BACKGROUND
superficial digital flexor tendon  injury affects up to 30-33% of thoroughbreds and event horses in racing or training   <cit> .
the consequent failure of regeneration with persistence of scar tissue leads to high rates of both re-injury  and retirement   <cit> .
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 , and reduced glycosaminoglycan levels  <cit> .
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  <cit> .
in the adult sdft the tenocytes are not highly active in collagen synthesis or turnover   <cit> .
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   <cit> .
additionally, direct or indirect damage to tenocytes themselves and/or their detachment from collagen fibrils  can instigate cell death or dysfunction; this dysfunctional activity may include upregulation of matrix metalloproteinase  activity, increased type iii collagen synthesis, and cytokine production  <cit> .
the result is a vicious cycle of cellular damage and production of mechanically weak matrix that is easily disrupted during normal activity  <cit> .
due to the limitations of in vivo work, appropriate cell culture models are required to more clearly define how tenocytes sense and respond to multiple environmental conditions occurring during galloping exercise, and how these processes might be modulated to reduce injury  <cit> .
tendon fibroblast monolayer  culture systems are frequently used as tractable and easily analysed primary systems for experimentation / manipulation  <cit> .
however, they are also necessary to obtain and expand these cells for use in  3-dimensional models, or for clinical purposes e.g.
autologous tenocyte implantation into tendon injury sites  <cit> .
there are many problems that might influence cellular stress and damage in these monolayers including the tissue extraction process: many researchers use enzymatic digestion rather than explant outgrowth due to the higher and more rapid yield of cells, without significant relative disadvantages in terms of phenotypic drift  <cit> .
importantly, levels of such damage can easily go unrecognized when using live/dead assays or simple phase contrast appearance for monitoring, as is common practice  <cit> .
in our monolayers we noted high numbers of binucleate  fibroblasts, a normally rare event in cell culture , that indicates cleavage failure during mitosis and has been associated with dna damage and matrix surface type  <cit> .
this prompted the present study, the objectives of which were to determine:
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
equine sdft fibroblast monolayers contain abnormally high percentages of binucleate cells, indicating cleavage failure during mitosis
specimens were obtained from an approved uk abattoir , and a veterinary post-mortem facility .
routine light microscopy examination of culture dishes and phase contrast microscopy of cells seeded onto collagen-coated coverslips revealed large numbers of bn  fibroblasts in all monolayers .
in dapi-labelled monolayers, these could only conclusively be identified where the nuclei were touching unless cytoplasmic or membrane elements were co-stained .
in confluent monolayers these comprised up to 7% of the total population and were not related to the age of the animal or the source group  .
however, in subconfluent cultures the numbers were significantly higher i.e.
up to 20%.
in all equine tendon fibroblast monolayers, numbers of bn cells were higher than routinely seen in human fibroblast monolayers  or in equine palate fibroblasts   <cit> , in which they did not exceed 3% .
the percentage of binucleate cells and those in prophase or metaphase , or anaphase/telophase  was calculated from counts of  <dig> fibroblasts in ~80% confluent fibroblast monolayers.
values for each parameter in tendon-derived monolayers could not be related to horse age or source.
data from fibroblasts derived from non equine  or equine non tendon  tissue sources are included for comparison.
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  <cit> , but can be induced by application of anti-cytoskeletal drugs including cytochalasin b  <cit> .
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  <cit> .
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 , and plasma membrane fission   <cit> .
in cells where defects occur in cytokinesis , 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  and metaphase ) with the remainder in anaphase  and telophase  when the daughter nuclei are formed and segregate  <cit> .
unusually, counts of mitotic indices in tendon-derived fibroblasts revealed a marked enrichment of the later mitotic phases; the a/t counts were 2– <dig> times those for p/m .
