Background
Cells can be influenced by different mechanostimuli, which lead to an activation of cellular and inter-cellular responses. These reactions may be caused by either a direct stimulation of the cell body (mechanoreception) or indirect cellular stimulation (response) [
1‐
3]. Extracellular fluid movement induces fluid shear stress (FSS) that can result in different cellular processes including proliferation, migration and gene expression [
4].
There are two different ways of cell stimulation by FSS, where both lead to extracellular signalling. First, fluid-induced cell stimulation occurs when the cell surface is in direct contact with the moving extracellular fluid as seen in the vascular endothelium. Second, it has been hypothesised that indirect stimulation occurs via fluid flow through the lacunar network as seen in bones such as close to loaded dental endosseous implants [
1‐
3]. This extracellular cell stimulation leads to an altered cell morphology as well as altered intracellular signal cascades such as changed gene and protein expression pattern [
4‐
7]. A reorganisation of actin fibres in accordance with the flow direction could be observed as well [
8].
To prove the theory of a FSS-triggered effect on different cell lines, several in vitro investigations using different flow chambers were conducted [
5,
9‐
12]. In osteoblasts, biochemical responses on FSS in form of an increased intracellular calcium production [
13‐
15] and an increased release of prostaglandins were reported [
15‐
19]. FSS stimulation of osteoblasts also improved the cell adhesion by enhancing the affinity of intracellular integrins to extracellular matrix ligands as well as to biomaterial surfaces [
20,
21]. Shear forces’ triggered effects on osteoblasts could be detected at a value of 10 dyn/cm
2, which almost reflects the in vivo situation [
4,
22,
23]. Todays’ frequently used flow chambers mainly simulate the in vivo formed shear forces. However, it is difficult to ensure the required reproducibility and linear flow conditions. The most distinctive feature of currently used flow chambers is a liquid flow along rigidly fixed cell-bearing surfaces. Some of the above mentioned flow devices are either operating with a constant flow velocity or using pulsating flow profiles, which should be applied in case of analysing blood flow characteristics [
9,
24]. Computerised investigations of flow chambers by Anderson et al. [
4] have shown that deviating shear forces occur in the same flow chamber after repeating the same experiment twice. Consequently, different results of stimulation and cells response are obtained. Another downside of reported flow chambers is the inability to simultaneously set different shear forces in a single experiment.
Therefore, the aim of the present study was to establish a new cell chamber model for FSS simulation and stimulation. In addition to its ease of use, the reported model in this study should meet the requirements of a simple design, generating reproducible flow characteristics next to laminar flows and clearly defined flow gradients on implant surfaces.
Discussion
The aim of this study was to establish a new FSS model that is easy to use as well as simple to assemble in order to create reproducible fluid shear forces on cells close to implant material surfaces. Todays’ commonly used commercial flow devices differ in geometry and function, which makes comparisons between experiments difficult [
4,
10,
26,
27]. The benefits of this novel testing device are reproducible laminar flows under controlled conditions (regulated temperature as well as steady partial pressure of CO
2). Due to its reproducibility, the stimulation of osteoblast cells by shear forces becomes assessable.
