|dc.description.abstract||Introduction: Adipose-derived stem cells (ADSC) have been investigated as a potential therapy for de novo bone formation around titanium devices. However, the osteogenic behaviour of ADSC grown in a clinical grade serum-free medium used for de novo bone formation in the presence of titanium devices is unknown. Moreover, the impact of the titanium surface on these cells remains elusive. Human ADSC (hADSC) and autologous ovine ADSC (oADSC) were cultured in serum-free conditions and used to evaluate in vitro and in vivo their de novo bone formation capacity on titanium discs with different roughness.
Hypothesis and research questions: This thesis presented the hypothesis that ADSC grown in serum-free conditions would promote de novo bone formation in the presence of titanium devices. Moreover, that titanium with micro-rough surfaces would further improve the de novo formation of bone. To address this hypothesis two main questions were formulated: 1. Do ADSC grown in serum-free conditions have the potential to regenerate bone in the presence titanium devices? 2. Does surface topography/roughness of titanium devices influence the amount of bone produced by serum-free grown ADSC?
Methods: A number of techniques were employed to answer the proposed questions. The machined (MTi) and alumina-blasted (ABTi) titanium surfaces were characterised using scanning electron microscopy, energy-dispersive x-ray spectroscopy, and contact angle analysis (goniometer). The human and ovine ADSC were cultured using serum-free and osteogenic conditions. The in vitro growth and osteogenic behaviour of these cells were analysed using cell proliferation and extracellular matrix mineralisation. The hADSC were further analysed in vitro using immunofluorescence for protein production and relative gene expression for genes associated to cell attachment (ITGA1, ITGA5, ITGAV, ITGB1, ITGB3), pluripotency and self-renewal (OCT-4 and TERT), and osteogenesis (MSX2, RUNX2, and BGLAP). The osteogenic capacity of autologous oADSC was further evaluated in vivo using a femur epicondyle de novo bone formation model containing titanium devices. This model included the in vivo tracking of oADSC using PKH26 cell tracking dye and the de novo bone formation in the defect sites using histology and histomorphometry.
Results: hOS-ADSC (osteogenically differentiated hADSC) proliferated less than hADSC and also produced more mineralised matrix. The immunofluorescence assays showed that hADSC cultured in serum-free and osteogenic conditions produced Runx-2 on titanium surfaces with different roughness, while only hOS-ADSC produced osteocalcin as shown by immunofluorescence. The gene expression analysis of hADSC demonstrated the stable relative gene expression of RUNX2, MSX2, and BGLAP over time. ITGA1, ITGB5, and ITGB1 were all highly expressed at day 0 in hADSC. However, only the expression of ITGAV and ITGB3 tended to increase over time in hADSC. In contrast, only hOS-ADSC showed an increased expression of ITGA1 over time. oADSC cultured in serum-free conditions showed variability in their in vitro differentiation potential with cells from only five out of seven animals differentiated into osteoblast-like cells. There was no statistically significant difference on the in vitro proliferation and mineralisation matrix deposition between oADSC and osteogenically induced oADSC (oOS-ADSC) regardless of surface type. The in vivo femoral epicondyle model showed that oADSC labelled with PKH26 remained at the defect site after one month of healing. However, there was no difference in de novo bone formation between the bone defects treated with oADSC and the ones with only blood clot after one month.
Conclusions: In summary, the culture condition had more effect on the osteogenic behaviour of hADSC than the titanium surface roughness. Also, the use in vivo of autologous oADSC did not improve the de novo bone formation in defects containing titanium discs with different surfaces after a period of 1 month.||