Indicators of replicative damage in equine tendon fibroblast monolayers
© Rich et al.; licensee BioMed Central Ltd. 2013
Received: 27 March 2013
Accepted: 10 September 2013
Published: 11 September 2013
Superficial digital flexor tendon (SDFT) injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. Relevant in vitro studies of this process require fibroblasts not already responding to stresses caused by the cell culture protocols. We investigated indicators of replicative damage in SDFT fibroblast monolayers, effects of this on their reparative ability, and measures that can be taken to reduce it.
We found significant evidence of replicative stress, initially observing consistently large numbers of binucleate (BN) cells. A more variable but prominent feature was the presence of numerous gammaH2AX (γH2AX) puncta in nuclei, this being a histone protein that is phosphorylated in response to DNA double-stranded breaks (DSBs). Enrichment for injury detection and cell cycle arrest factors (p53 (ser15) and p21) occurred most frequently in BN cells; however, their numbers did not correlate with DNA damage levels and it is likely that the two processes have different causative mechanisms. Such remarkable levels of injury and binucleation are usually associated with irradiation, or treatment with cytoskeletal-disrupting agents.
Both DSBs and BN cells were greatest in subconfluent (replicating) monolayers. The DNA-damaged cells co-expressed the replication markers TPX2/repp86 and centromere protein F. Once damaged in the early stages of culture establishment, fibroblasts continued to express DNA breaks with each replicative cycle. However, significant levels of cell death were not measured, suggesting that DNA repair was occurring. Comet assays showed that DNA repair was delayed in proportion to levels of genotoxic stress.
Researchers using tendon fibroblast monolayers should assess their “health” using γH2AX labelling. Continued use of early passage cultures expressing initially high levels of γH2AX puncta should be avoided for mechanistic studies and ex-vivo therapeutic applications, as this will not be resolved with further replicative cycling. Low density cell culture should be avoided as it enriches for both DNA damage and mitotic defects (polyploidy). As monolayers differing only slightly in baseline DNA damage levels showed markedly variable responses to a further injury, studies of effects of various stressors on tendon cells must be very carefully controlled.
KeywordsSuperficial digital flexor tendon Tendon Horses Cell culture Polyploidy DNA damage DNA repair Replicative stress GammaH2AX protein Comet assay
Superficial digital flexor tendon (SDFT) injury affects up to 30-33% of Thoroughbreds and event horses in racing or training (i.e. elite athletes) [1–3]. The consequent failure of regeneration with persistence of scar tissue leads to high rates of both re-injury (up to 50%) and retirement (up to 70%) [4–7]. It is generally agreed that injury usually follows a period of cumulative subclinical microdamage to the extracellular matrix that is both exercise- and age-associated; this damage includes reductions in collagen fibril diameter, increased amounts of weaker type III collagen (type I collagen predominates in normal adult tendon tissue), and reduced glycosaminoglycan levels [8–11].
Tenocytes are tendon fibroblasts with key responsibility for synthesis and degradation of the collagenous and noncollagenous matrix, and are thought to constantly repair microdamage under normal circumstances . In the adult SDFT the tenocytes are not highly active in collagen synthesis or turnover (relative to non-injury-prone digital tendons) [13–15]. This may explain why their reparative capacity is overwhelmed by factors occurring during high-speed exercise, including various potential combinations of high levels of mechanical strain, hypoxia, ischemia-reperfusion events, and hyperthermia (due to hysteresis in energy-storing tendons) [16–20]. Additionally, direct or indirect damage to tenocytes themselves and/or their detachment from collagen fibrils (resulting in mechanical understimulation) can instigate cell death or dysfunction; this dysfunctional activity may include upregulation of matrix metalloproteinase (MMP) activity, increased type III collagen synthesis, and cytokine production [18, 21–23]. The result is a vicious cycle of cellular damage and production of mechanically weak matrix that is easily disrupted during normal activity .
a reliable read-out for DNA damage in equine cells;
the relationship between DNA damage and the replicative fraction;
whether the relationship between DNA damage and cellular replication altered when fibronectin was used as a surface rather than collagen;
if reparative activity could overcome any or all of the damage.
Our ultimate aim was to achieve “healthy” tendon fibroblast monolayers i.e. a baseline comprised of cells that were not already responding to stresses introduced by the culture system itself.
