INTRODUCTION: The shear strength of attachment of the osteosarcoma cell line, MG63, has been investigated using a tailor made convergent flow chamber. The decreasing cross-sectional area for flow, incorporated in the design of the chamber, increases the linear velocity and thus the shear stress applied to cells in the chamber. Furukawa et al1. have altered a rheometer to allow shear to be applied to cells, and measured. However, their approach only allows one shear stress to be applied per experiment. In the current study the flow chamber geometry allows a range of shear stresses to be tested simultaneously. The aim of the study was therefore to measure the adhesion strength of an MG63 cell line in response to the application of two different volumetric flow rates. This work will contribute to the development of an experimental system and computational fluid dynamics (CFD) model of a fluidised bed bioreactor for the proliferation of MG63s in a porous hydroxyapatite scaffold. METHODS: MG63 cells were seeded at 20,000 cells/cm2 on to the tissue culture plastic base of the chamber and allowed to attach under static conditions. After 24 hours, media was pumped through the chamber at 80 ml/min for ten minutes. This first flow removed any unattached cells as well as applying shear stress to the attached cells. The media flowrate was then increased to 150 ml/min for a further ten minutes. To quantify the attached cells, images were recorded after each of the two flow regimes using an inverted microscope at 10x magnification. Images were taken at 5mm intervals along the length of the chamber; ten positions were assessed with three micrographs recorded at each position. RESULTS: The variation of cell number with shear stress for the two different flow rates of 80 ml/min and 150 ml/min is shown in Figure 1. At 80 ml/min the cell number decreased exponentially from a shear stress of 30 to 120 mPa. At 150 ml/min there was negligible variation in cell number between a shear stress of 60 and 230 mPa, once the standard deviation is considered. However the data from 140 mPa onwards suggest a continuation of the exponential decrease in cell number. Where the two sets of data overlap, in the range 60 - 125 mPa, the second, higher, flowrate resulted in a greater removal of cells at those shear stresses. Fig 1: Cell number post shearing at two volumetric flowrates. Error bars are ± 1 s.d., n = 3. DISCUSSION & CONCLUSIONS: Initially, at 80 ml/min, there is a graduation of cell detachment in the range 25 – 125 mPa with the number attached at 100 mPa only 30% of the number seen at 40 mPa. At 150 ml/min the number of cells remaining is similar across the range of shear stresses. The over-lapping data occurred at the downstream end of the chamber at 80 ml/min and upstream at 150 ml/min. Cell number at a given shear stress was expected to be comparable, so the differences suggest also time-dependency, or entrance effects in the chamber. An innovative convergent flow chamber has been developed which allows a range of shear stresses to be applied simultaneously to a cell population, allowing investigation of attachment and detachment kinetics. These results show that there was not a critical shear stress which resulted in the loss of all cells, in the range tested. The studies also suggest there is both shear rate and time dependency, and possible entrance effects. The next steps are to examine the rate- and time- dependencies of cell removal experimentally and using FLUENT. The experiments will then be repeated on a hydroxyapatite surface. REFERENCES: 1 K.S. Furukawa et al (2003) Int. J. Artif. Organs 26: 436-441. ACKNOWLEDGEMENTS: This work was funded by EPSRC Grant EP/I015922/1.</p
