La Pedagogía de Proyectos, una alternativa en la configuración

Data obtained from the cell growth experiment showed a difference in doubling rates between equine superficial digital flexor tendon-derived fibroblasts grown in different media with DMEM containing 100 U/mL penicillin/100 μg/mL streptomycin and 5% FBS giving more uniform growth than the specialised media. Whilst the media had an

effect, a greater difference was apparent between the fibroblasts from different tendons. Fibroblasts from the left thoracic limb superficial digital flexor tendon from horse G2 (G2LF) and left thoracic limb superficial digital flexor tendon from horse G3 (G3LF) showed much poorer growth in all of the media tested in comparison to the fibroblasts from the other tendons (Figure 28, Appendix 38).

Cells were extracted from the left and right superficial digital flexor tendons of each of the five horses. Of the 10 tendons cultured, the nine chosen for the cell growth study were those with better growth at the time of seeding. The fibroblasts from G2LF were not included in the cell growth study as they had the poorest growth at the time of seeding. Interestingly G2RF exhibited the poorest doubling rate of the cells tested. This suggests an in vivo cause. Within horse variations were apparent as well as between horse variations. The superficial digital flexor tendon fibroblasts from different tendons in the same horse G3LF and G3RF had different growth rates. This is likely to be a result of ex vivo and in vitro factors whereas between horse variations could be a component of in vivo differences as well.

Potential in vivo causes of differences in cell proliferation include genetic background, developmental factors such as diet and training and the presence of existing lesions within the tendon. Ex vivo influences would include collection technique and extraction protocol whilst in vitro factors such as expansion and cell culture environment are cell specific and fundamental for good cell survival.

Although the 2 year old colts were New Zealand thoroughbreds, the specific breeding was not available as they were unbranded. There is a reported gender predilection for superficial digital flexor tendon injury in racehorses in Japan with entire males being at greater risk than geldings or mares (Kasashima et al., 2004). In Hungary, blood group type O was found to be more highly represented in people with tendon ruptures than in the general population (Jozsa et al., 1989). There are over 30 blood groups in horses although only 8 are major systems. The most important are A and Q with approximately 85% of thoroughbreds being positive for Aa antigens and 61% for Qa antigens. No association between blood groups and tendon injury have been identified in horses,

however the high risk groups such as racehorses are likely to have the same blood group.

Sequence variants of the gene for Tenascin and the collagen V α 1 are associated with Achilles tendinopathies in people (Mokone et al., 2005, Mokone et al., 2006). A number of genetically determined factors have been identified as affecting the occurrence of Achilles tendon injury (September et al., 2006). It is therefore reasonable to expect that other, as yet unidentified, genetic factors closely involved with tendon structure and function also have an effect on the susceptibility of tendons to be damaged as well as their ability to repair. This is an area warranting further investigation in horses although genetic testing of affected individuals may be controversial.

The two year-old colts from which the superficial digital flexor tendon-derived fibroblasts used for these cell culture experiments were extracted had been raised under the same conditions and had similar stature and body condition at euthanasia with no gross evidence of undernourishment or developmental abnormalities. They were unhandled and in New Zealand such young thoroughbreds would have most likely have been managed at pasture (Morel et al., 2007). As the horses were young and unbroken, subclinical degeneration due to exercise was unlikely to be present. Ideally, clinical palpation and ultrasound examination of the tendons would have been performed prior to collection but this was not practicable.

The tendon collection technique was identical and performed under the same conditions for all tendons retrieved from the abattoir. The prolonged time between tendon collection and cell extraction would have resulted in survival of the more robust cells. This is discussed further in the section on collection and would have been similar for all the tendon derived cells. The main difference between individual tendons was time from

collection to extraction (between 12-15 hours), as the tendon collection technique was faster than the extraction technique. This did not appear to affect cell doubling time based on the results of the cell growth study using the xCelligence system (see Section 5.1.3.6). Tendons were processed in order of collection and there were no trends observed between cell growth and collection time. Tendon fibroblasts that grew well when seeded continued to grow well throughout expansion and after cryopreservation. A reduction in superficial digital flexor tendon cellularity, collagen production and gap junction quantities in tendon has been identified by comparing tendons from foals (1d-1 month) and those for young adult horses (2-5 years) (Young et al., 2009). The range of cell counts between the individuals within the young adult group in this study was narrow so the tendons collected would be expected to have similar cell numbers and be comparable to young adults.

The cell growth experiment used media containing 5% FBS to be consistent with conditions appropriate to the Sircol™ assay for the determination of total collagen content of the media. The cell growth rates were therefore not directly comparable to the final mimetic peptide and scrape-wound protocol in which DMEM containing 100 U/mL penicillin/100 μg/mL streptomycin and 10% FBS was used. Variations in cell doubling rates were observed between the tendon-derived fibroblasts from different horses as well as in different media. The fibroblasts from all tendons grew more quickly in DMEM containing 100 U/mL penicillin/100 μg/mL streptomycin and 5% FBS than in either of the specialist low serum media tested. The xCelligence system has reportedly less intra-assay variability and is more sensitive than the conventional colorimetric 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]- 2H-tetrazolium hydroxide XTT assay in the evaluation of cell viability (Karaca et al., 2012). Electrical impedance is measured using microelectrodes integrated into the

bottom of specially engineered tissue culture plastic. This provides quantitative information about the status of cells, including cell number, cell adhesion, cytotoxicity, cell viability, and cell morphology (Web Link 1).

Whilst superficial digital flexor tendon derived fibroblasts grew faster in DMEM containing 100 U/mL penicillin/100 μg/mL streptomycin and 10% FBS compared to DMEM containing 100 U/mL penicillin/100 μg/mL streptomycin and 5% FBS in general culture, cells from one tendon only (G5LF) were cultured with 10% FBS in the cell growth experiment so no meaningful comparisons can be made. The reduced serum concentration in the media was clearly important but not the sole reason for altered fibroblast growth rates.

5.2.2

M

C

43

C

Modulation of Connexin43 gap junction communication has been shown to improve healing in a number of tissues (Mori et al., 2006, Qiu et al., 2003) using either down- regulation of Connexin43 gap junction expression or functional blockade of the gap junctions. Down-regulation of Connexin43 expression using Connexin43 antisense oligodeoxynucleotides has been successfully used in a number of wound models in vivo (Coutinho et al., 2005, Qiu et al., 2003, Mori et al., 2006). The Connexin43 antisense oligodeoxynucleotides are rapidly broken down by serum and therefore they are administered in tissues within a pluronic gel which breaks down in a concentration- dependent rate (Becker, 1999) and therefore prolongs exposure. In this study the amount of serum in the media did not affect the dissolution of the pluronic gel and as expected the pluronic gel dissolved over a 1 hour period.

5.2.2.1USE OF ANTISENSE OLIGODEOXYNUCLEOTIDES TO REDUCE CONNEXIN43