Επίδραση οπτικής ακτινοβολίας στην τοπολογία κολλαγόνου με χρήση απεικονιστικής μικροσκοπίας ατομικής δύναμης και σάρωσης ηλεκτρονίων
Authors: Στυλιανού Ανδρέας 
Published: September 2021

The development of nano-materials and nanotechnology-based methods is a cutting edge research in a wide range of disciplines and applications and the models which allow the investigation of nanoscale interactions are of particular interest. Nowadays in nanotechnology a strong rising trend is the adoption of concepts from the nature for the development of high-efficiency bio-materials, which could be used as sensors, functional surfaces etc. Also these bio-materials are used as models for investigating the influence of external factors, such as electromagnetic radiation. The main aim of this PhD thesis was the development of collagen thin films and their use as biological interface models. Moreover, aim was the reproduction, control and identification of their topography with Atomic Force Microscopy imaging techniques. The films were used for the investigation and interpretation of the nanoscale effects of optical radiation on collagen. The understanding of the involved mechanisms in biological tissue-light (across the entire optical spectrum) interactions is crucial, as it can lead to the use of the minimally invasive light radiation for diagnostic and therapeutic purposes. Furthermore, the effects of optical radiation on biological tissue has been associated with various pathological conditions (for example, the relationship of ultraviolet radiation with photo-aging or several forms of skin cancer), while in many cases the optical radiation can be used as a method of treating biomaterials (e.g. for sterilization purposes). Although the interactions of the optical radiation with biological tissue have been investigated extensively, the governing mechanisms at the nanoscale have not been clarified and the effects on surface nano-features remain unclear. The effects on the nano-topography are of paramount importance as the majority of biological interactions take place on surfaces and/or interfaces.Collagen was selected for the development of the biological surface models as is the most abundant protein of the extracellular matrix (Extracellular Matrix, ECM) and covers the 25-30% of the total protein of mammals. From the 28 different types of collagen that have been identified so far, type I collagen is the most abundant and is found in tissues such as skin, bone, cartilage and tendons. Due to its particular characteristics (fibrous nature, ability for self-assembly, bio-compatibility, bio-degradability and non-toxicity) type I collagen is widely used as one of the main biomaterials in a wide range of applications in the fields of biomaterials and tissue engineering. Its abundance in the human body and its extensive use in biomedical applications, led to its selection for the investigation of the effects of optical radiation on it (and therefore on collagen-rich tissues) and the clarification of the involved mechanisms.The research objectives of this research were:i)the development of Atomic Force Microscopy imaging techniques for the nanoscale characterization and imaging of collagen thin filmsii)the quantification of surface changes occurring in collagen thin films due alterations in collagen physical and/or chemical propertiesa.the development of collagen thin films based biological surface models which were characterized by:b.the ability to control the nano-topography of the films, so as to pre-determine the surface features with repeatability andiii)the ability to support cell cultureiv)the investigation and subsequent control of cell behavior alterations due to the effect of surface changes,v)the study of second harmonic generation from thin films of collagen,vi)the investigation of the effect of ultraviolet radiation on collagen thin films,vii)the investigation of the effect of low level laser radiation in the red on collagen thin films, viii)the investigation of the alterations of cell behavior due to the induced changes on the biological surface model topography under the influence of ultraviolet and low level laser radiation in the red region. The selected wavelengths cover a wide range of the optical spectrum and have noticeable applications in biomedicine. More specifically, the mechanism of the Second Harmonic Generation (SHG), by near infrared (NIR) laser stimulation, from collagen thin films was investigated. The genesis of this signal can be used to develop non-linear techniques which can be applied for diagnostic purposes, so as to detect pathological conditions associated with collagen fibers differentiation. Moreover, the effects of ultraviolet radiation (UV) on collagen were investigated. The UV radiation has been associated with various pathological conditions (such as photo-aging) and is used for the treatment of collagen-based bio-materials for sterilization or cross-linking purposes. Furthermore, the effects of Low Level Laser radiation in the red region of the optical spectrum (Low-Level Red Laser, LLRL), which