In this FSS chamber, osteoblasts were cultured on the bottom of a rotating round glass panel that moves within a resting liquid. Computerised simulations determined a value of 200 rpm as the optimal system configuration in which a constant laminar flow occurs without pulsatile character. When creating laminar flows, induction of turbulences at boundary surfaces results in flow instability. To reduce this negative effect occurring in frequently used stationary devices, cells were cultured on a carrier plate, which is placed within the lower petri dish. In this context, the direct contact between the carrier plate and another interface was omitted. Laminar flows were chosen to achieve a good reproducibility. This required a flow profile that is characterised by parallel moving liquid layers [
26] that are present in the area in between the upper and lower plate. To define the most favourable position of the cell-bearing surface, computerised simulations were performed. Herein, it could be demonstrated that rising shear forces along the plate surfaces’ (0–2 dyn/cm
2) are too low for osteoblast test cell stimulation, which occurs at about 10 dyn/cm
2 [
28,
29]. The bottom of the glass plate generated enough shear forces (10 dyn/cm
2 in the periphery) to meet the requirements of an osteoblast-stimulating laminar flow chamber. Further on, the simulations indicated that the flow profile in between the two plates was not influenced by peripheral turbulences alongside the peripheral regions. To verify a cellular realignment towards the shear direction, cells were microscopically examined prior and after exposure to shear forces for 24 h upon a spinning disc at a speed level of 200 rpm. Even if not sufficiently meaningful alone, observing changes in osteoblast cell morphology are still appropriate methods to verify the good usability of a flow chamber for the generation of reproducible FSS. Although not statistically significant, a tendency of cellular realignment towards the liquid flow direction was demonstrated. Similarly, several studies have revealed characteristic changes of osteoblast morphology triggered by fluid shear stress, which depends on exposure time and strength [
22,
30]. Likewise to our findings, these changes are characterised by the formation of actin stress fibres, which in turn align towards the longitudinal cell axis and mainly appear near the nucleus [
8,
31]. However, the manual approach of analysing the actin fibres’ orientation has to be stated as a drawback of the present study, since it does not meet the requirements of a valid measurement. Immunofluorescence microscopy is largely a qualitative, or semiquantitative, approach with a limited capability of precise fibre differentiation and/or quantification since standard binary thresholds failed to exhaustively segment all fibres because of their wide variations in intensity and background levels [
32]. Hence, more objective measurements could be provided by the use of an automated software-assisted processing. In this context, the FibreScore Algorithm by Lichtenstein et al. presents a potential solution for quantification, since it allows the reliable segmentation of each actin fibre. The procedure itself is based on the acquisition of different pixel intensities, whereby it works through the correlation of pixel adjacent regions with synthetic fibre templates at different orientations and their assignment for the central pixel with the highest correlation coefficient among all orientations [
32].
Due to the fact that constant flows were generated within the parallel flow chamber only, the situations of in vitro experiments differ from in vivo setting where dynamic flow profiles are particular [
33]. As the constant laminar flow profile is not physiological in bones [
34], vessels and other tissues [
35], the informative value of the experimental setting is limited but it could be used for various cell proliferation and differentiation modulations. In accordance with this, constant laminar flows were rated to have more impact on target cells than pulsatile and oscillating flow profiles. With regard to these findings, the flow profile generated within the reported device meets the requirements to induce cell morphology changes by FSS.
In addition, when using the new flow chamber, an additive effect of FSS and centrifugal forces on the cells could be seen. Other flow chambers did not reveal this phenomenon due to the fact that the liquid flow moves along a stationary cell surface only [
4,
10,
22].
When comparing this new fluid flow chambers with other reported devices [
4,
10], several differences are seen. Commonly, test cells are placed on a fixed surface with the culture medium flowing along. According to the method of flow generation, they can be classified into open and sealed systems. Open systems, which are hydrostatically driven, are characterised by a fluid flow that passes the stationary phase once only [
36]. In sealed models, the culture medium recirculates pump driven through the system [
22]. Due to their inherent system-related drawbacks such as turbulent flow generation on boundary surfaces, those flow chambers are inappropriate for laminar flow creation. However, open systems have benefits of allowing the use of different culture media in a row without ceasing the fluid shear stress. Therefore, one can easily enable or disable different stimuli by exchanging the culture medium to evaluate its cellular impact. Sealed systems such as reported in this study do not provide this option. Initially added substances within the culture medium cannot be eliminated during the experimental process. Instead, stopping the flow and draining the cells would be necessary which would cause another unwanted influence to the test cells.
Besides, in the model reported in this study, microscopic examinations are possible after completing the experiment only. Nevertheless, an advantage of the new flow chamber is the possibility of testing different cell colonies simultaneously in one single experiment by placing cells in different radial locations on the spinning disc. Due to the current flow gradient from the centre to the periphery, different cell colonies are exposed to various levels of shear forces. To simplify the process of cell reaction examination, the use of a larger sized glass panel could be considered.
Biomaterial researchers are constantly looking for innovative materials like surface-binding ligands and implant materials, pursuing the aim of improving biocompatibility and healing into host tissues. For this purpose, this new developed flow chamber could provide an easy, as well as economic way to investigate material qualities in combination with tissue cells affected by FSS. A specific material to be tested could replace the cell-bearing glass panel. Alternatively, the glass panel could be coated with surface ligands in different ways [
37]. A potential use for evaluation of stem cell differentiation and/or proliferation with fluid shear stress as a mechanical stimulus may be assumed as well.
Acknowledgements
The authors thank the Department of Hydraulic Machines, Faculty of Mechanical Engineering, Technical University of Munich, Germany, for helping with the computerised simulations.