Results and discussion
Equine SDFT fibroblast monolayers contain abnormally high percentages of binucleate cells, indicating cleavage failure during mitosis
High binucleate cell numbers and prolongation of late stages of mitosis in tendon fibroblast monolayers
Horse age (y)
Binucleation indicates that there has been cleavage failure during mitosis; this is normally a rare event in cell culture for mammalian somatic cells with the exception of cardiomyocytes , but can be induced by application of anti-cytoskeletal drugs including cytochalasin B [31, 33]. During division a cell rounds up, disassembles the cytoskeleton and redistributes cytoskeletal proteins and other molecules into the cleavage furrow that forms by assembly and contraction of an actomyosin ring to partition the cytoplasm [34, 35]. The sister cells then re-spread and separate in the mid-body by removal of cytoskeletal structures from the intercellular bridge, constriction of the cell cortex (furrow ingression), and plasma membrane fission (the process of abscission) [34, 35]. In cells where defects occur in cytokinesis (separation of the cytoplasm), furrow regression leads to BN cell formation.
In normal diploid fibroblast monolayers, approximately 70% of the mitotic fraction should be in the early mitotic phases (prophase (P) and metaphase (M)) with the remainder in anaphase (A) and telophase (T) when the daughter nuclei are formed and segregate . Unusually, counts of mitotic indices in tendon-derived fibroblasts revealed a marked enrichment of the later mitotic phases; the A/T counts were 2–7 times those for P/M (Table 1; Figure 1D). This was particularly apparent in low-density cell cultures and was consistent with a delay in proceeding through the latter stages of mitosis. Late phase mitotic enrichment was not evident in human fibroblasts or in fibroblasts derived from equine palate. Previously it was shown that cells that could override a prolonged G2 arrest entered mitosis but remained tethered at the midbody at the end of cytokinesis . Two thirds of those tethered cells became binucleate and the remainder either apoptosed (11%), or re-entered mitosis (22%) whilst still bridged to generate similarly bridged daughter nuclei and eventually, multinucleate cells . Phase contrast images of our monolayer cultures revealed occasional multinucleated cells (Figure 1A), together with the binucleate fraction. The individual nuclei in each BN also showed a high correlation in terms of size (Figure 1B) and frequently the nuclei were enlarged, suggesting two tetraploid nuclei. A mechanism to describe the generation of tetraploid BN cells via successive rounds of failed cytokinesis has been described ; this together with the cell tethering described in  may be relevant to our monolayer cultures.
Normally, following furrow ingression there is a period when the (now) re-spreading and migrating daughter cells are still linked by the cytoplasmic bridge that narrows and then splits as they move apart , i.e. mechanical tension is involved . In one study, 3T3 fibroblasts that were strongly adherent to high-concentration (versus low) fibronectin showed extensive stretching of the intercellular bridge and failure of the midbody to disassemble . It was proposed that cells that cannot move apart from one another due to a too-adhesive substrate will be unlikely to undergo traction-mediated cytofission, i.e. this indicates a significant cell culture artefact . In contrast, researchers using rat embryonic fibroblasts relieved cytochalasin D-induced arrest by growing them on fibronectin, possibly via an integrin-linked effect on the cytoskeleton following failed cytokinesis; it has subsequently been suggested that species-specific differences may contribute to susceptibility to BN formation [40, 41]. These differences may be due to altered adhesion properties of fibroblasts on fibronectin versus collagen surfaces, and or their susceptibility to cytoskeletal disruption . To investigate the relationship between failed cytokinesis and matrix substrate for our equine cells, we compared numbers of BN fibroblasts on collagen and fibronectin-coated coverslips but did not see significant differences for these or for palate-derived equine fibroblasts (Figure 1E). If matrix adherence properties are a factor contributing to a greater failure of abscission (separation) for equine tendon fibroblasts versus equine palatine or human foreskin fibroblasts, the difference might also be explained by the difference in tissue-origin; tendon cells in tissue are located within very small niches where they interact intimately with surrounding, densely packed collagen fibrils and other collagenous and noncollagenous matrix components .
