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Impact of the exposure to ultraviolet light on the nanomechanical properties of collagen probed by atomic force microscopy and Raman spectroscopy

Schulze, Marcus (2020):
Impact of the exposure to ultraviolet light on the nanomechanical properties of collagen probed by atomic force microscopy and Raman spectroscopy.
Darmstadt, Technische Universität, DOI: 10.25534/tuprints-00011487,
[Online-Edition: https://tuprints.ulb.tu-darmstadt.de/11487],
[Ph.D. Thesis]

Abstract

Collagen is a body own protein that, due to its outstanding biocompatibility, is applicable as substrate for cell seeding in tissue engineering. Creating a substrate that mimics the characteristics of human tissue and, thus, allows for a sustainable proliferation of biological cells, requires a well-directed design of its mechanical properties. In the case of collagen, the exposure to ultraviolet (UV) light is a promising method for inducing stabilising or destabilising reactions that modify the mechanical properties, as for example, the tensile modulus. For enabling a targeted substrate design, the reaction of the collagen samples towards the UV light treatment needs to be understood and made predictable. Collagen fibrils, as small units of the collagen’s hierarchical structure, gained attention and are investigated regarding the response of their mechanical properties due to the exposure to UV light dependent on the kind of surrounding liquid. Atomic force microscopy (AFM) and Raman spectroscopy were applied to monitor the impact of UV light on collagen fibrils and hydrogels. Changes in the indentation modulus of single collagen fibrils were recorded for the exposure to UVA, UVB, or UVC light while being immersed in varied liquid environments in situ. Next, an experimental procedure for an AFM-based eased extraction of the tensile modulus from fibrillar structures as small as collagen fibrils was proposed, which was used for the acquisition of the changes in the tensile modulus of collagen fibrils, dependent on the combination of the applied kind of UV light and liquid environment. The observed modifications of the mechanical properties should be complemented by a description of structural changes induced by UV light irradiation for which Raman spectroscopy was used. Collagen fibrils responded to the exposure to UV light with a change in their mechanical properties and exhibited repeated patterns of behaviour dependent on the applied combination of UV light and the kind of liquid environment. A decrease in stability of up to 90% (tensile modulus) was detected for the immersion of the samples in deionised water. For gaining an increase in stability with advancing exposure time, the application of phosphate-buffered saline (PBS) was a necessary condition. A maximum averaged increase in indentation modulus of approximately 60% was seen during exposure of single collagen fibrils to UVB or UVC light. For the tensile tests, a fourfold increase of the tensile modulus was recorded as maximum value. The depicted dependence of the mechanical properties on the kind of liquid environment was confirmed by Raman spectroscopy on collagen hydrogels. The observed proliferation of biological cells on UV light exposed collagen hydrogels showed the applicability of the method.

Item Type: Ph.D. Thesis
Erschienen: 2020
Creators: Schulze, Marcus
Title: Impact of the exposure to ultraviolet light on the nanomechanical properties of collagen probed by atomic force microscopy and Raman spectroscopy
Language: English
Abstract:

Collagen is a body own protein that, due to its outstanding biocompatibility, is applicable as substrate for cell seeding in tissue engineering. Creating a substrate that mimics the characteristics of human tissue and, thus, allows for a sustainable proliferation of biological cells, requires a well-directed design of its mechanical properties. In the case of collagen, the exposure to ultraviolet (UV) light is a promising method for inducing stabilising or destabilising reactions that modify the mechanical properties, as for example, the tensile modulus. For enabling a targeted substrate design, the reaction of the collagen samples towards the UV light treatment needs to be understood and made predictable. Collagen fibrils, as small units of the collagen’s hierarchical structure, gained attention and are investigated regarding the response of their mechanical properties due to the exposure to UV light dependent on the kind of surrounding liquid. Atomic force microscopy (AFM) and Raman spectroscopy were applied to monitor the impact of UV light on collagen fibrils and hydrogels. Changes in the indentation modulus of single collagen fibrils were recorded for the exposure to UVA, UVB, or UVC light while being immersed in varied liquid environments in situ. Next, an experimental procedure for an AFM-based eased extraction of the tensile modulus from fibrillar structures as small as collagen fibrils was proposed, which was used for the acquisition of the changes in the tensile modulus of collagen fibrils, dependent on the combination of the applied kind of UV light and liquid environment. The observed modifications of the mechanical properties should be complemented by a description of structural changes induced by UV light irradiation for which Raman spectroscopy was used. Collagen fibrils responded to the exposure to UV light with a change in their mechanical properties and exhibited repeated patterns of behaviour dependent on the applied combination of UV light and the kind of liquid environment. A decrease in stability of up to 90% (tensile modulus) was detected for the immersion of the samples in deionised water. For gaining an increase in stability with advancing exposure time, the application of phosphate-buffered saline (PBS) was a necessary condition. A maximum averaged increase in indentation modulus of approximately 60% was seen during exposure of single collagen fibrils to UVB or UVC light. For the tensile tests, a fourfold increase of the tensile modulus was recorded as maximum value. The depicted dependence of the mechanical properties on the kind of liquid environment was confirmed by Raman spectroscopy on collagen hydrogels. The observed proliferation of biological cells on UV light exposed collagen hydrogels showed the applicability of the method.

