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Stimulation of mesenchymal stem cells osteogenic differentiation using perfusion bioreactor
Šljivnjak, Erna ; Rampichová, Michala (advisor) ; Rösel, Daniel (referee)
Bone cells in vivo reside in a dynamic environment exposed to constant chemical and mechanical stimuli caused by the interstitial fluid flow. It is hypothesized that perfusion of the medium through the scaffold increases the mass transport and creates at the same time shear stress, thereby in vitro simulating the interstitial fluid effects and bone tissue formation conditions. This work examined the effects of perfusion flow rates on cell viability, proliferation, migration and osteogenic differentiation of human mesenchymal stem cells within cell-seeded 3D poly-ε-caprolactone scaffolds. Scaffolds were perfused for 21 days at flow rates 1, 3 and 5 mL/min and were compared to the scaffolds from static culture. Cells were most viable, had upregulated expression of osteogenic markers collagen type I and highest alkaline phosphatase activity under flow rate 1 mL/min when compared to their static counterparts. Cells proliferated the most under flow rate 3 mL/min when compared to their static counterparts. Flow rate 5 mL/min did not significantly differ from the static culture in any of the examined parameters. Cell migration into the scaffold was not improved upon exposure to perfusion. This data confirms that medium perfusion may benefit cell proliferation and osteogenic differentiation by enhancing...
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Kompozitní pěnové nosiče pro tkáňové inženýrství kostí
Kuntová, Henrieta ; Rampichová, Michala (advisor) ; Krulová, Magdaléna (referee)
The goal of my work was to introduce the fast pacing field of tissue engineering with focus on bone regeneration. Tissue engineering could be a future alternative to the currently used conventional approaches that suffer from healing failures. Due to increasing demand for bone tissue replacement damaged by degenerative diseases or injuries, many laboratories have attempted to come up with solutions in a form of artificial constructs. In the present light of interest are composite scaffolds usually made of polymer and ceramic combinations. Their main advantage is that they combine elasticity and tensile strength of a polymer with bioactivity and mechanical hardness of a ceramic, while removing drawbacks of each material. Powered by TCPDF (www.tcpdf.org)
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Biomimetic modifications of titanium in bone tissue engineering.
Krýslová, Markéta ; Filová, Elena (advisor) ; Rampichová, Michala (referee)
When the big joints like a knee or hip joint are damaged, the solution of this problem is an artificial substitute. The replacement of damaged joints with endoprotesis helps to reduce the pain and to move normally. In the design of the implant is necessary to fulfil all requirements on the properties of the material. The surface of implant is important, because it is directly connected to bone tissue. After implantation, the negative effect include infection, inflammation or release of the implant due to limited osseointegration, may appear. The osseointegration can be improved by modifying the material surface. This thesis is focused on development and evaluation of advanced materials imitating the bone structure, especially nanoroughness and the presence of biomimetic component, such as hydroxyapatite. In this study is evaluated adhesion, proliferation, viability, differentiation, and synthesis of specific proteins of human osteoblasts like Saos-2 on titanium modified with nanotubes and plasma sprayed hydroxyapatite compared with smooth surfaces. Key words: titanium, nanotubes, osteoblasts, hydroxyapatite, nanoroughness
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Detection and visualization of native and artificial chondrogenous tissue using biophysical methods
Rampichová, Michala ; Amler, Evžen (advisor) ; Rosina, Jozef (referee) ; Kolářová, Hana (referee)
Tissue engineering is a rapidly developing field, based on using scaffolds made from natural or synthetic materials in combination with cells and stimulating factors for the replacement of damaged or lost tissue. It is very important to evaluate qualities of these scaffolds, which are necessary for cell proliferation and their mechanical support. The aim of this study was to develop a suitable scaffold for chondrocyte proliferation, scaffold functionalization and detection of cells and proteins of extracellular matrix (ECM) using methods of fluorescence and confocal microscopy. Another aim was to test an appropriate scaffold on a big animal model in vivo. Several scaffolds from natural and synthetic materials, in the form of microfibers, non-woven textiles, gels and foams were prepared for this study. Scaffolds were seeded with chondrocytes and cell adhesion, proliferation and synthesis of ECM proteins were detected. Methods of fluorescence microscopy, confocal microscopy and second harmonic generation (SHG) were used for visualization of cells and proteins. A hydrogel based on fibrin and hyaluronan was used as an scaffold for osteochondral defect regeneration in minipigs study. Prepared scaffolds showed high biocompatibility, good chondrocyte adhesion and ECM proteins synthesis. Moreover, microfibrous...
