the process of edc-nhs cross-linking of reconstituted collagen fibres increases collagen fibrillar...

Download The process of EDC-NHS cross-linking of reconstituted collagen fibres increases collagen fibrillar order and alignment

Post on 28-Mar-2017

213 views

Category:

Documents

1 download

Embed Size (px)

TRANSCRIPT

  • The process of EDC-NHS cross-linking of reconstituted collagen fibres increasescollagen fibrillar order and alignmentD. V. Shepherd, J. H. Shepherd, S. Ghose, S. J. Kew, R. E. Cameron, and S. M. Best Citation: APL Materials 3, 014902 (2015); doi: 10.1063/1.4900887 View online: http://dx.doi.org/10.1063/1.4900887 View Table of Contents: http://scitation.aip.org/content/aip/journal/aplmater/3/1?ver=pdfcov Published by the AIP Publishing Articles you may be interested in Frictional properties of native and functionalized type I collagen thin films Appl. Phys. Lett. 103, 143703 (2013); 10.1063/1.4824685 Longitudinal variations in the Poissons ratio of collagen fibrils Appl. Phys. Lett. 98, 163707 (2011); 10.1063/1.3567784 Self-aligned patterns of multiple biomolecules printed in one step Appl. Phys. Lett. 93, 133901 (2008); 10.1063/1.2990045 Detailed evaluation of the performance of microfluidic T mixers using fluorescence and ultraviolet resonanceRaman spectroscopy Rev. Sci. Instrum. 77, 055105 (2006); 10.1063/1.2198800 A minimal model for stabilization of biomolecules by hydrocarbon cross-linking J. Chem. Phys. 124, 164907 (2006); 10.1063/1.2185645

    This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://aplmaterials.aip.org/about/rights_and_permissions

    Downloaded to IP: 131.111.185.72 On: Sat, 22 Nov 2014 11:52:40

    http://scitation.aip.org/content/aip/journal/aplmater?ver=pdfcovhttp://oasc12039.247realmedia.com/RealMedia/ads/click_lx.ads/www.aip.org/pt/adcenter/pdfcover_test/L-37/1905342781/x01/AIP-PT/Goodfellow_APLMArticleDL_0914/New_Goodfellow_Banner0913.jpeg/47344656396c504a5a37344142416b75?xhttp://scitation.aip.org/search?value1=D.+V.+Shepherd&option1=authorhttp://scitation.aip.org/search?value1=J.+H.+Shepherd&option1=authorhttp://scitation.aip.org/search?value1=S.+Ghose&option1=authorhttp://scitation.aip.org/search?value1=S.+J.+Kew&option1=authorhttp://scitation.aip.org/search?value1=R.+E.+Cameron&option1=authorhttp://scitation.aip.org/search?value1=S.+M.+Best&option1=authorhttp://scitation.aip.org/content/aip/journal/aplmater?ver=pdfcovhttp://dx.doi.org/10.1063/1.4900887http://scitation.aip.org/content/aip/journal/aplmater/3/1?ver=pdfcovhttp://scitation.aip.org/content/aip?ver=pdfcovhttp://scitation.aip.org/content/aip/journal/apl/103/14/10.1063/1.4824685?ver=pdfcovhttp://scitation.aip.org/content/aip/journal/apl/98/16/10.1063/1.3567784?ver=pdfcovhttp://scitation.aip.org/content/aip/journal/apl/93/13/10.1063/1.2990045?ver=pdfcovhttp://scitation.aip.org/content/aip/journal/rsi/77/5/10.1063/1.2198800?ver=pdfcovhttp://scitation.aip.org/content/aip/journal/rsi/77/5/10.1063/1.2198800?ver=pdfcovhttp://scitation.aip.org/content/aip/journal/jcp/124/16/10.1063/1.2185645?ver=pdfcov

  • APL MATERIALS 3, 014902 (2015)

    The process of EDC-NHS cross-linking of reconstitutedcollagen fibres increases collagen fibrillar orderand alignment

    D. V. Shepherd,1,a J. H. Shepherd,1 S. Ghose,2 S. J. Kew,2 R. E. Cameron,1and S. M. Best11Department of Materials Science and Metallurgy, University of Cambridge,Cambridge, United Kingdom2Tigenix Ltd., Byron House, Cambridge Business Park,Cambridge, United Kingdom

    (Received 4 September 2014; accepted 22 October 2014; published online 5 November 2014)

    We describe the production of collagen fibre bundles through a multi-strand, semi-continuous extrusion process. Cross-linking using an EDC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide), NHS (N-hydroxysuccinimide) combination was consid-ered. Atomic Force Microscopy and Raman spectroscopy focused on how cross-linking affected the collagen fibrillar structure. In the cross-linked fibres, a clearfibrillar structure comparable to native collagen was observed which was notobserved in the non-cross-linked fibre. The amide III doublet in the Raman spectraprovided additional evidence of alignment in the cross-linked fibres. Raman spectros-copy also indicated no residual polyethylene glycol (from the fibre forming buffer)or water in any of the fibres. C 2014 Author(s). All article content, except whereotherwise noted, is licensed under a Creative Commons Attribution 3.0 UnportedLicense. [http://dx.doi.org/10.1063/1.4900887]

