Liu Y et al. (2016), Method to extract minimally damaged collagen fi...

ECB-ART-47397

J Biol Methods 2016 Sep 16;34:e54. doi: 10.14440/jbm.2016.121.

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Method to extract minimally damaged collagen fibrils from tendon.

Liu Y , Andarawis-Puri N , Eppell SJ .

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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.

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Genes referenced: LOC100887844 LOC115925415 LOC583082 LOC594261

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	Figure 2. Scanning electron microscopy images of an extracted single collagen fibril from rat patellar tendon (long thin strand running left-right) atop a mechanical test device. Scale bar above represents 30 Î¼m; scale bar below represent 500 nm. Master image at the top shows the entire fibril with length > 300 Î¼m (A); the images below show sections on the same collagen fibril at regions labeled with small arrow heads b, c and d, corresponding to B, C and D: fibril diameter ~200 nm imaged over viewing window (B); diameter ~200 nm over gap widens to ~300 nm on the silicon surface (C); and diameter tapering at end of fibril consistent with an intact fibril terminus (D).
	Figure 3. Dark-field microscopy images of rat patellar tendon collagen fibrils in solutions using the new method presented in this paper (A) and tissue homogenization (B) (scale bar represents 100 Î¼m). Small arrows point out some suspended collagen fibrils. Large arrow heads point out non-fibraller tissue components. The fibril extracted without using vigorous mechanical disruption (A) is > 1000 Î¼m. The fibrils extracted using tissue homogenization (B) vary in length from 30 Î¼m to 400 Î¼m.
	Figure 4. Length distribution. A. Sea cucumber dermis (SC) collagen fibrils extracted by deionized water. B. Sea cucumber dermis collagen fibrils extracted by the trypsin extraction medium. C. Sea cucumber dermis collagen fibrils extracted by tissue homogenization. D. Rat patellar tendon (RP) collagen fibrils extracted by the trypsin extraction medium. E. Rat patellar tendon collagen fibrils extracted by tissue homogenization. F. Cumulative (length) distribution frequency (CDF) of sea cucumber dermis collagen fibrils extracted by deionized water, trypsin and homogenization. G. Rat tendon collagen fibrils extracted by trypsin and homogenization.
	Figure 5. Scanning electron microscopy images of collagen fibrils showing that D-banding is not extinguished by trypsin treatment. A. An extracted rat patellar tendon collagen fibril obtained with the trypsin extraction method. B. An extracted sea cucumber dermis collagen fibril obtained with the trypsin-based extraction method. C. An extracted sea cucumber dermis collagen fibril obtained with the water extraction method. All three collagen fibril samples show clear D-banding patterns on the surface of the fibril, consistent with undamaged surface structure at the nano level. Scale bar is 500 nm.
	Figure 6. SDS-PAGE of collagen samples stained with Coomassie brilliant blue. Sample set A was prepared from the rat tail tendon collagen stock solution diluted by 1:4, and sample set B was prepared from synthetic collagen gel made from commerical bovine skin collagen solution (Purecol). All samples showed three band marks of molecular weights above 130 kDa. Lane A1: Rat tail tendon collagen molecules without trypsin treatment; A2: Rat tail tendon collagen molecules with trypsin treatment at the fibril level; B1: Bovine skin collagen without trypsin treatment; B2: Bovine skin collagen with trypsin treatment. No significant difference was observed within each sample set. No evidence of discrete digestion products between 15 and 130 kDa in any lane suggesting that trypsin does not cleave either rat tail tendon collagen or bovine skin collagen.

References [+] :

Bridgman, Comparisons in structure of tendon organs in the rat, cat and man. 1970, Pubmed