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Collagen secretion into the media is at a low level and close to the detection limit of the

Table 14 - Sircol™ assay to determine effects of culture conditions on absorbance reading. FBS Concentration Absorbance at 560nm Collagen (μg/L) Water 0 0 0 Water/RTV 0 0 0 DMEM 0 0 0 DMEM/RTV 0 0 0 WATER/SERUM 1 0 0 WATER/SERUM 2.5 0.57 3.68 WATER/SERUM 5 0.84 5.43 WATER/SERUM 10 1.80 11.69 RTV/DMEM/SERUM 1 0.42 2.69 RTV/DMEM/SERUM 2.5 0.86 5.59 RTV/DMEM/SERUM 5 1.45 9.36 RTV/DMEM/SERUM 10 3.55 23 DMEM/SERUM 1 0.1 0.64 DMEM/SERUM 2.5 0.38 2.44 DMEM/SERUM 5 1.35 8.77 DMEM/SERUM 10 3.49 22.57 Each reading is the mean of 4 different wells exposed to the same solutions.

Concentrations of serum above 1% in the water and serum groups gave absorbance’s

suggesting there was collagen in the media (Table 14). This is likely to be due to cross- reactivity of a serum component with the test and confirms the manufacturers’

recommendation that low serum containing media should be used. The effects of the higher concentrations of serum were increased when combined with DMEM containing phenol red as the culture media. This clarified how much serum and phenol red would impact the detection of collagen in the culture media. Exposure of the water to the RTV did not appear to affect the assay.

Standard DMEM containing 1%penicillin/streptomycin and 5% FBS, Opti-MEM™

containing 1% penicillin/streptomycin and 5% FBS and Advanced DMEM containing 1%penicillin/streptomycin and 5% FBS were evaluated for use in the Sircol assay. Known concentrations of collagen were added to aliquots of each of the media, from which a standard curve was constructed and against which the amount of collagen in the samples could be assayed. Media were collected at 18 hour intervals as this was when the media would need to be refreshed in the mimetic peptide experiments. Even using an overnight concentration step, there was insufficient collagen in any media that had been exposed to the tendon fibroblasts for 18 h for detection using the Sircol™ assay

(Table 15, Appendix 31). This suggested that the Sircol™ assay was not a useful means

of measuring total collagen in the mimetic peptide experiment. Subsequently in this study, it was shown that insufficient collagen was released into the media during 18 hours of cell culture to be detected by the Sircol™ assay.

Table 15 - Collagen Concentration in Media from Equine Superficial Digital Flexor Tendon-derived fibroblasts.

Time

(Hours) Opti-MEM Advanced DMEM DMEM

18 0.03 0.24 0.04

24 0.08 0.41 0.03

36 0.04 0.15 0.15

72 0.04 0.08 0.03

Time indicates length of media exposure to cells. Collagen concentration measured by Sircol™ assay at 560nm.

Each measurement is the average of four results. All media contained 5% FBS.

Waggett (2006) found that avian tendon fibroblasts release approximately 85% of the collagen produced into the media with the remainder being deposited in the cell layer. In a study of human tendon cells the concentration of collagen in the media was determined to be dependent on the duration the media remained in contact with the cells (Evans and Trail, 2001). Extrapolation of the graph of total collagen in the media from the human tendon cells suggested that a detectable amount of collagen would

potentially be released into the media between 18 and 24 hours although the levels would be close to the limits of the test. In the human flexor and extensor tendon cell study there was no significant difference between flexors and extensors with respect to total collagen production (Evans and Trail, 2001). Adult superficial digital flexor tendon fibroblasts, however have lower collagen synthesis than those fibroblasts from immature flexor tendons or from extensor tendons (Batson et al., 2003, Birch et al., 2008). Lower cell numbers and lower collagen synthetic activity may have contributed to the total collagen level being lower in the current study than in the human tendon fibroblast study.

Other techniques that could have been used to assess collagen secretion into the media include sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by western blotting or amino acid analysis. Dot Blot analysis (Bio-Rad, Hercules, California) and hydroxyproline determinations may also be used to determine collagen levels in the cells (Evans and Trail, 2001, Waggett et al., 2006).

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It was difficult to source locally, young thoroughbred horses that were undergoing euthanasia. Only seven were sourced in 2 years and of those, only four resulted in viable cells, Appendix 43. This paucity was unexpected considering the high attrition rate of young thoroughbred horses (Rogers et al., 2007) but needs to be considered when designing protocols around such source material. The most successful source was an abattoir where horses were killed for human consumption. The increased value of young horses for this market resulted in horses with less risk of injury being available. Photography of the horses brands at the abattoir allowed for identification via the New Zealand Thoroughbred Racing Website form (Appendix 45) so racing history and

performance could be checked. This was important as tendons could have chronic injury due to age and or exercise.

For the first set of cultures, tissue was sourced from thoroughbred horses between 2 and 5 years old to reduce variation due to age and exercise related changes in the tendon cells and connexin expression. Immature equine digital tendons have a higher cellularity than mature digital tendons, but no significant difference has been reported between mature and aged tendons (Stanley et al., 2007). Connexin expression reduces markedly in early maturity (Stanley et al., 2007). Collecting cells from horses less than 5 years old reduced the chances of existing damage being present due to age or exercise effects on the tendons. Exercise and age-related micro-damage in the human Achilles tendon and equine superficial digital flexor tendon is thought to develop due to insufficient cellular reparative ability (Kannus and Jozsa, 1991, Patterson-Kane et al., 1998a). Degenerative and older tendons also have a higher proportion of apoptotic cells than normal tendon and would therefore have been likely to result in a lower yield of viable cells (Yuan et al., 2003).

Following a loss of frozen cell stocks a second collection of tendons was made at the abattoir. This proved ideal as the tendons were from 5 unbroken 2 year old thoroughbred colts from the same stud farm. This provided an internal control as these colts had been raised under the same husbandry and at the same location so would have been exposed to similar environmental influences. Being unbroken and with no visible signs of tendon injury these tendons were also unlikely to have age or exercise related subclinical micro-damage.