is widely used for the treatment with low-power laser (Low-Level Laser Therapy, LLLT) were investigated. The LLLT can be used to treat a range of pathological conditions, including healing wounds. The investigation of UV and LLRL irradiation was further expanded in the study of the indirect effects of the optical radiation on cell behavior (particularly in dermal fibroblasts), by using irradiated collagen films as cell culture substrates. In order to carry out this investigation, the first step was the development of collagen thin films that could be used as a biological surface/interface model, with pre-determined (fixed) and/or controlled nanoscale topography. Appropriate protocols were developed for the development of thin films with pre-determined surface characteristics. These protocols allowed the investigation of the influence of different physicochemical factors (e.g. pH, temperature, time fibrillogenesis, adsorption time) on the surface characteristics of the developed collagen thin films. Furthermore, it has been studied how the different formation methodologies influence the thin films characteristics. AFM high resolution imaging techniques were developed for the qualitative and quantitative nano-characterization of the collagen thin films and were combined with the information that was provided by Scanning Electron Microscopy (SEM) imaging. The results showed that the two aforementioned microscopy techniques provided combinational information for a thorough characterization of films and the substrates that were used. The AFM techniques were shown to be suitable for imaging and characterizing of specific nanoscale features on the surfaces of the collagen films. Furthermore, the AFM imaging demanded minimal sample preparation, free of invasive techniques (such as the use of dyes or the coating of the sample with conductive films). As a result, samples did not accept any procedure that could alter their surface characteristics. The SEM imaging provided additional information for the characterization of larger sample areas (of the order of a few microns). Also, SEM had better performance on larger collagen structures and general in areas of samples where the AFM tip is unable to provide information. Moreover, advanced image analysis and processing techniques were used in order to obtain qualitative and quantitative data from the acquired images.The methods that were used to form collagen thin films were: drying in the air, the Spin Coating (SpC) process, the use of Hydrodynamic Flow (HF) and finally the combination SpC-HF. The results showed that each of these methods allowed the formation of films with different characteristics. By using the method of drying in the air, the formed films were relatively thick, homogeneous, consisting of collagen fiber/fibrils with random orientation and with collagen aggregations. The SpC process, allowed the formation of ultra-thin and homogeneous collagen films, consisting of collagen fibrils with physiological characteristics and random orientation. The HF leaded to the formation of collagen thin films with oriented fibrils, while the combination SpC-HF films allowed the formation of collagen structures with two major orientations (perpendicular to each other).Among the different substrates that were used, mica substrates presented to have better performance than glass substrates, as they allowed the formation of finer and more homogeneous films. The crystalline nature and the surface properties of mica appeared to allow better adsorption of collagen fibers and the formation of fibrils with physiological characteristics, such as the D-periodicity of ~ 67 nm. Additionally, of particular interest were the surfaces of polystyrene particles (Polystyrene Particle Surfaces, PPS) which were formed and used as substrates. By using polystyrene nano-particles and by optimizing the formation parameters, it was achieved the formation of uniform nanostructured surfaces. These surfaces are shown to be suitable substrates for the formation of thin collagen films exhibiting physiological characteristics. Moreover, they can be used as an excellent model/tool for investigating the relationship between specific surface nano-characteristics and the adsorption of collagen fibrils or other proteins.The investigation and use of different substrates and different thin films formation methodologies, allowed the development of collagen thin film with pre-determined surface characteristics. The control of surface nano-characteristics (like the fibers diameter, the D-band), the homogeneity of the samples and the orientation of the fibers was achieved by using appropriate substrate or/and formation methodology. The characterization, the quantification and the control of the collagen thin films topography allowed the films to be used as biological surface models for the investigation of the influence of the surface characteristics on cell behavior and the study of the effects of optical radiation. The development of biological surface models consisted from self-assembled fibrils/fibers with