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 g <dig> arrest entered mitosis but remained tethered at the midbody at the end of cytokinesis  <cit> .
two thirds of those tethered cells became binucleate and the remainder either apoptosed , or re-entered mitosis  whilst still bridged to generate similarly bridged daughter nuclei and eventually, multinucleate cells  <cit> .
phase contrast images of our monolayer cultures revealed occasional multinucleated cells , together with the binucleate fraction.
the individual nuclei in each bn also showed a high correlation in terms of size  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  <cit> ; this together with the cell tethering described in  <cit>  may be relevant to our monolayer cultures.
normally, following furrow ingression there is a period when the  re-spreading and migrating daughter cells are still linked by the cytoplasmic bridge that narrows and then splits as they move apart  <cit> , i.e.
mechanical tension is involved  <cit> .
in one study, 3t <dig> fibroblasts that were strongly adherent to high-concentration  fibronectin showed extensive stretching of the intercellular bridge and failure of the midbody to disassemble  <cit> .
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  <cit> .
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  <cit> .
these differences may be due to altered adhesion properties of fibroblasts on fibronectin versus collagen surfaces, and or their susceptibility to cytoskeletal disruption  <cit> .
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 .
if matrix adherence properties are a factor contributing to a greater failure of abscission  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  <cit> .
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 g <dig> arrest  <cit> .
however other researchers using human diploid fibroblasts showed that the p <dig> expression causing g <dig> arrest of bn cells was due to dna damage , rather than the tetraploidy itself  <cit> .
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  <cit> .
the activity of a dna damage checkpoint to prevent exit from mitosis has since been demonstrated  <cit> .
the full dna damage response  is suppressed in normal mammalian cells until the stages of telophase/cytokinesis , by high activity of cyclin-dependent kinase  <dig>  <cit> .
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  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  <cit> .
h2ax is phosphorylated by atm kinase in response to dna double-strand breaks ; this event results in the recruitment of downstream repair factors into a microscopically visible focus  <cit> .
an additional kinase, atr, has been shown to co-operate with atm/γh2ax to protect collapsed replication forks  <cit> .
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  and numbers of dsbs  <cit> .
phosphorylation of h2ax has also been shown in response to stress-induced chromatin reorganisation; this can occur without dna breaks.
the s-phase transition in fibroblasts usually results in reparable dna damage, visible as low numbers of large γh2ax puncta ; we only classified cells expressing  <dig> or more puncta as excessively damaged.
in monolayers from the pm group we detected occasional puncta together with micropuncta of γh2ax .
micropuncta have previously been shown to arise independently of dna damage  <cit> , possibly because of the reorganization of chromatin structure that accompanies replication and cell division; only the larger puncta have been associated with dna damage  <cit> .
in contrast, frequent large puncta were noted in fibroblast monolayers from the abattoir group , in 8-25% of both bn and mononuclear cells; the highest percentages were seen in the more sparse monolayers i.e.
the amount of dna damage reduced as the fibroblasts became confluent.
these levels of γh2ax puncta expression were remarkable, as they have previously only been demonstrated  in cells injured using genotoxic agents or ionising radiation  <cit> .
the difference between pm and abattoir groups may have related to the delay of at least 6–7 h prior to placement of specimens into medium  for the latter group, or subtle differences in digestion technique.
we feel that a tissue digestion effect was unlikely, given that comparable levels of dna damage and binucleation were evident in monolayer and explant cultures derived from the same tissue samples.
percentages of dna-damaged cells were  <dig> % ±  <dig>   and  <dig> %± <dig>  in ~70% confluent monolayer and explant cultures respectively, that were derived from an identical adult abattoir sample.
clearly the speed of cell processing post mortem requires further investigation, particularly as it has been considered acceptable to culture fibroblasts up to several days following death from both human and animal subjects  <cit> .
similar numbers of bn cells were detected in abattoir and pm group monolayers .
this suggested that bn cell formation was largely due to factors other than dna damage .
