Using Cell and Organ Culture Models to Analyze Responses of Bone Cells to Mechanical Stimulation

Abstract Bone cells are exposed to dynamic mechanical stimulation that is transduced into cellular responses by mechanotransduction mechanisms. The extracellular matrix (ECM) provides a physical link between loading and bone cells, where mechanoreceptors, such as integrins, initiate mechanosensation. Though this relationship is well studied, the dynamic interplay between mechanosensation, mechanotransduction and cellular responses is unclear. A hybrid-multiscale model combining molecular, cellular and tissue interactions was developed to examine links between integrins’ mechanosensation and effects on mechanotransduction, ECM modulation and cell-ECM interaction. The model shows that altering integrin mechanosensitivity threshold (MT) increases mechanotransduction durations from hours to beyond 4 days, where bone formation starts. This is relevant to bone, where it is known that a brief stimulating period provides persistent influences for over 24 hours. Furthermore, the model forecasts that integrin heterogeneity, with respect to MT, would be able to induce sustained increase in pERK baseline > 15% beyond 4 days. This is analogous to the emergence of molecular mechanical memory signalling dynamics. Therefore, the model can provide a greater understanding of mechanical adaptation to differential mechanical responses at different times. Given reduction of bone sensitivity to mechanical stimulation with age, these findings may lead towards useful therapeutic targets for upregulation of bone mass.

Download Full-text

Organic anion and cation transporter expression and function during embryonic kidney development and in organ culture models

Kidney International ◽

10.1038/sj.ki.5000170 ◽

2006 ◽

Vol 69 (5) ◽

pp. 837-845 ◽

Cited By ~ 45

Author(s):

D.H. Sweet ◽

S.A. Eraly ◽

D.A. Vaughn ◽

K.T. Bush ◽

S.K. Nigam

Keyword(s):

Organ Culture ◽

Kidney Development ◽

Organic Anion ◽

Culture Models ◽

And Function ◽

Transporter Expression

Download Full-text

Effects of Acceleration Amplitude and Frequency of Mechanical Vibration on Cultured Osteoblasts

Volume 2: Biomedical and Biotechnology Engineering ◽

10.1115/imece2008-67221 ◽

2008 ◽

Author(s):

Tetsuo Shikata ◽

Toshihiko Shiraishi ◽

Shin Morishita ◽

Ryohei Takeuchi

Keyword(s):

Real Time ◽

Mechanical Stimulation ◽

Bone Cells ◽

Bone Matrix ◽

Mechanical Vibration ◽

Housekeeping Gene ◽

Rt Pcr ◽

Calcium Salts ◽

Acceleration Amplitude ◽

Gene Level

This paper describes the effects of the frequency and acceleration amplitude of mechanical vibration on osteoblasts, the bone cells that generate the bone matrix. Their cell proliferation and bone matrix generation were investigated when sinusoidal inertia force was applied to the cells. Bone formation is subject in vivo to mechanical stimulation. Although many researches for bone cells of osteoblastic lineage sensing and responding to mechanical stimulation have been reported mainly in the biochemical field, effects of mechanical stimulation on bone cells are not well understood. After the cells were cultured in culture plates in a CO2 incubator for one day and adhered on the cultured plane, vibrating groups of the culture plates were set on an aluminum plate attached to a exciter and cultured under sinusoidal excitation in another incubator separated from non-vibrating groups of the culture plates. Acceleration amplitude and frequency were set to several kinds of conditions. The time evolution of cell density was obtained by counting the number of cells with a hemocytometer. Calcium salts generated by the cells were observed by being stained with alizarin red S solution and their images were captured with a CCD camera. The vibrating groups for the cell proliferation and the calcium salts staining were sinusoidally excited for 24 hours a day during 28 days of culture. Gene expression of alkaline phosphatase (ALP) and runt-related gene 2 (Runx2) was measured by a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method. After the vibrating groups for the PCR were excited for 4 days, the total RNAs were extracted. After reverse transcription, real-time RT-PCR was performed. Gene expression for ALP, Runx2, and a housekeeping gene were determined simultaneously for each sample. ALP and Runx2 gene level in each sample was normalized to the measured housekeeping gene level. The following experimental results of sinusoidal excitation of osteoblasts have been shown: (a) Cell density decreased at 0.5 G with increasing frequency in the range from 12.5 to 1000 Hz and increased at 25 Hz with increasing acceleration amplitude from 0 to 0.5 G at 14 days of culture. (b) No calcium salts were observed in the non-vibrating group and the areas of calcium salts observed in the 0.5 G vibration group were larger than those in the 0.25 G group at 25 Hz at 21 days of culture. (c) The mRNA level of ALP at 0.5 G showed the peak at 50 Hz in the range from 12.5 to 1000 Hz and that at 50 Hz showed the peak at 0.5 G in the range from 0.25 to 1 G at 4 days of culture. In the case of Runx2, the same tendency was found. It has been shown that it is important to consider mechanical vibration as well as biochemical aspects in studies of the functional adaptation of cells to mechanical stimulation.

Download Full-text

Viscoelastic Properties of a Cultured Osteoblast Under Adhesive Condition

Volume 1: 21st Biennial Conference on Mechanical Vibration and Noise, Parts A, B, and C ◽

10.1115/detc2007-34944 ◽

2007 ◽

Author(s):

Takafumi Onishi ◽

Toshihiko Shiraishi ◽

Shin Morishita ◽

Ryohei Takeuchi

Keyword(s):

Cell Surface ◽

Bone Formation ◽

Bone Mass ◽

Mechanical Stimulation ◽

Viscoelastic Properties ◽

Bone Cells ◽

Glass Plate ◽

Petri Dish ◽

Piezo Actuator ◽

Mechanical Dynamics

Bone cells are adaptive to surrounding mechanical conditions. Osteoblasts, one of bone cells, have been reported to be sensible to mechanical stimulation and change the generated bone mass. Viscoelastic properties of such cells are predicted to be related to this phenomenon in the view of mechanical dynamics. In order to find the effective stimulation on the bone formation, it is necessary to understand the viscoelastic properties of the cells. Especially in the case of bone cells, it is important to consider their adhesive condition because they attach on surfaces of bone matrices. In this study, we measured tensile, static and dynamic viscoelastic properties of a cultured osteoblast, MC3T3-E1, under adhesive condition. Using these experimental results, we derived a model for viscoelasticity of the cell and identified the value of each element in this model. The cells were seeded on a glass plate in a petri dish. After the cells were cultured for one day and adhered on the glass plate, it was vertically raised and fixed on a piezo actuator. The center of the cell surface was aspirated with an L-shaped micropipette to be held. The glass plate was moved with manual, an electric motor or the piezo actuator according to the types of tests. The load applied to the cell was obtained by measuring the deflection of the micropipette whose spring constant was calibrated after each test. Deflection of the micropipette and elongation of the cell were measured by captured image during the test. As a result, the viscoelasticity of the cells was measured and modeled, and the value of each element in this model was identified.

Download Full-text