All the sections stained well with DAPI and the majority had some procollagen I and procollagen III staining (Figure 22) although procollagen III was predominant. Sections in areas of no grossly visible lesions had either some staining for both procollagen I and III (5/7) or no staining for procollagen I (2/7) (Table 9). This could suggest either that low levels of procollagen are produced in un-injured adult horse tendon or that the grossly non-injured areas were responding at a cellular level to injury elsewhere in the tendon. Based on the minimal histological findings even close to injury areas the latter is less likely. Methods to increase procollagen I production, especially in response to injury, require investigation as a means to promote improved quality of tendon healing.
Table 9 - Detection of procollagen types I and III in tendon sections
Identification Lesion
Y or N 488 procollagen I Fluorescence of 568 procollagen III Fluorescence of DAPI stained nuclei Fluorescence of
ALFPa 12-16 4 N + ++ + ALFPa 16-20 1 Y + + + ARFPa 8-12 4 Y ++ ++ + ARFPa 12-16 2 N 0 ++ + BLFPa8-13 3 N + + + BLFPa 8-13 5 Y + + + BLFPa 13-20 9 Y + ++ + BRFPa 11-16 1 N + + + BRFPA 11-16 4 Y + + + CLFPa 6-12 2 N + + + CLFPa 12-16 5 Y + + + CLFPa 16-20 3 Y + + + DLFPa 4-8 4 N 0 + + DLFPa 8-12 1 Y + + + DLFPa 8-12 2 Y + ++ + DRFPa 8-12 4 Y 0 0 + DRFPa 16-20 2 N + ++ +
0 = fluorescence less than or equal to negative control, + = fluorescence more than negative control and less than positive control and ++ fluorescence equal to positive control.
Figure 22 - Examples of immunofluorescent staining for procollagen
DAPI stained nuclei (blue); procollagen III with Alexa 568 secondary (red) and procollagen I with Alexa 488 secondary (green). Bar on images = 50 μm
a) Positive control horse skin wound – the staining shows cell nuclei and procollagen I and III are present in the wound.
b) Negative controls of mouse brain – cell nuclei are present but no procollagen I and III.
c) Example of positive staining for nuclei; some staining for procollagen III and minimal staining for procollagen I. d) Example of sparse but positive staining for procollagen I and III.
4.4.5
L
IMITATIONSThe horses in this study had a relatively wide age range and therefore this could have increased the inter-horse variation. Horse B was 11 years old and would therefore be expected to have some underlying chronic micro trauma within the tendon although these were not detectable on clinical or ultrasonographic examination nor on histological sections. 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 and this would not have been ideal had the horses been followed for a longer period.
Ultrasonography was used as a screening tool to confirm no significant underlying pathology existed in the tendons although as the study showed, this is a relatively insensitive tool. Computer assisted tomography may have been more sensitive but required general anaesthesia and would have been significantly more costly.
The short study period allowed investigation of the response to acute tearing injury in the core of the superficial digital flexor tendon. It would have been ideal to have sufficient horses to follow some out to a longer time point to determine if the lesion healed in a similar fashion to clinical injury.
4.4.6
C
ONCLUSIONx The aim of this study to create a lesion in the core of the equine SDFT that would mimic the acute phase of tendon injury was achieved.
x Furthermore, the lesion proved technically easy to create in standing sedated horses and resulted in minimal post-operative discomfort.
x The study showed that even in the hands of experienced equine surgeons grossly apparent tendon lesions could prove difficult to detect on ultrasound examination and to confirm histologically.
x New collagen was detectable in lesions less than or equal to four days old and type III collagen was the predominant procollagen detected in these lesions. The use of this model has the potential to improve the understanding and investigation of tendon injury and healing.
C
HAPTER5
EVALUATING THE ROLE OF CONNEXIN43 IN WOUND
HEALING USING A CELL CULTURE SYSTEM.
5.0
INTRODUCTION
The equine superficial digital flexor tendon is an energy-storing tendon that is commonly injured in equine athletes and has similarities to the human Achilles tendon (Firth, 2000). The major problems arising from tendon injury are the slow rate of tendon healing and poor biomechanical properties of the healed tendon (Birch et al., 2008). Tendon fibroblasts are responsible for collagen and extra-cellular matrix production and therefore are largely responsible for tendon healing. The low tendon fibroblast numbers in adult tendon and specialisation for anaerobic metabolism and therefore for efficient locomotion limit the ability of these cells to effectively respond to injury and therefore the ability of tendon to heal (Stanley et al., 2007). In horses, healing is further complicated as they remain standing most of the time (David et al., 2012), thus their injured tendons are under constant and varying load.
Tendon fibroblasts produce different types of collagen. The mechanically superior collagen type I is produced in normal healthy tendon and in injured areas of tendon that have undergone effective remodelling. Collagen type III is produced during repair in
injured tendons and if it is not remodelled persists as a fibrous scar (Smith et al., 2002). The reduced ratio of type I to type III collagen in healed tendon is thought to be a considerable component of the predisposition of tendons to re-injury (Kannus et al., 1997).
Connexin43 gap junctions are one of the two types of gap junction linking fibroblasts in the same tendon fibril and also fibroblasts on different fibrils (Ralphs et al., 1998). Gap junctions aid intercellular communication including the spread of cell death signals in injured tissue (Cusato et al., 2003). Uncoupling the gap junctions in injured tissue may reduce this effect and also aid in cell migration to close the defect (Wright et al., 2009), thus modulation of gap junction expression may enhance tissue repair.
Cell culture models have been established to investigate the effect of modifying connexion 43 gap junctions on healing cells. Modulation of connexion 43 gap junction action, by mimetic peptides for example, has been shown to improve the rate of scrape- wound healing in cultured human dermal fibroblasts (Wright et al., 2009). Consequently cell culture studies were undertaken to evaluate the following hypothesis: Modulation of gap junction communication in cultured equine superficial digital flexor tendon-derived fibroblasts would improve the rate of scrape-wound healing and would increase the ratio of production of collagen type I to collagen type III.
The aim of these studies was to:
x Determine the effect of modulation of gap junction communication on superficial digital flexor tendon-derived fibroblasts in vitro.
x Determine the effects of modulation of gap junction communication on rate of closure of a scrape-wound as a measure of healing potential.
x Determine the effects of modulation of gap junction communication on the type of collagen produced as an indicator of biomechanical strength.
x Measure collagen production and secretion into the media and deposited on the
cell monolayer using the Sircol™ assay.
x This assay requires low serum media that does not support good tendon fibroblast growth therefore cell doubling rates in three specialised low serum media were therefore measured to determine the best media to use in the scrape- wound experiment.
x Develop a method to reproduce uniaxial load in cell culture that mimicked the in vivo situation.
5.1
MATERIALS AND METHODS
5.1.1
S
OURCE OFT
ENDONS FORC
ELLE
XTRACTIONThe thoracic limb superficial digital flexor tendons of thoroughbred horses 2-5 years old were used as a source of tendon fibroblasts. Preparative studies were performed on cells obtained from thoroughbred horses of any age undergoing euthanasia at Massey University Veterinary Teaching Hospital (Palmerston North, New Zealand) or the local hunt clubs for a reason unrelated to the thoracic limb superficial digital flexor tendon. The cells for the data generating studies were from thoroughbred horses between 2-5 years old, with no history of tendon injury, undergoing humane slaughter for human consumption at an abattoir in the South Island of New Zealand. Thoracic limb superficial digital flexor tendon tissue was collected and cells extracted as described (Appendices 18 to 20).