Modulation of growth and differentiation of mesenchymal cells for cartilage and bone tissue engineering

Tissue engineering is a highly promising technology for the treatment of challenging cartilage and bone lesions for which no adequate therapeutic options are available yet. However for their widespread use, engineered tissues will first have to prove a predictable clinical success. To reach this objective, a reproducibly high product quality will be required, which can be achieved by a better knowledge and a continuous control of the cell phenotype during all phases of the tissue engineering process. The aim of this thesis is therefore to demonstrate how two types of mesenchymal cells, chondrocytes and bone marrow-derived mesenchymal/stromal cells (BMSC), can be modulated during growth and differentiation in order to conserve and fully exploit their potential. The present work is divided into 4 chapters. Chapter I will reveal how different chondrocyte subpopulations change their phenotype during in vitro proliferation and how this can lead to the detection of cells with increased differentiation capacity. In chapter II, the parameters governing the maintenance of osteogenic potential of BMSC during expansion will be analyzed. Chapter III will demonstrate how the coculture of BMSC with macrophages can result in a better cartilage-forming capacity. Finally in chapter IV, the effect of ascorbic acid on chondrogenic differentiation will be established. Chapter I: The De-differentiation of Human Chondrocytes is Linked to Individual Cell Divisions The relationship between proliferation and de-differentiation of chondrocytes during in vitro culture remains poorly understood. It was hypothesized here that cell proliferation tracking could reveal differences in the progression of de-differentiation and chondrogenic potential among subpopulations proliferating at different rates. Results showed that changes in the expression of cell surface markers and extracellular matrix genes were linked to individual cell divisions. Different culture conditions influenced cell doubling rates but not the relationship between cell divisions and phenotypic alterations, which indicated a strong coupling between both phenomena. Interestingly the highest chondrogenic potential was measured for slowly growing chondrocytes, even after a same number of total doublings was reached for all subpopulations. The increased understanding of the link between proliferation, de-differentiation and re-differentiation capacity will lead to innovative ways to maintain chondrogenic differentiation potential during chondrocyte expansion. It will also facilitate the identification of progenitor populations with intrinsically superior capacity for the generation of enhanced engineered cartilage grafts. Chapter II: Perfused 3D Scaffolds and Hydroxyapatite Substrate Maintain the Osteogenic Potential of Human Bone Marrow-Derived Mesenchymal Stromal Cells during Expansion In previous studies it was repeatedly shown that the expansion of bone marrow-derived mesenchymal/stromal cells (BMSC) on 3D ceramic scaffolds resulted in increased maintenance of osteogenic potential as compared to culture on 2D polystyrene (PS). Since several culture parameters completely differ between 3D ceramic and 2D PS culture, the individual influences of the 3D scaffold and the ceramic material, as well as of the extracellular matrix deposition were investigated here. Results revealed that BMSC expanded on 2D PS only yielded bone matrix if the culture time was not longer than 2 weeks. Cells cultured for 3 weeks on both 3D PS and 3D ceramic scaffolds produced a dense bone matrix. The number of explants containing bone was higher with cells expanded on 3D ceramic compared to 3D PS. However there were no significant differences between cells extracted from 3D ceramic and directly implanted constructs. These findings suggest that the bone-forming capacity of BMSC can be maintained by a 3D environment and further improved by a ceramic substrate material, but that a preexisting 3D niche is not required for bone formation. The preservation of BMSC with osteogenic potential during 3D expansion in bioreactors opens the perspective for a streamlined production of large-scale bone grafts for clinical use. Chapter III: Anti-Inflammatory/Tissue Repair Macrophages Enhance the Cartilage-Forming Capacity of Human Bone Marrow-Derived Mesenchymal Stromal Cells Macrophages play a key role in healing processes, by regulating inflammation and stimulating tissue repair. However their influence on the tissue formation potential of BMSC is unknown. The effect of the coculture of macrophages with either pro-inflammatory or tissue-remodeling traits on the chondrogenic differentiation capacity of BMSC was therefore tested here. Results showed that the coculture of BMSC with tissue-repair but not with pro-inflammatory macrophages resulted in significantly higher glycosaminoglycan content and type II collagen expression, while type X collagen expression was unaffected. This chondro-inductive effect was found to be caused by an increased survival and higher clonogenic and chondrogenic capacity of BMSC that were cocultured with tissue-repair macrophages. No difference was detected however in the cartilage tissue maturation in nude mice, as evidenced by similar accumulation of type X collagen and calcified tissue. These results demonstrated that a coculture with tissue-repair macrophages can improve the chondrogenic differentiation capacity of BMSC. This increased knowledge can lead to new coculture strategies for the manufacturing of cartilage grafts with enhanced quality. Chapter IV: Chondrogenic Differentiation and Collagen Synthesis of Human Chondrocytes in the Absence of Ascorbic Acid Ascorbic acid is considered to be an important supplement for cartilage tissue engineering because of its role in collagen hydroxylation in vivo. Due to its instability, ascorbic acid requires specific liquid handling conditions, which poses significant challenges to the automation of cartilage graft manufacturing. The aim of this study was to investigate the effect of ascorbic acid on chondrogenesis in vitro, with special regard to collagen synthesis and hydroxylation. Results showed that cartilage gene expression, tissue formation, and production of glycosaminoglycans were indistinguishable whether chondrocyte micromass pellets were cultured with or without ascorbic acid. Not adding ascorbic acid caused a reduction of collagen deposition, but collagen hydroxylation was not significantly different. Collagen secretion was unaffected and collagens showed a similar fibril structure in the absence of ascorbic acid. In conclusion, ascorbic acid did not influence chondrogenesis except for a small effect on collagen quantity, and can thus be omitted to simplify automation for a more cost-efficient cartilage graft manufacturing. Conclusion: In this work, four different approaches to modulate the growth and differentiation of chondrocytes and BMSC were presented. With the gained knowledge the cell phenotype can be better controlled during manufacturing processes, which will be required for the production of engineered tissue grafts with reproducibly high quality for clinical translation