Binucleation occurs in tendon fibroblast monolayers regardless of the presence of DNA damage
Binucleation was originally proposed to be a “tetraploidy checkpoint” to protect against cell overgrowth and neoplastic transformation, and therefore invariably resulting in permanent p53-dependent G1 arrest . However other researchers using human diploid fibroblasts showed that the p53 expression causing G1 arrest of BN cells was due to DNA damage (in those experiments caused by cell synchronisation treatments), rather than the tetraploidy itself . It was suggested that cells that have failed to undergo cytokinesis are more likely to arrest because they simply have twice as much DNA in which the damage may be present . The activity of a DNA damage checkpoint to prevent exit from mitosis has since been demonstrated . The full DNA damage response (DDR) is suppressed in normal mammalian cells until the stages of telophase/cytokinesis (i.e. the late stages of mitosis, as enriched in our monolayers), by high activity of cyclin-dependent kinase 1 .
Because we documented abnormal accumulation of our equine tendon fibroblasts in the latter phases of mitosis, and due to this known link between DNA damage and binucleation, we immunolabelled the monolayers for the phosphorylated form of the H2AX histone protein variant (γH2AX) using the vertebrate specific monoclonal antibody, clone JBW301; this antibody has previously been used to stain the γH2AX fraction involved in chromatin remodelling in equine sex chromosomes . H2AX is phosphorylated by ATM kinase in response to DNA double-strand breaks (DSBs; the most lethal lesion); this event results in the recruitment of downstream repair factors into a microscopically visible focus [48, 49]. An additional kinase, ATR, has been shown to co-operate with ATM/γH2AX to protect collapsed replication forks . The growing γH2AX focus is thought to open up the chromatin structure and forms a “platform” to stabilise accumulating damage response and repair factors; there is close correlation between numbers of foci (or puncta, each containing several hundred γH2AX molecules) and numbers of DSBs . Phosphorylation of H2AX has also been shown in response to stress-induced chromatin reorganisation; this can occur without DNA breaks.
Similar numbers of BN cells were detected in abattoir and PM group monolayers (and explant cultures). This suggested that BN cell formation was largely due to factors other than DNA damage (e.g. matrix substrate, as discussed above). In a previous study of human fibroblasts, not all arrested BN cells contained γH2AX puncta, again suggesting that DNA damage is not a necessary factor . Interestingly, in both abattoir and PM tendon fibroblasts (i.e. those with and without large numbers of γH2AX puncta), diffuse or occasionally punctate γH2AX labelling was noted at mitosis at the spindle poles and microtubules (Figure 2C). In some interphase cells γH2AX labelling was also noted in small perinuclear foci consistent with localisation to the centrosome; this has been reported previously (in tandem with other DNA repair proteins) but is of unknown significance . These distributions of γH2AX (puncta/micropuncta/centrosome-like and spindle) are identical to those previously described in other species .
DNA double-strand breaks in tendon fibroblast cultures are replication-associated but do not result in significant cell death
Another potential cause of DNA damage detectable by γH2AX staining is Mycoplasma sp. infection. Although this damage has never been linked to replicative status, it can be largely reversed by treatment with the free radical quenching agent N-acetyl cysteine (NAC) . We routinely screen our cells for Mycoplasma infection and, additionally, we sent cells to a microbiology laboratory for testing (Veterinary Diagnostic Services, University of Glasgow). All isolates were negative. Despite this, we additionally ‘treated’ our cultures with a bacteriocidal agent (plasmocin) that targets mycoplasma protein synthesis and DNA replication, to see whether levels of DNA damage would be reduced. Following several weeks of treatment (i.e. the recommended time to achieve mycoplasma removal) we did not detect a reduction in the levels of γH2AX staining. Treatment with 1 mM NAC also failed to reduce levels of DNA damage. We therefore concluded that DNA damage in our cultures was not because of Mycoplasma contamination.
Despite the large amounts of replication-associated DNA damage in the abattoir fibroblast monolayers, there were not similarly significant levels of cell death. Morphologically, few apoptotic bodies were noted, and additionally there was no evidence of the diffuse, nuclear-wide γH2AX labelling that has been associated with apoptosis . Importantly, because most researchers monitor the success of their monolayer cultures by observing and/or measuring cell death and viability, these differences in replication stress and DNA damage would not be identified routinely. We also rarely saw γH2AX puncta in mitotic figures as can occur when collapsed replication forks result in under-replicated loci . These observations indicated that the tendon fibroblasts were largely repairing DNA lesions prior to mitosis.
