9385

27. Raiskup F., Spoerl E. Corneal crosslinking with ribofl avin and ultraviolet AI Principles. The Ocular Surface, 2013, vol. 11, no. 2, pp. 65–74. DOI: https://doi.org/10.1016/j.jtos.2013.01.002

28. McCall A. S., Kraft S., Edelhauser H. F., Kidder G. W., Lundquist R. R., Bradshaw H. E., Dedeic Z., Dionne M. J. C., Clement E. M., Conrad G. W. Mechanisms of corneal tissue cross-linking in response to treatment with topical ribofl avin and long-wavelength ultraviolet radiation (UVA). Invest Ophthalmol Vis Sci., 2010, vol. 51, no. 1, pp. 129–138. DOI: https://doi.org/10.1167/iovs.09-3738

29. Wang C., Fomovsky M., Miao G., Zyablitskaya M., Vukelic S. Femtosecond laser crosslinking of the cornea for non-invasive vision correction. Nature Photonics, 2018, vol. 12, no. 7, pp. 416–422. DOI: https://doi.org/10.1038/s41566-018-0174-8

30. Zhang Y., Conrad A. H., Conrad G. W. Effects of ultraviolet-A and ribofl avin on the interaction of collagen and proteoglycans during corneal cross-linking. Journal of Biological Chemistry, 2011, vol. 286, no. 15, pp. 13011–13022. DOI: https://doi.org/10.1074/jbc.M110.169813

31. Wollensak G., Aurich H., Pham D. T., Wirbelauer C. Hydration behavior of porcine cornea crosslinked with ribofl avin and ultraviolet A. Journal of Cataract & Refractive Surgery, 2007, vol. 33, no. 3, pp. 516–521. DOI: https://doi.org/10.1016/j.jcrs.2006.11.015

32. Charulatha V., Rajaram A. Infl uence of different crosslinking treatments on the physical properties of collagen membranes. Biomaterials, 2003, vol. 24, no. 5, pp. 759–767. DOI: https://doi.org/10.1016/S0142-9612(02)00412-X

33. Wollensak G., Wilsch M., Spoerl E., Seiler T. Collagen fi ber diameter in the rabbit cornea after collagen crosslinking by ribofl avin/UVA. Cornea, 2004, vol. 23, no. 5, pp. 503–507. DOI: https://doi.org/10.1097/01.ico.0000105827.85025.7f

34. Choi S., Lee S. C., Lee H. J., Cheong Y., Jung G. B., Jin K. H., Park H. K. Structural response of human corneal and scleral tissues to collagen cross-linking treatment with ribofl avin and ultraviolet A light. Lasers in Medical Science, 2013, vol. 28, no. 5, pp. 1289–1296. DOI: https://doi.org/10.1007/s10103-012-1237-6

35. Shvachkina M. E., Pravdin A. B. On the Use of Optical Clearing in Strengthening the Sclera by Collagen Photocrosslinking. Izv. Saratov Univ. (N. S.), Ser. Physics, 2015, vol. 15, iss. 4, pp. 37–41 (in Russian). DOI: https://doi.org/10.18500/1817-3020-2015-15-4-37-41

36. Tuchin V. V. Tissue optics: light scattering methods and instruments for medical diagnosis. Bellingham, Washington, SPIE Press, 2015. 812 p.

37. Zhu D., Larin K. V., Luo Q., Tuchin V. V. Recent progress in tissue optical clearing. Laser & Photonics Reviews, 2013, vol. 7, no. 5, pp. 732–757. DOI: https://doi.org/10.1002/lpor.201200056

38. Maksimova I. L., Zimnyakov D. A., Tuchin V. V. Control of biotissue optical properties. Opt. Spektrosk., 2000, vol. 89, no. 1, pp. 86–95 (in Russian).

39. Tuchin V. V., Bashkatov A. N., Genina E. A., Sinichkin Y. P. Scleral tissue clearing effects. Proceedings of SPIE, 2002, vol. 4611, pp. 54–58.

40. Bashkatov A. N., Genina E. A., Kochubey V. I., Kamenskikh T. G., Tuchin V. V. Optical Clearing of Human Eye Sclera by Aqueous 30%-Glucose Solution. Izv. Saratov Univ. (N. S.), Ser. Physics, 2015, vol. 15, iss. 3, pp. 18–24 (in Russian). DOI: https://doi.org/10.18500/1817-3020-2015-15-3-18-24

41. Zaman R. T., Rajaram N., Nichols B. S., Rylander H. G., Wang T., Tunnell J. W., Welch A. J. Changes in morphology and optical properties of sclera and choroidal layers due to hyperosmotic agent. Journal of Biomedical Optics, 2011, vol. 16, no. 7, pp. 077008-1–077008-14. DOI: https://doi.org/10.1117/1.3599985

42. Tuchina D. K., Genin V. D., Bashkatov A. N., Genina E. A., Tuchin V. V. Optical clearing of skin tissue ex vivo with polyethylene glycol. Opt. Spectrosc., 2016, vol. 120, no. 1, pp. 28–37. DOI: https://doi.org/10.1134/S0030400X16010215

43. Meek K. M., Fullwood N. J., Cooke P. H., Elliott G. F., Maurice D. M., Quantock A. J., Wall R. S. Worthington C. R. Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration. Biophysical Journal, 1991, vol. 60, no. 2, pp. 467–474. DOI: https://doi.org/10.1016/S0006-3495(91)82073-2

44. Rowe R. W. D. The structure of rat tail tendon. Connective Tissue Research, 1985, vol. 14, no. 1, pp. 9–20. DOI: https://doi.org/10.3109/03008208509089839

45. Svensson L., Aszodi A., Reinholt F. P., Fassler R., Heinegard D., Oldberg A. Fibromodulin-null mice have abnormal collagen fi brils, tissue organization, and altered lumican deposition in tendon. Journal of Biological Chemistry, 1999, vol. 274, no. 14, pp. 9636–9647. DOI: https://doi.org/10.1074/jbc.274.14.9636

46. Fratzl P. Collagen: structure and mechanics, an introduction. New York, USA, Springer Science+Business Media, LLC, 2008. 506 p.

47. Shvachkina M. E., Yakovlev D. D., Lazareva E. N., Pravdin A. B., Yakovlev D. A. Monitoring of immersion optical clearing of collagen fi bers using optical coherence tomography. Opt. Spektrosk., 2019, vol. 127, iss. 2, pp. 337–346 (in Russian). DOI: https://doi.org/10.21883/OS.2019.08.48052.302-18

48. Shvachkina M. E., Yakovlev D. D., Pravdin A. B., Yakovlev D. A. Average refractive index of tendon as a function of water content. Journal of Biomedical Photonics & Engineering, 2018, vol. 4, no. 1, pp. 010302-1–010302-7. DOI: https://doi.org/10.18287/JBPE18.04.010302

49. Tanaka Y., Kubota A., Yamato M., Okano T., Nishida K. Irreversible optical clearing of sclera by dehydration and cross-linking. Biomaterials, 2011, vol. 32, no. 4, pp. 1080–1090. DOI: https://doi.org/10.1016/j.biomaterials.2010.10.002