Methods : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
For constant and fully developed laminar flow between the two parallel plates, the magnitude of the wall shear stress (τ) in between was calculated by formula 1:
in which η is the dynamic fluid viscosity (dyn/cm2), r is the radius of the plate (cm), ω stands for angular velocity and H for height (vertical distance in between the two plates).
To get information whether the flow is laminar or turbulent, Reynolds numbers (Re) were calculated for all flow regimes using formula 2 [25]:
in which ρ is the liquids’ density, η is its dynamic viscosity, v stands for average angular velocity and A is the characteristic area within which liquids flow (vertical distance between the two plates). Re values of >1500 are commonly considered illustrative of turbulent flow as well as values <1500 create laminar flows.
Numerous computerised simulations were performed to verify flow characteristics occurring within the plate/plate flow chamber assisted by the Department of Hydraulic Machines, Faculty of Mechanical Engineering, Technical University of Munich, Germany. For simulation of flow profiles to assess a potential cellular impact by fluid shear stress inside the chamber, graphical illustrations were created by using Ansys CFX® software (Ansys Germany GmbH, Otterfing, Germany).
The experimental process involved three steps. First, a count of n = 50.000 commercially available osteoblasts (PromoCell, Heidelberg, Germany) per millilitre of culture medium were cultured on the bottom of the cell-bearing surface (glass panel). Therefore, cells were seeded in a culture medium (cf. Appendix 2 for a detailed composition) at 37 °C. Prior to the test procedure, the cells were manually removed from the culture bottles’ bottom by gentle movements while adding 5 ml of Trypsin followed by 10 min of incubation. Finally, Trypsin residues were removed with, first, centrifugation (1600 rpm/5 min) of the cell fluid (culture medium with additives and loose osteoblast cells) and, second, by subsequently adding 10 ml of culture medium. After 24 h of incubation, cells showed adherent to the glass panel. A conventional petri dish was filled to 70% of its capacity with cell fluid (culture medium and additives (Appendix 2). The petri dishs' bottom formed the lower plate and the round glass panel the upper plate placed within the culture medium. Directly after, the circulation process for FSS induction was initiated. In brief, after 24 h of incubation at 37 °C and 5% CO2 concentration, cells adhered to under side of the glass panel and the glass panel was incorporated into the device as described above. The circulation process (speed level = 200 rpm) started for 24 h under sterile incubation conditions (37 °C and 5% of CO2). Via repeated computational simulations, a rotational speed level of 200 rpm was found as adequate to provide 10 dyn/cm2 of shear force at the plates’ peripheral region. Lastly, light microscopic examination was conducted (Leica DC480®, Leica Microsystems, Wetzlar, Germany) to verify the cell orientation (after 24 h with and without rotation), followed by phallacidin fluorescence staining according to the manufacturer’s protocol (Appendix 3) (BODYPY® FL Phallacidin, ThermoFisher Scientific, MA, USA) and fluorescence microscopy with Leica/Leitz DM RBE® (Leica Microsystems, Wetzlar, Germany). The cell body and its longitudinal actin fibre orientation was put in relation to the total-force-vector (Fig. 5) (resulted from the flow velocity-vector and the centrifugal force-vector) which was calculated by formula 3. For differentiation into an oriented and non-oriented cell formation, an angle of 90° was set as threshold value.
Serial posts:
- Background : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [1]
- Background : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
- Methods : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [1]
- Methods : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
- Results : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [1]
- Results : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
- Discussion : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [1]
- Discussion : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
- Discussion : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [3]
- Conclusions : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- Abbreviations : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- References : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [1]
- References : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
- References : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [3]
- References : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [4]
- Acknowledgements : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- Author information : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [1]
- Author information : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up [2]
- Rights and permissions : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- About this article : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- Table 1 Listing of the single components of the flow chamber together with manufacturers’ data : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- Table 2 Listing of the culture media and additives together with manufacturers’ data : Cellular fluid shear stress on implant surfaces—establishment of a novel experimental set up
- Fig. 1. Three-dimensional illustration (a–e) and photography (f) of the experimental setup with the components marked numerical. a1 Lower petri dish (s’ bottom serving as the lower plate); 2 Rotating glass panel [60 mm diameter (cell bearing)]; 3 Titanium axis. b4 Liquid medium (red). c5 Reversed upper petri dish. d6 Gearwheel with set screw. e7 Closing; 8 Electronic motor device and adjusting ring with additional set screw : Cellular fluid shear stress on implant
- Fig. 2. Side view of a computerized simulation, showing the flow chambers’ lower compartment and the flow profile in between the two plates; shearing gap and bottom plate are shown on the left side; rotation speed = 200 rpm; colour code bar (left edge) showing shear force values [Pa] [1 Pa = 10 dyn/cm2]; flow direction presented by arrows : Cellular fluid shear stress on implant
- Fig. 3. Diagram for visualisation of the calculation of shear stress rates taking into account the centrifugal force and the glass plates’ dimensions. For example, at a distance of 25 mm from the centre of the upper plate, the shear forces’ value is 8.33 dyn/cm2, together with an additional centrifugal force that has a value of 0.55 dyn/cm2 : Cellular fluid shear stress on implant
- Fig. 4. Randomly orientated osteoblasts without influence of rotation (phallacidin fluorescence staining). On the left side with 200× and on the right side with 400× magnification. The white X on the coloured circle marks the location upon the plate where the osteoblasts were located. The red X marks the centre of the plate : Cellular fluid shear stress on implant
- Fig. 5. Osteoblasts with an orientation tendency after 24 h of rotation (phallacidin fluorescence staining). On the left side with 200× and on the right side with 400× magnification. The yellow arrows show the orientation of the cells. The red arched arrow within the coloured circle shows the direction of rotation. The dashed white line oriented to the right stands for the resulting centrifugal force. The dashed white line pointing upwards shows the direction of the resulting flow resistance. The solid white arrow stands for the vectorial sum of the abovementioned forces : Cellular fluid shear stress on implant