Equine tendon fibroblasts have robust DNA repair activity, but the integrity of this repair is uncertain
There were now two reasons for investigating the reparative ability of these tendon fibroblasts: firstly the evidence that DNA damage even at remarkably high levels was not proceeding in most cases to cell death (or permanent arrest of BN cells in those monolayers) i.e. it was being repaired; and secondly due to the potential role of defective repair processes in the formation of DSBs in the first place. Given that SDFT fibroblasts have been shown to have low synthetic and degradative activity levels post-maturation and that they frequently allow accumulation of matrix microdamage [8–15], it was of interest to know if this extended to a poor ability to repair their own DNA.
Because γH2AX labelling only indicates DSB-type damage  and repair intermediates we used alkaline comet assays to assess damage and repair in these tendon cells. This assay involves lysing agarose-embedded cells and performing DNA unwinding and electrophoresis at an alkaline pH (12.1-12.4), to detect multiple types of DNA damage i.e. single and double strand breaks, incomplete excision repair sites and cross-links. These lesions often precede DSB formation. DNA migrates out of each cell in response to application of an electric field, and is visualised when fluorescently labelled, as a ‘comet’; the comet tail size and shape correlate with the degree of DNA damage . The equine genome is 2.7 Gb i.e. it is slightly smaller than the human genome at 3.2 Gb. As comet analysis is capable of measuring 0.06 to 3 breaks per 109 daltons, the lowest level of sensitivity would be approximately 100 breaks per equine genome; this is more than adequate to measure levels of background DNA damage in control cells . We used hydrogen peroxide (H2O2) as a damaging agent because it is known to predominantly cause non-DSB lesions in DNA, particularly in replicating cells. Reactive oxygen species are also of practical relevance in tendon fibroblast research, as they are likely to be increased within the tissue of energy-storing tendons during high-speed exercise [18, 19].
Interestingly, pairwise analyses to compare cells on either matrix surface within each of the EP and LP groups (i.e. comparing the EP controls on collagen and fibronectin) showed that DNA damage levels were not significantly different between cells on collagen versus fibronectin surfaces with one exception; for the LP cells there was a lower DNA damage level of 20.6% in the recovery group on fibronectin, versus 32.8% for collagen (p < 0.001). This finding is in keeping with previous work that indicates potentiation of DNA damage responses in cells growing on fibronectin , which might result in the altered repair kinetics that we observed. However, the integrity of this more rapid re-ligation is not certain given our previous measurements of high levels of γH2AX in cells growing on that surface, that occurred regardless of their replication status. High levels of γH2AX labelling have been shown to persist following DNA break re-ligation in situations where DNA repair has been inaccurate . Our demonstration of a DNA damage-replication link would suggest that repair processes that act in replicating cells, for example, HR, may be defective in the tendon fibroblasts. It is possible that this defect is enriched in monolayer cultures of tendon-derived cells due to the fact that they are generally post-replicative in their tissue niche . This possibility will be investigated in future work.
Culturing equine tendon fibroblasts in monolayers resulted in two forms of cellular injury: numbers of binucleate cells at levels previously only documented in cells irradiated or treated with cytoskeletal poisons; and in cells from some sources, high initial levels of DNA damage including double strand breaks that did not resolve with further replicative cycling but did not result in significant cell death. We demonstrated that both forms of injury were replication-linked on collagen substrates, with uncoupling of the replication-DNA damage link on fibronectin. DNA repair mechanisms were robust, but increased DNA damage delayed repair; this effect was mitigated for cells carrying high levels of DNA damage but cultured on fibronectin. We would recommend that cells expressing these damage phenotypes at an early passage not be injected into injury sites or used for mechanistic injury studies due to problems with comparability. Additionally, ex-vivo cell expansion should be minimised in order to limit replication-induced cellular injury and mitotic disruption. The effects of fibronectin in uncoupling DNA damage from replication and accelerating DNA re-ligation (in monolayers with high DNA damage levels) require further investigation, given the high levels of this matrix glycoprotein in injury sites.
Mid-metacarpal segments (5 cm) of the SDFT were harvested under aseptic conditions from the forelimbs of Thoroughbred horses euthanized for reasons unrelated to tendon disease. Specimens from 5 adults (3, 5, 14, 15 years of age) and one foal (6 months of age) were obtained from an approved UK abattoir. Specimens from one adult (3-year-old) and one foal (full-term stillborn) were obtained from a veterinary post-mortem facility. All specimens were obtained and used in accordance with guidelines reviewed and approved by the Ethics and Welfare Committee, School of Veterinary Medicine, University of Glasgow. Equine palate fibroblasts were donated by Dr. L. Nasir (University of Glasgow) . Human primary foreskin fibroblasts (Hs68) were purchased (CRL 1635; ATCC, Teddington, Middlesex, UK).
