Andreas Stylianou

Surface nanoscale imaging of collagen thin films by Atomic Force Microscopy

Collagen, the most abundant protein in mammals, due to its unique properties is widely used as biomaterial, scaffold and culture substrate for cell and tissue regeneration studies. Since the majority of biological reactions occur on surfaces and structures at the nanoscale level it is of great importance to image the nanostructural surface of collagen based materials. The aim of this paper was to characterize, with Atomic Force Microscopy (AFM), collagen thin films formed on different substrates (glass, mica, polystyrene latex particle surfaces) and correlate their morphology with the used substrates, formation methodologies (spin coating, hydrodynamic flow) and original collagen solution. The results demonstrated that, by altering a number of parameters, it was possible to control the formation of collagen nanostructured films consisting of naturally occurring fibrils. The spin coating procedure enabled the formation of films with random oriented fibrils, while substrates influenced the fibril packing and surface roughness. The hydrodynamic flow was used for guiding fibril major orientation, while adsorption time, rinsing with buffer and solution concentration influenced the fibril orientation. The clarification of the contribution that different parameters had on thin film formation will enable the design and control of collagen nanobiomaterials with pre-determined characteristics.

Combined information from AFM imaging and SHG signal analysis of collagen thin films

Abstract Collagen being the most abundant protein in mammals is important for a variety of functions and its structure, concentration and orientation disturbance is associated with different pathological states. The use of the optical second harmonic generation (SHG) is emerging as a powerful non-invasive tool for assessing collagen modification in a variety of pathological conditions. The properties of second harmonic light from collagen structures have not yet been fully clarified due to a number of limitations, such as the difficulty to prepare collagen samples with well-known characteristics and optical properties, at a nanoscale resolution. The results of this paper suggest that some of these limitations can be overcome by using thin collagen films with pre-determined characteristics (PDC), which maintain or/and enhance their NLO properties capacities, as they have been checked by using atomic force microscopy (AFM). The collagen fiber structure and orientation was systematically altered by using thermal denaturation or different preparation methodologies (spin coating procedure, use of collagen solution hydrodynamic flow). These films can be used as prototypes and the combined information from AFM imaging and the one included in SHG signal, delivered from them, can significantly contribute to further understanding of the NLO properties of collagen and in the long term to take advantage as a non-invasive tool.

Investigation of the influence of UV irradiation on collagen thin films by AFM imaging

Collagen is the major fibrous extracellular matrix protein and due to its unique properties, it has been widely used as biomaterial, scaffold and cell-substrate. The aim of the paper was to use Atomic Force Microscopy (AFM) in order to investigate well-characterized collagen thin films after ultraviolet light (UV) irradiation. The films were also used as in vitro culturing substrates in order to investigate the UV-induced alterations to fibroblasts. A special attention was given in the alteration on collagen D-periodicity. For short irradiation times, spectroscopy (fluorescence/absorption) studies demonstrated that photodegradation took place and AFM imaging showed alterations in surface roughness. Also, it was highlighted that UV-irradiation had different effects when it was applied on collagen solution than on films. Concerning fibroblast culturing, it was shown that fibroblast behavior was affected after UV irradiation of both collagen solution and films. Furthermore, after a long irradiation time, collagen fibrils were deformed revealing that collagen fibrils are consisting of multiple shells and D-periodicity occurred on both outer and inner shells. The clarification of the effects of UV light on collagen and the induced modifications of cell behavior on UV-irradiated collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist in the appropriate use of UV light for sterilizing and photo-cross-linking applications.

The effects of UV irradiation on collagen D‐band revealed by atomic force microscopy

The objective of this paper was to investigate the influence of UV irradiation on collagen D-band periodicity by using the AFM imaging and nanoindentation methods. It is well known than UV irradiation is one of the main factors inducing destabilization of collagen molecules. Due to the humans skin chronic exposure to sun light, the research concerning the influence of UV radiation on collagen is of great interest. The impact of UV irradiation on collagen can be studied in nanoscale using Atomic Force Microscopy (AFM). AFM is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Hence, high resolution images of individual collagen fibrils and load-displacement curves on the overlapping and gap regions, under various time intervals of UV exposure, were obtained. The results demonstrated that the UV rays affect the height level differences between the overlapping and gap regions. Under various time intervals of UV exposure, the height difference between overlaps and gaps reduced from ~3.7 nm to ~0.8 nm and the fibril diameters showed an average of 8-10% reduction. In addition, the irradiation influenced the mechanical properties of collagen fibrils. The Youngs modulus values were reduced per 66% (overlaps) and 61% (gaps) compared to their initial values. The observed alterations on the structural and the mechanical properties of collagen fibrils are probably a consequence of the polypeptide chain scission due to the impact of the UV irradiation.

Atomic Force Microscopy surface nanocharacterization of UV-irradiated collagen thin films

Collagen, the most abundant protein in mammals, is a basic component of the extracellular matrix and due to its unique properties it is widely used as biomaterial, scaffold and culture substrate for cell and tissue regeneration studies. Due to human skin chronic exposure to sun light and since UV rays are used as sterilizing and cross-linking methods the clarification of the UV light-collagen interactions are very crucial. Moreover, since the majority of the biological reactions occur on surfaces or interfaces the influence of UV light on the surface of collagen-based materials attracts the scientific interest, especially in the biomaterials science. Surface-nanoscale characterization could be performed with Atomic Force Microscopy (AFM), which is a powerful tool and offers quantitative and qualitative information. Its ability of high resolution imaging and non-destructive characterization makes it very attractive for biological samples investigation. The aim of this paper was to determine the surface properties and alterations of collagen thin films after UV-irradiations using AFM techniques. Furthermore, it was aimed to investigate the possible different influence on the surface when the collagen solution or the thin films were irradiated. In this paper topographic AFM images were acquired from thin films, formed from both irradiated and non-irradiated collagen solutions, with spin coating procedure. The results demonstrated that the UV irradiation have different results when it is applied in the collagen solution or in the film after the spin coating methodology. For short irradiation times (<;120 min) UV caused only rather small changes in the morphology of the studied films although fluorescence and absorption studies confirmed collagen photodegradation. The surface roughness and topography altered after 3 and 7 hours, respectively, while the fibrous structure was completely destroyed after 15 hours. Surface roughness of the films depends on whether the solution was irradiated or the film and on the time irradiation. The fully clarification of the role of the UV light on collagen thin films will enable the proper design and control of collagen based nanobiomaterials with appropriate and improved surface properties.