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  <cit> .
interestingly, in both abattoir and pm tendon fibroblasts , diffuse or occasionally punctate γh2ax labelling was noted at mitosis at the spindle poles and microtubules .
in some interphase cells γh2ax labelling was also noted in small perinuclear foci consistent with localisation to the centrosome; this has been reported previously  but is of unknown significance  <cit> .
these distributions of γh2ax  are identical to those previously described in other species  <cit> .
in abattoir fibroblast monolayers labelled for p <dig> phosphorylated at serine  <dig> , most of the positive nuclei were seen in bn cells i.e.
although the numbers of bn formed were not influenced by dna damage, they were more likely to contain an amount of damage visible in terms of stabilised p <dig> enrichment .
we did not see prominent p <dig> stabilisation in bn cells in monolayers from the pm group.
additional positive labelling for p <dig>  confirmed cell cycle arrest in the p53-positive cells ; p <dig> mediates the cell cycle arrest , initiated and tightly controlled by stabilised p <dig>  some researchers have demonstrated the involvement of p <dig> in elevation of reactive oxygen species  levels in cultured  fibroblasts, feeding back to result in further γh2ax accumulation, continuation of cell cycle arrest and activation of cellular senescence  <cit> .
not all of the bn cells in the abattoir monolayers had p53-positive nuclei , and some of them were both p53- and p21-negative.
together with the fact that their numbers did not accumulate with time, this indicates survival and eventual re-entry into the cell cycle to complete mitosis.
it has previously been demonstrated that if dna damage does not reach a certain level, most bn cells will eventually finish mitosis and repair the dna in the next g <dig> phase  <cit> .
dna double-strand breaks in tendon fibroblast cultures are replication-associated but do not result in significant cell death
because in the abattoir group the numbers of large γh2ax puncta per nucleus were greatest in subconfluent monolayers, we proposed that dna damage could be occurring in response to replication stress.
during dna replication the replication fork forms a y-shaped structure at which dna is unwound and copied.
erroneous replication can stall the replication fork complex, and the single-stranded dna generated at that point activates cell cycle arrest  <cit> .
if the stalling is prolonged, dna damage will occur, and if the fork collapses, dsbs are generated that cannot be repaired by simply restarting the fork.
however, the precise relationship between the formation of γh2ax puncta and dsbs at blocked replication forks is controversial.
some studies have shown that γh2ax puncta form some hours prior to fork breakage, after many hours of cell cycle arrest, i.e.
the puncta may not necessarily represent breaks directly, but they do indicate replication stress and possibly the activities of different repair and chromatin modifying complexes  <cit> .
because we needed to determine if the high numbers of γh2ax puncta in abattoir fibroblasts were actually due to dsbs, we double immunolabelled the cells for γh2ax and two markers of replication-induced dna damage, promyelocytic leukaemia protein  and rad <dig>  we previously demonstrated that dsbs juxtapose  with nuclear puncta of pml; pml has since been shown to play a prominent role in the homologous recombination  of dna during dsb repair  <cit> .
this repair process is favoured uniquely in the replicative fraction.
the rad <dig> protein searches for homologous sequence to act as a template for repair, having first bound to single stranded dna generated by dna resection at the breakage site  <cit> .
our co-staining experiments revealed the stereotypic juxtaposition of nuclear domains of pml protein with large γh2ax puncta in the abattoir fibroblast monolayers  with co-localised foci of rad <dig> .
the majority of the large γh2ax puncta in abattoir cells expressed co-localised puncta of γh2ax and rad <dig> .
rarer, large puncta of γh2ax in pm cells also co-localised with rad <dig>  however, not all γh2ax puncta contained rad <dig> protein; these may have represented  sites of γh2ax accumulation prior to breakage.
alternatively, some researchers have observed continued marking of resealed but poorly repaired dsb sites by γh2ax in order to recruit the more accurate hr repair machinery  <cit> .
having confirmed a strong association between γh2ax puncta and multiple dna damage markers in these tendon fibroblasts, we then investigated the link with replication status.
