Effects of Silica Based Biomaterials on Bone Marrow Derived Cells - Material Aspects of Bone Regeneration

Silica based biomaterials, such as melt-derived bioactive glasses and sol-gel glasses, have been used for a long time in bone healing applications because of their ability to form hydroxyapatite and to stimulate stem cell proliferation and differentiation. In this study, bone marrow derived cells were cultured with bioactive glass and sol-gel silica, and seeded into porous polymer composite scaffolds that were then implanted femorally and subcutaneously in rats to monitor their migration inside host tissue. Bone marrow derived cells were also injected intraperitoneally. Transplanted cells migrated to various tissues inside the host, including the lung, liver spleen, thymus and bone marrow. The method of transplantation affected the time frame of cell migration, with intraperitoneal injection being the fastest and femoral implantation the slowest, but not the target tissues of migration. Transplanted donor cells had a limited lifetime in the host and were later eliminated from all tested tissues. Bioactive glass, however, affected the implanted cells negatively. When it was present in the scaffold no donor cells were found in any of the tested host tissues. Bioactive glass S53P4 was found to support both osteoblastic and osteoclastic phenotype of bone marrow derived cells, but it was resistant to the resorbing effect of osteoclastic bone marrow derived cells, showing that bioactive glass is rather dissolved through physicochemical reactions than resorbed by cells. Fast-dissolving silica sol gel in microparticulate form was found to increase collagen formation by bone marrow derived cells, while slow dissolving silica microparticles enhanced their proliferation, suggesting that the dissolution rate of silica controls the response of bone marrow derived cells.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.