| Περίληψη: | Nowadays, bone diseases are constantly on the rise either because of lifestyle change – population aging, increased obesity rates, minimal physical exercise - or because of permanent bone damage. To date, methods for defective bone treatment, such as autografting and allografting present short-term or long-term problems - painful and long-term processes, unpredictable bone resorption, risk of disease and / or infection that may cause a reduction or complete loss of limb - . For this reason, it is necessary to study and apply new biomaterials and techniques for bone regeneration. In this thesis, osteoblasts were differentiated from bone marrow cells isolated from hip and knee bones and cultured under suitable conditions similar to human’s body (37 ° C, 5% CO2, nutrient). Then in vitro studies were performed, and the development of osteoblasts on different types of substrates - polystyrene, smooth titanium, titanium dioxide nanotubes, rough titanium - was evaluated. Finally, through an electric stimulation device, manufactured in the laboratory, it was examined whether electrical stimulation of cells on the same substrates, as above, improves their proliferation and differentiation.
The first chapter consists of two parts. The first part analyses the structure and composition of the bones, as well as the types of bone cells. The second part analyses the types of mesenchymal cells, isolated from bone marrow, and the indices of osteogenic differentiation - alkaline phosphatase, total protein. The second chapter refers to biomaterials. Firstly, the desired properties of biomaterials associated with bone engineering are presented. Then the biomaterials used in the present study, are presented, the polystyrene - its evolution over years and its conversion from plain polystyrene to bio- and the titanium - its applications in medicine and its modifications to improve its properties -. The third chapter presents the cell - material interaction. At first, the cellular environment, the extracellular matrix - synthesis and interactions - and the function of adhesion-dependent cells are described. Then the properties of the bio-interface - chemistry, topography, substrate elasticity - and cell growth factor are presented. Finally, a look at the evolution of biomaterials over the years is made.
The fourth chapter is about in vitro cell culture. It first describes a cell culture, its usefulness and its conditions - nutrient, CO2, temperature -. The conditions for cell passaging are also reported, as well as the method for cell counting. Finally, a reference is made to osteoblasts, the type of cells used in our study. The fifth chapter includes electrical stimulation of cells. The human body by itself emits an electrical stimulus that enhances cellular communication. However, there is the possibility of external electrical stimulation of bone - regenerating tissues - and this chapter refers to electrical stimulation applications in the body. The sixth chapter includes the experimental part. The materials and methods used as well as the preparation of the substrates are presented. The procedure for isolation and differentiation of osteoblasts from their receipt from the hospital to their culture in the laboratory is described below. Moreover, the process of cell degradation and subculturing as well as cell counting is being explained. After that, the experimental part is divided into two sub-sections. The first section describes the cell culture in polystyrene flasks, and then the biocompatibility test on different substrates - polystyrene, titanium, titanium dioxide nanotubes, rough titanium - . The second section describes the osteoblast culture on the same substrates - polystyrene, titanium, titanium dioxide nanotubes, rough titanium - under electrical current stimuli through an electric stimulation device manufactured in the laboratory.
The present investigation aimed to find an appropriate combination of parameters related to substrate characteristics (nature and topography) and an induced electrical field that leads to the proliferation and enhanced activity of osteoblast bone cells. With this purpose, a cell carrying device has been designed and used for the electrical stimulation of the human osteoblast cells. It was found that a combined effect of both applied electrical field (0.3V, 3 hours) and specific substrate nature and topography considerably increase the ration ‘alkaline phosphatase/ total protein’ (ALP/TP), which has been translated as the degree of biocompatibility. When no electric field is applied, the cells tend to increase ALP levels associated to their multiplication and proliferation on soft surfaces, while a light decrease is observed when they are seeded on rough surfaces.
On the opposite, the total protein level is increased cells seeded on rough substrates, since this is related to their adhesion process. The ALP/TP is decisive for the faith osteoblasts population. Generally, a light increase in proliferation on TNTs compared to other substrates is associated with the highly organized nature of the TNTs. Results agree with already reported studies and confirm that substrate nature which includes its chemical composition, its mechanical properties, and surface topography are very important factors, decisive for osteoblasts well-functioning. When an electric field is applied, cells functions are improved. Titanium dioxide nanotubes, due to their nature and highly organized architecture, contribute to the uniform distribution of the electric field in the medium and under the bone cell population. Finally, the effect of an adjusted intensity of the electric field and the appropriate topography of the substrate on cells behaviour demonstrates the importance of these two in vitro parameters for the activity of bone forming cells and may be considered crucial in the manufacturing of advanced biomaterials for orthopaedic applications.
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