double immunolabelling was performed for γh2ax and tpx2/repp <dig> or centromere protein f  antigens respectively; both of these markers are expressed diffusely in the nucleus during the s and g <dig> phases of the cell cycle, with relocation to the spindle during mitosis .
there was almost exact matching of nuclei expressing increased numbers of γh2ax puncta with positive labelling for each of the replication markers , with the exception of bn cells .
other evidence of replication stress was noted morphologically, including the generation of nuclear blebs, microbodies, and aberrant multipolar mitoses .
there was one exception to this rule, found by comparing replicative and damaged fractions in coverslips from each of the abattoir horses  at different levels of confluence on collagen or fibronectin surfaces .
an effect of age of the donor  was not shown, but for all horses, in the monolayers of the highest confluence level , the damage levels on fibronectin remained high i.e.
there was damage-replication uncoupling on that matrix substrate.
confluence was not noted to be reached more rapidly on fibronectin versus collagen.
interestingly, recent data have indicated that fibronectin promotes dna damage recognition  in an α <dig> integrin-dependent fashion  <cit> .
this requires further investigation in three-dimensional cell culture systems and in tissue, as it has relevance for cellular therapies: fibronectin expression is markedly upregulated in damaged  tendon tissue, where it occurs pericellularly  <cit> .
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   <cit> .
we routinely screen our cells for mycoplasma infection and, additionally, we sent cells to a microbiology laboratory for testing .
all isolates were negative.
despite this, we additionally ‘treated’ our cultures with a bacteriocidal agent  that targets mycoplasma protein synthesis and dna replication, to see whether levels of dna damage would be reduced.
following several weeks of treatment  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  <cit> .
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  <cit> .
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  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  <cit> , 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  <cit>  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 , 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  <cit> .
the equine genome is  <dig>  gb i.e.
it is slightly smaller than the human genome at  <dig>  gb.
as comet analysis is capable of measuring  <dig>  to  <dig> breaks per  <dig> daltons, the lowest level of sensitivity would be approximately  <dig> breaks per equine genome; this is more than adequate to measure levels of background dna damage in control cells  <cit> .
we used hydrogen peroxide  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  <cit> .
significant damage was recorded for both abattoir and pm group cells following h2o <dig> treatment, but the majority of comet-tails  were lost after 1 h recovery i.e.
these data showed robust repair activity for both groups .
we were particularly interested to see how subtle variation in initial dna damage levels could alter damage and repair following the h2o <dig> treatment; to achieve this we reduced the biologic noise in our assay by comparing cells derived from the same animal.
we used pm cells as they carried a lower amount of endogenous damage.
our assay involved two sets of cells; one early passage  and the second, derived after several weeks in continuous culture .
to more accurately identify changes in repair capacity we used the cometscore software that allows analysis of individual comets with definition of their dna distributions .
the cometscore software generates histograms of fluorescence intensity across each comet which can then be quantified .
cometscore analyses from our two groups of cells  are shown in the box-whisker charts in figure 7c.
the upper chart shows ep cells; the lower, lp.
the median values for control  levels of dna damage  in ep and lp cells were  <dig> % and  <dig> % respectively on collagen-coated surfaces and  <dig> % and  <dig> % on fibronectin.
following h2o <dig> treatment, the median values of these two groups diverged by more than 20% i.e.
<dig> % for ep versus  <dig> % for lp on collagen, with similar differentials when grown on fibronectin .
this difference was maintained following the 1 h recovery period.
we could speculate that the increased injury generated by peroxide treatment of the lp cells delayed repair; the contribution of a minor increase in basal dna injury prior to peroxide treatment remains to be established.
however, if small differences in basal dna damage can alter the injury thresholds for tendon fibroblasts, researchers will have to be wary of this possibility and carefully control for genotoxic stress in their experiments.
interestingly, pairwise analyses to compare cells on either matrix surface within each of the ep and lp groups  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  <dig> % in the recovery group on fibronectin, versus  <dig> % for collagen .
this finding is in keeping with previous work that indicates potentiation of dna damage responses in cells growing on fibronectin  <cit> , 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  <cit> .