natural characteristics, like D-band periodicity. These films were formed with different formation methodologies and on different substrates (mica, glass, PPS) depending on the required surface characteristics. For the investigation of the optical properties the films that were formed on mica were used due to its crystalline nature and its extremely flat surface enable the nanoscale imaging. The other two substrates (glass, PPS) allowed to be applied for the investigation of the adsorption of collagen fibers/fibrils on different substrates.The developed biological models were used as substrates for the growth of primary culture of human dermal fibroblasts. The results showed that the films can be used as cell culture substrates and that fibroblasts respond to the topography of the films. After films with oriented collagen fibers/fibrils were used, it was demonstrated that the fibroblasts followed the main orientation of the collagen fibers. Moreover, on films with randomly oriented fibers, the fibroblasts showed a no standard arrangement in the available space during their growth. In this PhD Thesis a method for simultaneous imaging of both the AFM fibroblasts and the collagen fibers was developed, so as to qualitatively present the relationship among the fibroblast orientation and the characteristics of the substrate. Also, in this specific field of research, this thesis presents for the first time the quantitative measurement and the graphical representation of the orientation (Circular histograms of local orientation (CHLO)) of collagen fibers in the nanoscale by applying AFM imaging methods and image processing techniques. In the case of studying the SHG signal, the biological surface/interface model and the experimental setups that were developed, they were used to investigate the possibility of producing SHG from thin films. Moreover, it was investigated the relationship between the polarization angle of the stimulation laser radiation and the orientation of the collagen fibers. In addition, thin films that were formed with thermal demodulated collagen, were used to correlate the SHG signal with the effects of thermal demodulation of the collagen fibers/fibrils. The results showed that the thin films are suitable for the detection and investigation of weak SHG signals, when the laser stimulation is performed with low intensities. Through the use of a polarizer, it was demonstrated that it was possible to detect the orientation of collagen fibers. Moreover, it was presented that the SHG signals were decreasing by the thermal denaturation of the collagen fibers. Therefore, the results demonstrated that measurements of the SHG signal can be used as a tool for detecting the orientation or alteration in the orientation of the collagen fiber/fibrils. These alterations may be observed in vivo in various pathological conditions and the SHG can be used as a diagnostic tool. Furthermore, the collagen-based biological surface model can be used as a tool for simulating these pathological alterations in order to clarify the exact relationship among the emitted SHG signal and the collagen characteristics.In the case of the ultraviolet radiation (UV, 254 nm), the thin collagen films allowed the exploration of the effects of UV irradiation on collagen optical properties of collagen (fluorescence, absorption) and its topography. Moreover, the use of the UV-irradiated collagen films as cell culture substrate was also investigated results showed that the used doses and irradiation times (which are in the range of those that are used for sterilization or for crosslinking processes) the fluorescence of collagen increases, while also the collagen absorption increase. These alterations in the fluorescence and absorption spectrum of collagen are due to the photo-degradation that was caused by UV radiation on fibrous collagen. The photo-degradation induced structural changes in aromatic amino acids residues or in the amino acids. More specifically, it caused the breaking of the peptides bonds and subsequently increased the formation of photoproducts from tyrosine and phenylalanine. When the irradiation was carried out directly on the collagen thin film for short irradiation intervals, the surface topography was not distorted (as no modifications were detected in the collagen fibers, like fibrous form, D-periodicity and diameter) or in the surface roughness. In the case that the films were formed by UV-irradiated collagen no lesions were observed in the collagen fibers/fibrils. Also, the surface roughness showed fluctuations, which suggests that the photo-degradation is a reversible process when the UV-irradiation takes place in wet environment. These results suggested that this method can be used as a technique for controlling/manipulating the surface roughness, which plays an important role in biomedical applications, especially when the surfaces are contacted with cells. Furthermore, the effects of UV irradiation for long irradiation intervals on nanoscale characteristics of collagen fibers and on the D-periodicity, were investigated. The