The paratenon and peripheral tissue were removed from each tendon segment and the remaining tissue diced into 2 mm3 pieces. These were incubated with constant agitation for 1 h at 37°C in sterile DMEM medium (10 mL per 1 g tissue) containing 1 mg/mL pronase and supplemented with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin [13, 21]. A second digestion for 1 h at 37°C with complete DMEM containing 0.25 mg/mL collagenase type VIII and 0.55 mg/mL dispase (40 mL per 1 g of tissue) was then performed [13, 21]. Digested material was passed through a 70 μm cell strainer, and cells collected by centrifugation at 1000 rpm for 5 min; cell pellets were re-suspended in complete DMEM. The collagenase/dispase digestion was repeated to yield more cells. Routine expansion was carried out in flasks coated with 10 μg/cm2 type I collagen. For experiments the cells were seeded onto glass coverslips coated with either 10 μg/cm2 type I collagen or 1 μg/cm2 fibronectin (as per manufacturers’ instructions) and grown to 30-80% confluence in a standard humidified incubator (37°C, 21% O2, 5% CO2). Equine palate fibroblasts were processed as described previously . Human fibroblasts were seeded onto collagen-coated coverslips in complete DMEM (10% serum; 1 g/L glucose) supplemented with penicillin/streptomycin, and maintained in the same incubator.
Culture of tendon explants
A 5 cm mid-metacarpal segment of the SDFT were aseptically retrieved and placed into pre-warmed DMEM (37°C). To retrieve tendon fibroblasts from the core of the mid-metacarpal region, a sharp single edged blade (HD Hardware) was used to excise peripheral tissue. The exposed core was diced into 2 mm3 pieces that were placed onto collagen-coated 60 mm plastic culture dishes. The bottom of each dish was scored with a scalpel blade to create 5 or 6 parallel lines; this facilitated surface-adhesion of the tissue pieces. Pre-warmed (37°C) complete DMEM containing 10% bovine serum albumin and 1% penicillin / streptomycin was added to the dishes.
Imaging of binucleate fibroblasts and calculation of mitotic indices
Binucleate (BN) cells were quantified by phase contrast and fluorescent microscopy using an inverted Leica DM IRB microscope; CellMask (plasma membrane dye/Invitrogen) and AlexaFluor 488 Phalloidin were used to demarcate the cytoplasm. Mitotic indices were counted in multiple random fields by fluorescence microscopy using the DNA co-stain DAPI and the replicative marker TPX2. Numbers of prophase (P) and metaphase (M) cells were counted as one cohort and anaphase (A) and telophase (T) as the second.
Cells were fixed in 4% paraformaldehyde for 5 min, followed by permeabilization using 0.25% Triton-X 100 for 5 min. Non-specific binding was blocked using 10% milk powder/PBS for 30 min at room temperature (RT). Primary and secondary antibody incubations were carried out overnight at 4°C and 1 h at RT, respectively. Alexa-fluor conjugates (1:1000) were used to detect the primary antibodies. Slides were mounted using Prolong-Gold anti-fade containing DAPI (Invitrogen). Levels of DNA damage were assessed by labelling for phospho-H2AX (γH2AX) (clone JBW301 (1:250) Millipore, Watford, UK). p53 phosphorylated at serine 15 was immunolabelled using ab1431 (1:200; Abcam, Cambridge, UK). Cells were additionally labelled for p21 using EA10 (1:100; Calbiochem, Watford, UK). Double-stranded DNA breaks were more definitively identified by co-labelling for phospho-H2AX and proteins that respond to DNA damage, i.e. either promyelocytic leukaemia protein (PML; H-238 (1:100), Santa Cruz, Dallas, TX, USA) or Rad51 (H-92 (1:100), Santa Cruz, Dallas, TX, USA). Replication was assessed by labelling for the TPX/repp86 and centromere protein F (CENPF) antigens (ab71816 (1:100) and ab5 (1:100), Abcam, Cambridge UK), these being expressed diffusely in the nucleus during S and G2 phases of the cell cycle with relocation to the spindle during mitosis. Cells were examined and imaged using the 63x objective lens of an upright Axiophot2 (Zeiss) or inverted DM IRB microscope (Leica). Fluorescent micrographs were overlaid using GIMP digital software (version 2.6.1).
