Method to extract minimally damaged collagen fibrils from tendon
A new method is presented to extract collagen fibrils from mammalian tendon tissue. Mammalian tendons are treated with a trypsin-based extraction medium and gently separated with tweezers in an aqueous solution. Collagen fibrils released in the solution are imaged using both dark-field light microscopy and scanning electron microscopy. The method successfully extracts isolated fibrils from rat tail and patellar tendons. To examine whether the method is likely to damage fibrils during extraction, sea cucumber dermis fibril lengths are compared against those obtained using only distilled water. The two methods produce fibrils of similar lengths. This is contrasted with fibrils being shortened when extracted using a tissue homogenizer. Scanning electron microscopy shows the new method preserves D-banding features on fibril surfaces and that fibril diameter does not vary substantially compared with water extracted fibrils.
1. Shoulders MD, Raines RT (2009) Collagen structure and stability. Annu Rev Biochem 78: 929-958.
2. Fratzl P (2008) Collagen Structure and Mechanics. In: Fratzl P, editor. Collagen. New York, Philadelphia: Springer+Business Media LLC. pp.1-13
3. Kadler KE, Baldock C, Bella J, Boot-Handford RP (2007) Collagens at a glance. J Cell Sci 120: 1955-1958.
4. Gross J, Schmitt FO (1948) The structure of human skin collagen as studied with the electron microscope. J Exp Med 88: 555-568.
5. Glimcher MJ (1959) Molecular Biology of Mineralized Tissues with Particular Reference to Bone. Reviews of Modern Physics 31: 359-393.
6. Young RD, Knupp C, Pinali C, Png KM, Ralphs JR, et al. (2014) Three-dimensional aspects of matrix assembly by cells in the developing cornea. Proc Natl Acad Sci USA 111: 687-692.
7. Buehler MJ (2008) Hierarchical nanomechanics of collagen fibrils: Atomistic and molecular modeling. In: Fratzl P, editor. Collagen. New York, Philadelphia: Springer Science+Business Media LLC. pp. 175-247
8. Gautieri A, Vesentini S, Redaelli A, Buehler MJ (2011) Hierarchical Structure and Nanomechanics of Collagen Microfibrils from the Atomistic Scale Up. Nano Letters 11: 757-766.
9. Grant CA, Phillips MA, Thomson NH (2012) Dynamic mechanical analysis of collagen fibrils at the nanoscale. Journal of the Mechanical Behavior of Biomedical Materials 5: 165-170.
10. Heim AJ, Koob TJ, Matthews WG (2007) Low strain nanomechanics of collagen fibrils. Biomacromolecules 8: 3298-3301.
11. Shen ZL, Dodge MR, Kahn H, Ballarini R, Eppell SJ (2010) In vitro fracture testing of submicron diameter collagen fibril specimens. Biophys J 99: 1986-1995.
12. Shen ZL, Kahn H, Ballarini R, Eppell SJ (2011) Viscoelastic properties of isolated collagen fibrils. Biophys J 100: 3008-3015.
13. Tong W (2002) Synthesis of nanometer-sized apatites using highly ordered collagen structure. Cleveland: Case Western Reserve University. 228 p.
14. Wenger MPE, Bozec L, Horton MA, Mesquida P (2007) Mechanical properties of collagen fibrils. Biophysical Journal 93: 1255-1263.
15. Francis MJ, MacMillan DC (1971) Extraction of polymeric collagen from human skin. Biochem J 122: 34p.
16. Wilson JA (1929) The Chemistry of Leather Manufacture. New York: J.J. Little and Ives. pp. 94-132.
17. Eppell SJ, Smith BN, Kahn H, Ballarini R (2006) Nano measurements with micro-devices: mechanical properties of hydrated collagen fibrils. J R Soc Interface 3: 117-121.
18. Thurmond FA, Trotter JA (1996) Morphology and biomechanics of the microfibrillar network of sea cucumber dermis. Journal of Experimental Biology 199: 1817-1828.
