Osteoblast responses one hour after load-induced fluid flow in a three-dimensional porous matrix.
When bone is loaded, substrate strain is generated by the external force and this strain induces fluid flow that creates fluid shear stress on bone cells. Our current understanding of load-driven gene regulation of osteoblasts is based primarily on in vitro studies on planer two-dimensional tissue culture substrates. However, differences between a flat layer of cells and cells in 3-dimensional (3D) ECM are being recognized for signal transduction. Proliferation and differentiation of osteoblasts are affected by substrate geometry. Here we developed a novel 3D culture system that would mimic physiologically relevant substrate strain as well as strain-induced fluid flow in a 3D porous collagen matrix. The system allowed us to evaluate the responses of osteoblasts in a 3D stress-strain environment similar to a mechanical field to which bone is exposed. Using MC3T3-E1 osteoblasts grown in the 3D collagen matrix with and without hydroxyapatite deposition, we tested the role of strain and the strain-induced fluid flow in the expression of the load-responsive genes such as c-fos, egr1, cox2, osteopontin, and mmp1B involved in transcriptional regulation, osteogenesis, and rearrangement of ECM. Strain-induced fluid flow was visualized with a microspheres approximately 3 microm in diameter in real time, and three viscoelastic parameters were determined. The results obtained by semi-quantitative PCR, immunoblot assay, enzymatic activity assays for collagenase and gelatinase, and mechanical characterization of collagen matrices supported the dominant role of strain-induced fluid flow in expression of the selected genes one hour after the mechanical treatment.
Tanaka
Shigeo M
SM
Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, Japan.
Sun
Hui B
HB
Roeder
Ryan K
RK
Burr
David B
DB
Turner
Charles H
CH
Yokota
Hiroki
H
D016475:3T3 Cells; D000818:Animals; D003094:Collagen / Q000378:metabolism; D017886:Durapatite / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D015870:Gene Expression; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013314:Stress, Mechanical; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D016474:Weight-Bearing
D012333:RNA, Messenger; D014157:Transcription Factors; D003094:Collagen; D017886:Durapatite