    In 1989, Kato and Silver first described the extrusion of an acid swollen collagen gel underaqueous conditions on a laboratory scale.1 Since then there have been several studies of this processof self-assembly,2 with production parameters and cross-linking treatments36 considered in orderto optimise mechanical and biological characteristics. The acidic collagen suspension is typicallyextruded into a bath containing neutral fibre forming buffer, the collagen gels on contact with neutralpH as the fibrils, reconstitute and the macroscopic fibres form. Along the length of the bath, the threaddehydrates due to the osmotic gradient between the collagen and the fibre forming buffer.7

    When collagen is generated in vivo, cross-linking occurs enzymatically and covalent inter- andintra-molecular bonds are formed that provide sufficient mechanical strength and proteolytic resis-tance.3 These same cross-links are not formed during the self-assembly of collagen in neutral pHand as such cross-linking routes are a particular area of consideration in the production of artificialcollagen fibre based structures. Cross-linking approaches include chemical (glutaraldehyde, isocy-anates, or carbodiimide based),6 physical (dehydrothermal),8,9 and enzymatic.10 The authors havepreviously settled upon the use of the zero length cross-linker carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS) as an effectiveroute for the cross-linking of extruded collagen fibres.1113 This route results in a lower cross-linkingdensity than other chemical and physical cross-linking routes, but it has been shown to exhibit afavourable biological response14 unlike more aggressive techniques that have been associated withinflammatory response and reduced bioactivity.15

    Whilst the mechanical properties,3,4,6,16 degradative characteristics,14,17,18 and surface micro-structure3,6 of extruded fibres have been considered quite extensively, any rigorous investigation

    aAuthor to whom correspondence should be addressed. Electronic mail: dvs23@cam.ac.uk

    2166-532X/2015/3(1)/014902/8 3, 014902-1 Author(s) 2014

    This article is copyrighted as indicated in the article. Reuse of AIP content is subject to the terms at: http://aplmaterials.aip.org/about/rights_and_permissions

    Downloaded to IP: 131.111.185.72 On: Sat, 22 Nov 2014 11:52:40

    http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887http://dx.doi.org/10.1063/1.4900887mailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukmailto:dvs23@cam.ac.ukhttp://crossmark.crossref.org/dialog/?doi=10.1063/1.4900887&domain=pdf&date_stamp=2014-11-05

  • 014902-2 Shepherd et al. APL Mater. 3, 014902 (2015)

    into the fibrillar structure and the effect of cross-linking is more limited. Polarised light microscopyhas been carried out to provide a qualitative indication of alignment11,19 and Transmission Elect-ron Microscopy (TEM)6 and Small Angle X-Ray Scattering (SAXS)11,19 have also been applied toa limited extent, however, not with a view of investigating the effect of cross-linking on collagenstructure. Atomic force microscopy (AFM) has been considered quite extensively for the character-isation of native collagen2024 but can also provide evidence as to the effect of cross-linking on thecollagen order and alignment in extruded collagen fibres. Raman spectroscopy may also provide auseful route for investigating orientation and alignment within the collagen structure.2527 A recentstudy investigating bundles of collagen fibres from bovine Achilles tendon demonstrated that evennon-polarized Raman measurements (i.e., where a polarisation analyser is not present between thesample and detector) are sensitive to the laser polarisation direction.28 The amide III doublet at 1245and approximately 1270 cm1 was shown to be highly sensitive to the orientation of the fibre.28

    The aim of the work was to study the effects of cross-linking of collagen fibres extruded through asemi-continuous process13 using AFM and Raman spectroscopy. These techniques allowed the align-ment of the collagen fibres as well as the banding and chemistry to be analysed. This understandingof the basic structure is key to better understand fibre mechanics, degradation, and ultimately thebiological response to the material.

    Collagen fibre bundles were extruded using a method applied previously by the authors13 andbased upon the route of Silver et al.29 The collagen source was an acid swollen gel type I collagenderived from bovine dermis (Devro Medical, Scotland). Briefly, the collagen was added to 2 mMHCl in a concentration of 6 mg/ml and left to swell overnight before blending and degassing. Sixchannels of the collagen slurry were then extruded into a long trough containing a fibre forming bufferof 20% by weight polyethylene glycol (PEG) (molecular weight 8000, Sigma Aldrich, UK) in 0.01Mphosphate buffered saline (PBS) solution. A peristaltic pump provided a constant flow rate of thefibre forming buffer, the 6 strands were brought together at the end of the tr

Recommended

View more >