19. Trotter JA, Thurmond FA, Koob TJ (1994) Molecular structure and functional morphology of echinoderm collagen fibrils. Cell Tissue Res 275: 451-458.
20. Shen ZL, Dodge MR, Kahn H, Ballarini R, Eppell SJ (2008) Stress-strain experiments on individual collagen fibrils. Biophys J 95: 3956-3963.
21. Kongsgaard M, Qvortrup K, Larsen J, Aagaard P, Doessing S, et al. (2010) Fibril morphology and tendon mechanical properties in patellar tendinopathy: effects of heavy slow resistance training. Am J Sports Med 38: 749-756.
22. DeVente JE, Lester GE, Trotter JA, Dahners LE (1997) Isolation of intact collagen fibrils from healing ligament. Journal of Electron Microscopy 46: 353-356.
23. Gevorkian SG, Allahverdyan AE, Gevorgyan DS, Simonian AL (2009) Thermal (in)stability of type I collagen fibrils. Phys Rev Lett 102: 048101.
24. van der Rijt JAJ, van der Werf KO, Bennink ML, Dijkstra PJ, Feijen J (2006) Micromechanical testing of individual collagen fibrils. Macromolecular Bioscience 6: 697-702.
25. Yang L, Van der Werf KO, Fitie CFC, Bennink ML, Dijkstra PJ, et al. (2008) Mechanical properties of native and cross-linked type I collagen fibrils. Biophysical Journal 94: 2204-2211.
26. Svensson RB, Hassenkam T, Hansen P, Magnusson SP (2010) Viscoelastic behavior of discrete human collagen fibrils. Journal of the Mechanical Behavior of Biomedical Materials 3: 112-115.
27. Trotter JA, LyonsLevy G, Thurmond FA, Koob TJ (1995) Covalent composition of collagen fibrils from the dermis of the sea cucumber, Cucumaria frondosa, a tissue with mutable mechanical properties. Comparative Biochemistry and Physiology a-Physiology 112: 463-478.
28. Rajan N, Habermehl J, Cote M-F, Doillon CJ, Mantovani D (2007) Preparation of ready-to-use, storable and reconstituted type I collagen from rat tail tendon for tissue engineering applications. Nat Protocols 1: 2753-2758.
29. Kadler KE, Hojima Y, Prockop DJ (1990) Collagen fibrils in vitro grow from pointed tips in the C- to N-terminal direction. Biochem J 268: 339-343.
30. Bornstein.P, Kang AH, Piez KA (1966) Limited Cleavage Of Native Collagen With Chymyotrypsin Trypsin And Cyanogen Bromide. Biochemistry 5: 3803-&.
31. Powers KL, Gilmore LA, Monroy JA, Uyeno TA, Nishikawa KC (2010) Using trypsin digestion to determine the relative contributions of titin and collagen to passive elastic properties of whole muscles. Integrative and Comparative Biology 50: E139-E139.
32. Zhang M, Wang K, Lin W, inventors; Zhang M, assignee. Collagen preparing process, China. patent NO. CN1190499-C, Feb 23 2005, 3, Collagen preparing process involves water washing, pre-treating, mechanical crushing, enzymolysis with trypsin, filtering, desalting, vacuum concentration, spraying to dry, collection and packing.
33. Standard FA (2008) Standard Guide for Characterization of Type I Collagen as Starting Material for Surgical Implants and Substrates for Tissue Engineered Medical Products (TEMPs). West Conshohocken: ASTM International.
34. Yoon JH, Halper J (2005) Tendon proteoglycans: biochemistry and function. J Musculoskelet Neuronal Interact 5: 22-34.
35. Kannus P (2000) Structure of the tendon connective tissue. Scand J Med Sci Sports 10: 312-320.
36. Thorpe CT, Birch HL, Clegg PD, Screen HR (2013) The role of the non-collagenous matrix in tendon function. Int J Exp Pathol 94: 248-259.
37. Bridgman CF (1970) Comparisons in structure of tendon organs in the rat, cat and man. The Journal of Comparative Neurology 138: 369-372.