Nanotopography of collagen thin films in correlation with fibroblast response

Abstract. Collagen thin films consisting of randomly oriented and oriented collagen fibrils/fibers are fabricated by hydrodynamic flow and spin coating, and then they are characterized by atomic force microscopy (AFM). Fibroblasts are cultured on these films in order to correlate their morphology and alignment, which are assessed with fluorescence and AFM imaging with different film characteristics. The results showed that the formed films could be used as substrates for culturing cells. Furthermore, cells reacted to film nanocharacteristics and especially to the orientation of fibrils/fibers. The investigation of the influence that the substrate nanotopography has on cells will help to elucidate the mechanisms of cell–biomaterial interactions, and will enable the design of intelligent coatings for implants and tissue engineering purposes.

Atomic Force Microscopy Imaging of the Nanoscale Assembly of Type I Collagen on Controlled Polystyrene Particles Surfaces

The adsorption of collagen and the morphology of its assemblies at solid surfaces play an important role in a variety of research areas and applications, such as biomaterials, biocompatibility, tissue mechanics and cell studies. In this paper the nanoscale organization of type I collagen on polystyrene particle surfaces with controlled patterns was investigated by high-resolution atomic force microscopy. The results showed that patterned surfaces with a well-ordered morphology could be formed by spin coating polystyrene particles dispersions (20 sec at 1000 rpm) and appropriate annealing conditions (at 90°C for 2h). Collagen thin films consisting of fibers with natural characteristics, such as the typical 67nm periodicity (D-band), were formed on these nano-patterns. This kind of surfaces provides a useful substrate for studying the regulation of collagen assembly on substrates with different nano-topographical and environmental conditions and offers a potential way to create surfaces of functionalized and nano-patterend materials for biotechnological and biomedical applications.

Mechanical properties of collagen fibrils on thin films by Atomic Force Microscopy nanoindentation

Atomic Force Microscopy (AFM) is a powerful tool as far as surface characterization is concerned, due to its ability to relate high resolution imaging with mechanical properties. Furthermore biological samples, such as collagen, can be studied by AFM with a non-destructive manner. Collagen is the most abundant protein in mammals and because of its unique properties is widely used as biomaterial. Due to the human skin chronic exposure to sun light and since UV-rays are used in sterilizing and cross linking methods of collagen based biomaterials, the investigation of the influence of UV light on collagen, is crucial. The purpose of this paper was to investigate the topographic features and the mechanical properties of collagen fibrils prior and post ultraviolet (UV) by using the AFM indentation method. Hence, load-displacement curves were obtained on collagen fibrils in order to calculate the Young modulus for each case. Each curve presented, was the average of 10 curves. The results showed that Young modulus value increased after 4 hours of UV irradiation from 0.5 GPa to 1.53 GPa. After 8 hours of UV irradiation the Young modulus value was calculated equal to 3.2 GPa. These experiments yielded a clear stiffening of collagen fibrils as a result of UV exposure. Moreover, after 8 hours of UV exposure, collagen fibrillar structure started to deform and the characteristic D-band of collagen fibrils deteriorated. The investigation of the alterations of the modulus under UV irradiation, will contribute to the clarification of the impact that different physical and chemical parameters have on the mechanical properties of collagen-based materials. In addition, this will enable the design and development of biomaterials with improved properties.

Combined SHG signal information with AFM imaging to assess conformational changes in collagen

Collagen is the most abundant protein in mammals and is important for a variety of functions and its concentration, structure and function is associated with different pathological states. In this research we correlate structural changes with those changes in the Second Harmonic Generation (SHG) signal. The combination of Atomic Force Microscopy (AFM) imaging with information included in SHG signal can significantly contribute to further understanding of the nonlinear optical properties of collagen.

Atomic force microscopy investigation of the interaction of low-level laser irradiation of collagen thin films in correlation with fibroblast response

Low-level red laser (LLRL)–tissue interactions have a wide range of medical applications and are garnering increased attention. Although the positive effects of low-level laser therapy (LLLT) have frequently been reported and enhanced collagen accumulation has been identified as one of the most important mechanisms involved, little is known about LLRL–collagen interactions. In this study, we aimed to investigate the influence of LLRL irradiation on collagen, in correlation with fibroblast response. Atomic force microscopy (AFM) and fluorescence spectroscopy were used to characterize surfaces and identify conformational changes in collagen before and after LLRL irradiation. Irradiated and non-irradiated collagen thin films were used as culturing substrates to investigate fibroblast response with fluorescence microscopy. The results demonstrated that LLRL induced small alterations in fluorescence emission and had a negligible effect on the topography of collagen thin films. However, fibroblasts cultured on LLRL-irradiated collagen thin films responded to LRLL. The results of this study show for the first time the effect of LLRL irradiation on pure collagen. Although irradiation did not affect the nanotopography of collagen, it influenced cell behavior. The role of collagen appears to be crucial in the LLLT mechanism, and our results demonstrated that LLRL directly affects collagen and indirectly affects cell behavior.