The Synergistic Effect of Bone Graft Substitute Architecture and Mechanical Environment on hMSCs Responses in vitro

Abstract

Porous silicate substituted hydroxyapatite (SiHA) as synthetic bone graft substitute (BGS) shows excellent bone repair in vivo. It is accepted that the mechanical environment to which cells are exposed regulates cellular differentiation. One mechanism by which BGS architecture may regulate bone formation could be through influencing the shear stress distribution of interstitial fluid. The aim of this study was to investigate the combined influence of BGS architecture and fluid shear environment on human mesenchymal stem cells (hMSCs) responses. hMSCs were cultured on SiHA BGS with defined porosity. A 3D in-house perfusion bioreactor system was established, and two shear stress profiles were applied in this study: 1) continuous basal perfusion rate (BPR) at 0.07 ml/min; 2) BPR with a period of high perfusion rate (pHPR) every day at 2.5 ml/min. The cytoskeleton of hMSCs was reorganized under perfusion conditions compared with under static condition. Shear stress induced both ERK1/2 and pEKR1/2 translocation from the cytoplasm to nucleus. hMSCs cultured in BPR profile differentiated towards osteogenic lineage, while pHPR induced hMSCs to differentiate towards chondrogenic lineage. Gene expression of osx, sox9, runx2 and col ii was not dependent on BGS micro-porosity under static condition. However, the expression of osteogenic transcription factor osx increased significantly with increasing BGS micro-porosity under BPR condition, whereas the expression of chondrogenic markers like sox9, runx2 and col ii decreased with increasing BGS micro-porosity under pHPR condition after 3 days. Nifedipine was used to block L-type voltage-sensitive Ca2+ channel (VSCC) activity. The translocation of ERK1/2 and pEKR1/2 from the cytoplasm to nucleus was found to be dependent on L-type VSCCs. Both BPR induced osteogenic differentiation and pHPR induced chondrogenic differentiation were found to be modulated by L-type VSCCs. The findings of this PhD thesis demonstrate that the future evaluation of porous BGS bioactivity should be conducted under carefully selected perfusion conditions, and the results of this thesis suggest that chondrogenic markers should also be used as one of the indicators for BGS performance in addition to conventional osteogenic markers, as early chondrogenic activity may denote the onset of osteochondral bone formation. This would also argue for longer term culture to further monitor cell fate and the development of any extracellular matrix (ECM) produced.