LA GRATUIDAD DE LA JUSTICIA MILITAR

Equine superficial digital flexor tendon-derived fibroblasts demonstrated a preference for collagen gel substrate for attachment and growth. Whilst it was possible to make a collagen gel substrate from horse tail tendon this was not the preferred substrate when compared with rat tail derived collagen gel. Intuitively, collagen gel made from the same species would have been thought to be preferred, however, this was not found to be the case in the present study. The high concentration of acetic acid needed to soften the equine tendon tissue may have affected the collagen gel and reduced the subsequent attachment of cells.

Equine superficial digital flexor tendon-derived fibroblasts preferred DMEM with 4.5 mg/mL glucose over Advanced™ DMEM or Opti-MEM™ supplemented with 1% penicillin/streptomycin and 5% FBS. Cell doubling rates as determined by using the xCelligence electrical impedance system confirmed within and between horse variations. This warrants more investigation as the differences could indicate a component of the variation in the ability of different horses to respond to injury and heal effectively.

6.3.2

E

QUINE

S

UPERFICIAL

D

IGITAL

F

LEXOR

T

ENDON

D

ERIVED

F

IBROBLASTS HAVE

F

UNCTIONAL

C

ONNEXIN

43

G

AP

J

UNCTIONS AND

H

EMI

-

CHANNELS

.

Connexin43 gap junctions in equine superficial digital flexor tendon-derived fibroblasts in cell culture transported Lucifer yellow dye between cells and this transport was blocked by culturing with 500 μM / mL / mL mimetic peptide. Connexin43 hemi- channels in the membrane of equine superficial digital flexor tendon-derived fibroblasts

allowed propidium iodide to enter living cells in a low calcium environment. This action was blocked by culturing with 10 μM / mL mimetic peptide. This cell culture model is therefore appropriate for testing effects on Connexin43 gap junctions and hemi-channels in equine tendon fibroblasts.

6.3.3

G

AP JUNCTION MODULATION IN CULTURE

Unmodified Connexin43 antisense oligodeoxynucleotides did not down-regulate numbers of Connexin43 gap junctions as detected by immunofluorescence staining of equine superficial digital flexor tendon-derived fibroblasts when administered in culture media. This may be due to poor uptake into the cells in the absence of a transfection agent. As pluronic gel is required as a reservoir to overcome the limitations of using antisense oligodeoxynucleotides in a serum containing environment, other means of altering connexin43 gap junction actions, such as mimetic peptides, are likely to be more clinically useful.

The mimetic peptide, Peptide5 did not alter the migration of superficial digital flexor tendon-derived cells to close a monolayer scrape wound. Peptide5 had been shown to be effective in reducing inflammation and cell death in an ex vivo sheep spinal cord model. This mimetic peptide may therefore be better as an in vivo treatment.

6.3.4

C

OLLAGEN

D

ETECTION

Quantification of total collagen produced by scrape-wounded monolayer cultures of superficial digital flexor tendons was not achieved. Under the present study conditions equine superficial digital flexor tendon-derived fibroblasts did not produce sufficient total collagen into the culture media over 18 hours for detection using the Sircol™

Similarly Herovici staining of new collagen in equine superficial digital flexor tendon fibroblasts cultures did not produce a strong enough stain for quantification.

Immunofluorescent staining of procollagen types I and III successfully stained the cytoplasm of equine superficial digital flexor tendon-derived fibroblasts in monolayer cultures with and without scrape-wounds. Accurate comparison of the ratios of collagen types I and collagen type III with and without mimetic peptide treatment was not achieved in this study due to fixation problems in the final experiment.

6.3.5

RTV

DISHES

A novel method to expose the superficial digital flexor tendon derived fibroblasts to unilateral loading in culture was developed from an existing method using loading of a silastic, RTV, dish (Neidlingerwilke et al., 1994). The dishes designed could withstand repetitive loads of up to 20% strain, and consistent with strains measured in galloping horses. Strain rates and speed of loading can be altered to closely mimic tendon loading in vivo. Injured horses remain standing most of the time and therefore their tendons remain under load. Most cell loading devices use radial load which is less consistent with clinical loading. The apparatus developed in the present study fixed the dish at one end and stretched from the other similar to the in vivo situation. A limitation of the apparatus was differences in material properties between the RTV dishes; this would have made interpretation of results of loading problematic, therefore dish construction must be standardised further before loading experiments can be undertaken.

6.4

LIMITATIONS

The major limitations of the study performed on the sheep were caused by external factors. The sheep were part of a surgery teaching practical and therefore the time they

were anaesthetised and when they underwent euthanasia were controlled by the speed at which the students worked. The situation exposed the sheep to the effects of the non- selective gap junction blocker isoflurane that may have influenced lesion expansion. It also prevented any of the sheep being recovered and the tendons subject to weight- bearing. The teaching situation prevented ultrasound imaging of the lesions although the early time course of the model made this less of a concern as lesion expansion would have continued for up to a week post injury. The sheep were initially just to be used to test the model and were ideal due to the low welfare cost. The technique and situation proved so successful that the treatment groups were subsequently included in the study. It was not possible to confirm perfect sealing of the tendon by the cyanoacrylate glue although it appeared well adhered at sample collection.

The lack of reduction of the Connexin43 levels in response to antisense oligodeoxynucleotides may be due to the short time period of the study or problems with the antisense oligodeoxynucleotide action in this site or species. Samples are stored for future reverse transcriptase polymerase chain reaction to determine if actual expression of Connexin43 was down-regulated by the treatment.

The wide age range of the horses used for the in vivo study described in chapter 4 is likely to have increased the inter-horse variation. Also as Horse B was 11 years old she would be expected to have some underlying chronic micro trauma within the tendon (PattersonKane et al., 1997a). Ideally, young untrained horses would have been used to minimise the risk of chronic micro trauma. The horses were also unshod making accurate detection of lameness challenging.

Ultrasonography was used as a screening tool to confirm no significant underlying pathology existed in the tendons although as the study has proven, ultrasonography

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