Browsing by Author "Kleinhans, Claudia"

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Evaluation of human bone and fat derived stem cells for their application in bone tissue engineering
(2014) Kleinhans, Claudia; Tovar, Günter (apl. Prof. Dr.)
Bone tissue engineering, an emerging interdisciplinary research field, sets its focus on the development of biological substitutes to regenerate, maintain, or improve bone tissue function. The improvement and examination of biomaterials in combination with cellular components is a major aim of recent research activities. Essential factors that should be considered are the availability of a suitable matrix, a sufficient amount of cells and culture conditions that allow the nourishment of three-dimensional (3D) artificial tissue equivalents. The first part of this thesis focuses on the evaluation of chemically modified surfaces, created by low-pressure plasma discharge and the effect of these modifications on the morphological and physiological behavior of human mesenchymal stem cells derived from bone marrow (hMSCs) and adipose tissue (hASCs). Polystyrene substrates modified with ammonia- (NH3), carbon dioxide- (CO2), and acrylic acid-plasma (Acc) revealed significant changes in their chemical surface composition and changes in surface wettability. In order to identify the most suitable surface functionalization for each cell type, the adhesion kinetics and proliferation rates were investigated which revealed a significantly higher cell number of adherent hMSCs in comparison to tissue culture polystyrene (TCPS) on aminated surfaces. Consistent with an initial accelerated cell adhesion, a significantly increased enzymatic activity was detected for hMSCs. The faster adhesion process of hMSCs on aminated samples in comparison to the other modified surfaces was associated with an outspread phenotype. A similar adhesion and proliferation tendency was observed for hASCs, however, only a minor preference to the aminated surfaces was detected. The NH3-plasma treatment was applied to modify a common used biomaterial, Poly (lactic acid) (PLA) blended with hydroxyapatite (HA). The modification of the atomic composition was associated with an increased wettability, promoted adhesion of hMSCs and hASCs, which resulted in significant higher cell numbers on the modified surfaces. In addition to human mesenchymal stem cells derived from bone marrow, another promising cell source presents adipose tissue which is beneficial due to the minimal invasive operative procedure for the patient. Within the scope of this thesis, the evaluation of primary human adipose derived stem cells with regard to their differentiation capacity was investigated, particularly for their osteogenic differentiation. Cell fractions, obtained by gradient centrifugation after enzymatic digestion of lipoaspirate, and an enriched cell fraction separated against the surface antigen CD271, were isolated and characterized. These cell fractions were compared to hMSCs and skin dermal cells (hSDCs). hMSCs demonstrated the potential to differentiate into adipocytes, osteoblasts and chondrocytes. Whereas hASCs and hASCs(CD271+), showing a high differentiation capacity towards adipocytes, fostered an osteoblastic phenotype, but lacked the capacity to form hyaline cartilage. The comparison of hMSCs and hASCs differentiation within a 3D environment was conducted by seeding and culturing the cells in porous, amino functionalized PLA-HA composite scaffolds. The expression of osteogenic related marker genes and the formation of a mineralized extracellular matrix were verified, whereas hASCs reveal a similar expression pattern of osteogenic related marker genes in comparison to hMSCs. hASCs exhibited an osteoblastic phenotype by synthesizing and depositing a mineralized matrix. Finally, to enable the culture of larger 3D-tissue-substitutes, the implementation of a perfusion bio-reactor system was used to achieve and maintain sufficient cell nourishment in the core regions of a scaffold. An increased flow rate was associated with an enhanced seeding outcome within the beta-tricalcium phosphate scaffold, but also accompanied by an accumulation of lactate dehydrogenase in the culture media. For subsequent dynamic culture conditions, a continuous flow was adjusted, which led to the induction of stress related genes analyzed on day one and seven. Nevertheless, after a culture period of seven days, similar gene expression levels of the apoptosis marker FAS were measured in static and dynamic settings. Furthermore, viable cells were detected within the scaffold and cell exposition to shear stress resulted in the induction of osteogenic marker genes. Even without osteogenic supplementation higher mRNA level were detected in the dynamic culture in comparison to the static culture set-up. The results presented in this thesis provide fundamental knowledge and the establishment of parameters for the culture of human mesenchymal stem cells isolated from bone or fat tissue concerning surface modification, differentiation capacity and perfusion culture for their application in bone tissue engineering.