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Cell-free scaffolds functionalized with bioactive compounds in bone tissue engineering
Lukášová, Věra ; Rampichová, Michala (advisor) ; Hubálek Kalbáčová, Marie (referee) ; Libusová, Lenka (referee)
There are good prospects for bone tissue engineering and therefore researcher is aimed towards the development of cell-free scaffolds. A cell-free scaffold serves as a temporal filling for critical size defects that do not heal spontaneously. Nevertheless, a suitable scaffold composition is yet to be discovered. Moreover, modifications of cell-free scaffolds with a drug delivery system activate the internal healing capacity. Platelets occur in the healing cascade as a natural source of growth factors (GFs), chemokines and cytokines. This autologous source of bioactive compounds enables the substitution of synthetic GFs. The aim of this thesis is to develop a bioactive cell-free scaffold with a drug delivery system supporting the physiological healing of bone defects. The centrifugal spinning method was used to produce nanofibrous poly-ε-caprolactone (PCL) scaffolds. PCL scaffolds were functionalized with different platelet concentrations. Bioactive compounds released from activated platelets were trapped within the formed fibrin net, enabling their gradual release. Improved metabolic activity, proliferation and alkaline phosphatase activity of MG-63 cells and human mesenchymal stem cells (hMSCs) were detected. The release of compounds lasted for two weeks and nearly reached the plateau phase,...
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Coaxial nanofibers with incorporated suplements for regulated chondrogenic differentiation
Korbelová, Gabriela ; Rampichová, Michala (advisor) ; Vištejnová, Lucie (referee)
In the field of regenerative medicine, regeneration of cartilage defects (caused either by injury or age-related degeneration) has become a widely discussed topic. Nanofibrous scaffolds provide a suitable environment for cell adhesion, proliferation, differentiation, and also for the local involvement of bioactive substances. Nanofibrous scaffolds mimic the extracellular matrix (ECM) of hyaline cartilage. These scaffolds are seeded with autologous chondrocytes. After having been isolated from the patient, the cells must be cultivated in vitro in order to obtain a sufficient amount of chondrocytes. Scaffolds with cultivated chondrocytes are later implanted back into the pacient. Chondrocytes, however, when grown on a 2D tissue culture plastic rapidly de-differentiate and thus lose the ability to synthesize ECM molecules. The aim of the work was modulation of chondrogenic differentiation medium through finding the ideal concentration of chondrogenic supplements, composed of L-ascorbate-2-phosphate (A2P) and dexamethasone (DEX), in the culture of primary chondrocytes seeded on a nanofibrous polycaprolactone (PCL) scaffold. The effect of different concentrations of the chondrogenic supplements on chondrocyte adhesion to the scaffold and their proliferation and differentiation was studied. The influence...