results of the growth of primary cultures of human dermal fibroblasts on the thin collagen films -in both cases-, showed that the behavior of cells was affected by the UV irradiation. By increasing the irradiation time period, the body of fibroblasts and their nuclei became increasingly globular, which demonstrates abnormal growth of the fibroblasts. Consequently, the results of the thesis presented that UV-irradiated collagen-based biomaterials should be avoided or kept to the minimum possible level, when the bio-materials are intended to be used in applications where the biomaterials come into contact with cells. The last optical radiation that was investigated -with the use of the thin collagen films as a model, was the Low Level Laser radiation in the Red region (LLRL, 661 nm). The experimental results showed that although a slight increase in the emitted fluorescence intensity was measured, no alterations were observed in the nano-topography of collagen films after irradiation. It should be noted that the parameters of irradiation (wavelength, dose, irradiation time intervals) were of the order of magnitude as those used for the treatment with a low power laser (LLLT). The fact that the fibroblasts’ behavior was altered when the LLRL-irradiated films were used as substrate for the cultivation of cells is particularly interesting. By increasing the irradiation treatments (and consequently the irradiation dose offered in the collagen thin films), fibroblasts became more and more spherical. This is an evidence of abnormal growth of the fibroblasts, as it was also observed in the UV-irradiated films. This doctoral dissertation focused on the effects that LLRL had directly on collagen and it was the first time in the international literature that it was demonstrated that irradiation of LLRL may have a negative effect on cell growth due to the effects of irradiation on collagen. This result represents important and novel information that offers new data toward the clarification of the LLLT mechanism. Since the modulation of cellular behavior was observed in the absence of measurable surface changes, the alteration of cell behavior due to LLRL irradiation is not affected by the ‘surface guidance mechanism’ (which relies on the surface properties of the substrate). The results lead to a new dimension for investigating the involved phenomena and suggest that for the clarification of the LLLT mechanisms the investigation of the effects of LLRL (or other low power radiation) on other tissue properties (like the mechanicals) and on each separate tissue element is demanded.From this PhD thesis, important results concerning the model of the collagen fiber were obtained. Although the fibrous type I collagen has extensively been investigated and various models of its structure have been proposed, none of these models can explain all of the in vivo and in vitro observed structures. The results that were obtained showed that the developed protocols were suitable for the formation of collagen fibers presenting polymorphism, i.e. the existence of more than one periodicities along the fibers (e.g., the simultaneously occurrence of D- periodicities of the order of ~ 67 and ~ 100 nm). It is interesting that in the developed protocol the initial collagen solution consisted of a high concentration of collagen, without the addition of any other kind of substances that are traditionally used for the development of collagen fibers with polymorphism. Moreover, the use of the Spin Coating procedure allowed the formation of collagen fibers with kicks (areas along the fiber where the fiber is abruptly changes direction). The kicks were formed because of the large centrifugal forces that were exerted on the fibers and these mechanical deformations are similar to those of the tubes (structures of elongated shape and which are either empty inside or consist of a harder outer shell). Finally, upon irradiation with UV for long time intervals on the surface of the fibers ‘volcano-like’ holes were formed. AFM imaging allowed the visualization of the D-band within the fiber, i.e. below the damaged outer shell. The combination of these results leaded to a ‘multiple shells’ model of the collagen fiber according to which collagen fibers are behave as ‘tubes’ and consist of multiple shells, with the outside shell to be harder, and the D-periodicity to continue into the inner shells.The results of the research were qualitatively, quantitatively and theoretically evaluated and compared with the results presented in the relevant international literature. Based on the conclusions that were reached, it was shown that this dissertation contributes significantly to a wide range of research fields, concerning nano-characterization/nano-imaging of biomaterials and the optical radiation-biological tissue interactions. The obtained results received international recognition, while the developed techniques and models will be important tools in a number of research or biomedical applications, especially in the fields of nano-biomaterials and tissue engineering.