Place of Publication: Darmstadt
Divisions: 11 Department of Materials and Earth Sciences
11 Department of Materials and Earth Sciences > Material Science
11 Department of Materials and Earth Sciences > Material Science > Physics of Surfaces
Date Deposited: 05 Apr 2020 19:55
DOI: 10.25534/tuprints-00011487
Official URL: https://tuprints.ulb.tu-darmstadt.de/11487
URN: urn:nbn:de:tuda-tuprints-114873
Referees: Stark, Prof. Dr. Robert and von Klitzing, Prof. Dr. Regine
Refereed / Verteidigung / mdl. Prüfung: 29 November 2019
Alternative Abstract:
Alternative abstract Language
Kollagen ist ein körpereigenes Protein, das aufgrund seiner hervorragenden Biokompatibilität als Substrat beim Tissue Engineering eingesetzt wird. Bei der Herstellung eines Substrates, das die Eigenschaften menschlichen Gewebes imitiert und dadurch eine nachhaltige Proliferation der biologischen Zellen ermöglicht, müssen unter anderem die mechanischen Eigenschaften angepasst werden. Im Fall von Kollagen bietet die Bestrahlung mit ultraviolettem (UV) Licht eine vielversprechende Möglichkeit, mechanische Eigenschaften, wie z.B. den Zugmodul, durch photochemische Prozesse zu modifizieren. Für ein gezieltes Substrat-Design mit UV-Bestrahlung muss man deshalb den Einfluss von UV-Licht auf Kollagenproben besser verstehen. Der Einfluss von UV-Licht auf Kollagenfibrillen und -hydrogele wurde mittels Rasterkraftmikroskopie (englisch: atomic force microscopy, AFM) und Raman Spektroskopie an gequollenem Kollagen untersucht. UVA-, UVB-, oder UVC-Strahlungs-induzierte Änderungen des Indentationsmoduls von Kollagenfibrillen wurden für unterschiedliche Salzkonzentrationen aufgezeichnet. Für diese Untersuchungen waren vor allem Kollagenfibrillen von Interesse, da sie eine der kleinsten Kollageneinheiten darstellen. Anschließend wurde ein Vorgehen für eine AFM-basierte Messung des Zugmoduls von nanofibrillären Strukturen etabliert. Die so nachgewiesenen UV-Licht-induzierten Veränderungen der mechanischen Eigenschaften wurden durch eine Untersuchung der strukturellen Änderungen mittels Raman Spektroskopie ergänzt. Im Zuge der Bestrahlung mit UV-Licht veränderte sich die Elastizität der Kollagenfibrillen in Abhängigkeit der verwendeten Kombination von UV-Licht und Umgebungsflüssigkeit. Dabei konnten sowohl stabilisierende als auch destabilisierende Effekte beobachtet werden. Eine Abnahme der Stabilität von bis zu 90 % (Zugmodul) wurde für Proben ermittelt, die in deionisiertem Wasser behandelt wurden. Eine Zunahme der Steifigkeit wurde dagegen bei UV-Bestrahlung in phosphatgepufferter Salzlösung (PBS) erreicht. Hier konnte für den Indentationsmodul von einzelnen Kollagenfibrillen eine mittlere Erhöhung der Steifigkeit um etwa 60% gemessen werden. Dagegen konnte in Zugversuchen ein bis zu vierfacher Anstieg des Zugmoduls nachgewiesen werden. Die gemessene Abhängigkeit der mechanischen Eigenschaften von der Umgebungsflüssigkeit wurde durch die Raman Spektroskopie-Ergebnisse unterstützt. Eine Proliferation von Zellen auf Kollagenhydrogelen, die mit UV-Licht bestrahlt waren, zeigte das Anwendungspotential der Methode auf.German
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