5.2.1.2.1 Scrape-Wounding and Imaging
Scoring of the underlying culture substrate during scrape-wounding could influence the ability of the tendon fibroblasts to close the wound. Therefore, it was important to determine which method of scrape wounding was preferable. Pipette tips proved to be the best means to make the scrape-wound and this is consistent with reported data (Wright et al., 2009, Maffulli et al., 2000). The ideal sized tip depended on the size of the wound required and in this study when photographing the scrape-wounds at 20x magnification the P10 pipette tip produced a wound with a width less than the visual field of the microscope. It was therefore easy to image the entire wound over 2-4 visual fields and combine the images to create a single collated image encompassing the complete scrape-wound. This was important as the appearance of the wounds changed rapidly and when using width measurements the determination of the exact site of previous measurements was problematic.
5.2.1.2.2 Identification of Sampling Intervals for Scrape-Wounds
A repeated measures general linear model with a Bonferroni post hoc test identified the appropriate sampling intervals from the second pilot study on scrape wounding. The times with significant changes in wound width were approximately 3, 9, 22 and 47 hours to the nearest hour (Appendix 32).
In the final experiment a minimum of 12 hours between imaging reduced the time the culture dishes were out of the incubator. This was important because the incubator environment maintains the cells at 37oC, 5% CO
2 in a humidified atmosphere and the
microscope used for imaging did not have an environmental chamber to replicate those conditions. Therefore, during imaging the tendon fibroblasts were exposed to a
temperature of 20oC for 30-40 minutes each time the plate was removed from the
incubator. Cells will tolerate a reduction in temperature better than an increase; however, the reduction of temperature of the media also results in a reduction in pH. In this situation the buffers in the media should have compensated for this (Kramer et al., 2010). Most normal fibroblasts grow well between pH 7.4 and 7.7 and buffers in the culture medium equilibrate with atmospheric CO2 such that an increase in CO2 results in
a lower pH (Freshney et al., 2007). Room air contains less CO2 (approximately 394
ppm) than the culture incubator (50,000 ppm) and the phenol red in the media changes colour as the pH alters. Phenol red in DMEM is purple at pH 7.8, pink at 7.6, red at 7.4, orange at 7.0 and yellow at 6.5. The cultures changed from orange to pink the more time they were out of the incubator.
Cells in culture prefer low oxygen tensions and this is partially recreated with the addition of CO2 to the air in the incubator. Obviously the addition of 5% CO2 is only
going to drop the percentage of oxygen by 5% but this is nearly 25% of the total oxygen (21%) in average room air (Kramer et al., 2010). In room air, the oxygen tension is higher and may result in more free radical damage to the cells. Oxygen tolerance may be provided by serum and control of oxygen tension is more critical when low or serum free media are used (Freshney et al., 2007). Tendon cells normally exist in a hypoxic environment and the fewer capillaries and thicker endothelial walls present at sites prone to injury in adult people are thought to be a contributory factor to hypoxic damage (Tuite et al., 1997). This is supported by histological evidence that hypoxic damage is a major component of tendon rupture in people (Jozsa and Kannus, 1997). Culturing of tendon cells in a more hypoxic environment may have made them more tolerant to other manipulations. The ideal oxygen and other gas tensions for tendon fibroblast growth is an area of tendon culture that has not been reported and
warrants investigation. Cell culture conditions that mimic in vivo conditions closely must always be the gold standard.
The use of DMEM supplemented with only 5% FBS was required for the Sircol™ assay
for total collagen measurement. This low serum media changed the response of the tendon fibroblasts considerably with many lifting off and dying after 5 days rather than closing the scrape-wound. This is inconsistent with data reported by (Wright et al., 2012).
Oxygen tolerance is influenced by serum and therefore control of oxygen tension is likely to be more critical in low serum media (Freshney et al., 2007). Changes in oxygen tension during imaging and the metabolic demands of dividing in a low serum environment may well have been factors contributing to the cell death (Freshney et al., 2007) experienced in the initial mimetic peptide experiment. This prompted the investigation to identify the optimum low serum media to support cell growth and proliferation for these studies. Advanced™ DMEM and Opti-MEM™ both containing 100 U/mL penicillin/100 μg/mL streptomycin and 5% FBS are specialised media designed for low serum situations (Web Link 2). Cell growth in these media were compared to cell growth in DMEM containing 100 U/mL penicillin/100 μg/mL streptomycin and 5% FBS using the xCelligence™ instrument and software (Appendix 37).