Cell culture samples were added to both solid and liquid mycoplasma media (Mycoplasma Experience, Reigate, UK), and incubated under microaerophilic conditions for up to 7 d. Cell cultures were also tested using the MycoProbe® Mycoplasma Detection Kit (R&D Systems Europe Inc., Abingdon, UK). Plasmocin (Invivogen, Toulouse, France) was used at a curative (rather than prophylactic) concentration of 25 μg/mL for two weeks.
The alkaline comet assay was performed as per the manufacturers’ instructions using triplicate test samples (Trevigen Inc., Gaithersburg, MD, USA). DNA damage was induced by incubation with 200 μM H2O2 for 5 min on ice. To assess repair capacity, the H2O2 treatment was followed by a 1 h recovery period. The untreated controls were trypsinized immediately, counted and embedded in agarose. The “treatment” group was processed in this way directly following H2O2 incubation. The “recovery” group cells were rinsed with PBS, fresh DMEM was added, and they were then maintained for 1 h at 37°C in a 5% CO2 incubator prior to processing. The processing was done as rapidly as possible to limit any further DNA repair that would result in under-estimation of the damage at that point. Cells at 1 × 105/mL were added to molten agarose (1:10 v/v). The CometSlides™ were evenly warmed to 37°C prior to application of cells in agarose (to improve adherence of cells); it was important to avoid spreading the cell mixture over the margins of each sample area (delineated in red). The lysis stage was performed at 4°C overnight to increase sensitivity; if the lysis solution crystallized over this period it was discarded. Electrophoresis (21 V, 30 min) at a high pH resulted in structures resembling comets as observed by fluorescence microscopy (using SybrGreen to stain DNA); comets form due to electrophoretic migration of broken DNA strands away from the nucleoid. Electrophoresis is one of the principal factors that can affect data reproducibility. If using a Trevigen CometAssay ES II tank it is crucial to add only 850 mL electrophoresis buffer such that the buffer height is held at 0.4 cm over the slide tray; changing this parameter will alter the efficiency of electrophoresis. Each run was standardized using control ‘damaged’ cells with expected comet tail grades. Images were viewed and captured using a Leica DM IRB microscope. Etoposide-damaged cells (Trevigen Inc., Gaithersburg, MD, USA) were used to establish damage reference groups and ensure that the control conditions were not themselves genotoxic. Treatment and recovery groups were electrophoresed at the same time as controls to reduce experimental variability; failure to visualize comet tails in etoposide-damaged cells was used to indicate failed electrophoresis, in which case the data was not used.
Analysis was performed using CometScore version 18.104.22.168 (TriTek Corp., Sumerduck, VA, USA). Individual “comets” were manually selected from monochrome bitmap images with all metrics saved. Overlapping comets in the images were not scored. Imaging and noise cut-off was as described in . Values plotted were derived from ‘percent DNA in the tail’ as suggested by the COMET assay users group . Each data point represented independent triplicate experiments with at least 50 comets scored per experiment.
All statistical analyses were performed using SPSS Statistics version 9 (IBM, New York, USA). Data normality was assessed using the Kolmogorov-Smirnov statistic. Probability values for significance between the treatment groups were assessed using the non-parametric Mann–Whitney U Test.
Superficial digital flexor tendon
Ataxia telangiectasia mutated
Ataxia telangiectasia and Rad3-related protein
DNA damage response
Reactive oxygen species
Promyelocytic leukaemia protein
Centromere protein F
Dulbecco’s modified eagle medium
Foetal calf serum
We gratefully acknowledge the support of this study by The Equine Trust (Massey University, New Zealand), Petplan Charitable Trust, the Biotechnology and Biosciences Research Council (BBSRC; project grant BB/J000655/1) and the Horserace Betting Levy Board. None of the funding sources played any role in the study design, data collection, analysis, data interpretation, manuscript writing or the decision to submit the manuscript for publication. We also wish to thank the staff of the equine abattoir, and at The Animal Health Trust including Dr. Debbie Guest. We would also like to thank Elizabeth Gault for her assistance with primary culture; including handling of equine palate cells.
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