The Synergistic Effect of Bone Graft Substitute Architecture and Mechanical Environment on HMSCs Responses in Vitro

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.

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Year

Entry

1993

Lanyon LE. Osteocytes, strain detection, bone modeling and remodeling. Calcif Tiss Int. February 1993;53(suppl 1):S102-S107.

2006

Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, Deans RJ, Keating A, Prockop DJ, Horwitz EM. Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement. Cytotherapy. August 2006;8(4):315-317.

2003

Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a post‐natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. April 2003;88(5):873-884.

2006

Ingber DE. Cellular mechanotransduction: putting all the pieces together again. FASEB J. May 2006;20(7):811-827.

2006

Robinson JA, Chatterjee-Kishore M, Yaworsky PJ, Cullen DM, Zhao W, Li C, Kharode Y, Sauter L, Babij P, Brown EL, Hill AA, Akhter MP, Johnson ML, Recker RR, Komm BS, Bex FJ. Wnt/β-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem. October 20, 2006;281(42):31720-31728.