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Coaxial nanofibers with incorporated supplements for regulated chondrogenic differentiation
Korbelová, Gabriela ; Rampichová, Michala (advisor) ; Vištejnová, Lucie (referee)
In the field of regenerative medicine, regeneration of cartilage defects (caused either by injury or age-related degeneration, such as osteoporosis) has become a widely discussed topic. Nanofibrous scaffolds provide a suitable environment for cell adhesion, proliferation, differentiation, and also local involvement of bioactive substances. Nanofibrous scaffolds mimic the extracellular matrix (ECM) of hyaline cartilage and thus have the potential to treat cartilage defects. The aim of the work was modulation of chondrogenic differentiation medium through finding the ideal concentration of chondrogenic supplements, composed of ascorbate-2- phosphate and dexamethasone, in the culture of primary chondrocytes of pig origin seeded on a nanofibrous polycaprolactone (PCL) scaffold. The effect of different concentrations of the chondrogenic supplements on chondrocyte adhesion to the scaffold and their proliferation and differentiation was studied. Firstly, the influence of each of the supplements alone in the medium was studied, followed by study of effects of their combinations. Then, the supplements were incorporated into the nanofibers and their effect upon their release from the nanofibers was investiaged. The supplements were studied in 21-day experiments. The chondrogenic re- differentiation was best...
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Growth factors and other bioactive substances for osteogenic differentiation of mesenchymal stem cells
Blahnová, Veronika ; Rampichová, Michala (advisor) ; Vandrovcová, Marta (referee)
The main function of mesenchymal stem cells in the body is to facilitate the restoration and regeneration of damaged tissues. They are known for the ability to differentiate into tissue originating from the mesoderm, which among others includes connective tissues. Due to this feature are MSCs being intensively examined. Different directions of differentiation can be induced by treatment of specific polypeptides, so called growth factors. In the field of tissue engineering are growth factors used to induce and accelerate the healing processes. They may be incorporated into the nanofiber carrier which is inserted into the site of injury. Cells in this area would thus be stimulated by surrounding 3D microenvironment. At the same time the scaffold provides a supply of growth factors which are able to affect metabolism, motility and differentiation of present cells. In order to induce osteogenic differentiation of human MSCs the following bioactive substances were used: TGF-β, bFGF, HGF, IGF-1, VEGF and the BMP-2 and the organic acid taurine. During 21 days lasting experiments, were these molecules added to the medium in various combinations and in the case of taurine also at two different concentrations. Cells were cultured on plastic. The best effect on cellular metabolism of MSCs, evaluated by MTS...
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Kompozitní pěnové nosiče pro tkáňové inženýrství kostí
Kuntová, Henrieta ; Rampichová, Michala (advisor) ; Krulová, Magdaléna (referee)
The goal of my work was to introduce the fast pacing field of tissue engineering with focus on bone regeneration. Tissue engineering could be a future alternative to the currently used conventional approaches that suffer from healing failures. Due to increasing demand for bone tissue replacement damaged by degenerative diseases or injuries, many laboratories have attempted to come up with solutions in a form of artificial constructs. In the present light of interest are composite scaffolds usually made of polymer and ceramic combinations. Their main advantage is that they combine elasticity and tensile strength of a polymer with bioactivity and mechanical hardness of a ceramic, while removing drawbacks of each material. Powered by TCPDF (www.tcpdf.org)
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Biomimetic modifications of titanium in bone tissue engineering.
Krýslová, Markéta ; Filová, Elena (advisor) ; Rampichová, Michala (referee)
When the big joints like a knee or hip joint are damaged, the solution of this problem is an artificial substitute. The replacement of damaged joints with endoprotesis helps to reduce the pain and to move normally. In the design of the implant is necessary to fulfil all requirements on the properties of the material. The surface of implant is important, because it is directly connected to bone tissue. After implantation, the negative effect include infection, inflammation or release of the implant due to limited osseointegration, may appear. The osseointegration can be improved by modifying the material surface. This thesis is focused on development and evaluation of advanced materials imitating the bone structure, especially nanoroughness and the presence of biomimetic component, such as hydroxyapatite. In this study is evaluated adhesion, proliferation, viability, differentiation, and synthesis of specific proteins of human osteoblasts like Saos-2 on titanium modified with nanotubes and plasma sprayed hydroxyapatite compared with smooth surfaces. Key words: titanium, nanotubes, osteoblasts, hydroxyapatite, nanoroughness
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