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  <cit> .
this possibility will be investigated in future work.
CONCLUSIONS
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  require further investigation, given the high levels of this matrix glycoprotein in injury sites.

 superficial digital flexor tendon injury affects up to 30-33% of thoroughbreds and event horses in racing or training. the consequent failure of regeneration with persistence of scar tissue leads to high rates of both re-injury and retirement. 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, and reduced glycosaminoglycan levels. 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. 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. additionally, direct or indirect damage to tenocytes themselves and/or their detachment from collagen fibrils can instigate cell death or dysfunction; this dysfunctional activity may include upregulation of matrix metalloproteinase activity, increased type iii collagen synthesis, and cytokine production. the result is a vicious cycle of cellular damage and production of mechanically weak matrix that is easily disrupted during normal activity. due to the limitations of in vivo work, appropriate cell culture models are required to more clearly define how tenocytes sense and respond to multiple environmental conditions occurring during galloping exercise, and how these processes might be modulated to reduce injury. tendon fibroblast monolayer culture systems are frequently used as tractable and easily analysed primary systems for experimentation / manipulation. however, they are also necessary to obtain and expand these cells for use in 3-dimensional models, or for clinical purposes e.g. autologous tenocyte implantation into tendon injury sites. there are many problems that might influence cellular stress and damage in these monolayers including the tissue extraction process: many researchers use enzymatic digestion rather than explant outgrowth due to the higher and more rapid yield of cells, without significant relative disadvantages in terms of phenotypic drift. importantly, levels of such damage can easily go unrecognized when using live/dead assays or simple phase contrast appearance for monitoring, as is common practice. in our monolayers we noted high numbers of binucleate fibroblasts, a normally rare event in cell culture, that indicates cleavage failure during mitosis and has been associated with dna damage and matrix surface type. this prompted the present study, the objectives of which were to determine: 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.  equine sdft fibroblast monolayers contain abnormally high percentages of binucleate cells, indicating cleavage failure during mitosis specimens were obtained from an approved uk abattoir, and a veterinary post-mortem facility. routine light microscopy examination of culture dishes and phase contrast microscopy of cells seeded onto collagen-coated coverslips revealed large numbers of bn fibroblasts in all monolayers. in dapi-labelled monolayers, these could only conclusively be identified where the nuclei were touching unless cytoplasmic or membrane elements were co-stained. in confluent monolayers these comprised up to 7% of the total population and were not related to the age of the animal or the source group. however, in subconfluent cultures the numbers were significantly higher i.e. up to 20%. in all equine tendon fibroblast monolayers, numbers of bn cells were higher than routinely seen in human fibroblast monolayers or in equine palate fibroblasts, in which they did not exceed 3%. the percentage of binucleate cells and those in prophase or metaphase, or anaphase/telophase was calculated from counts of fibroblasts in ~80% confluent fibroblast monolayers. values for each parameter in tendon-derived monolayers could not be related to horse age or source. data from fibroblasts derived from non equine or equine non tendon tissue sources are included for comparison. 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. 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. 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, and plasma membrane fission. in cells where defects occur in cytokinesis, 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 and metaphase ) with the remainder in anaphase and telophase 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– times those for p/m. 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 g 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, or re-entered mitosis whilst still bridged to generate similarly bridged daughter nuclei and eventually, multinucleate cells. phase contrast images of our monolayer cultures revealed occasional multinucleated cells, together with the binucleate fraction. the individual nuclei in each bn also showed a high correlation in terms of size 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 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, 3t fibroblasts that were strongly adherent to high-concentration 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. 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. if matrix adherence properties are a factor contributing to a greater failure of abscission 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 g arrest. however other researchers using human diploid fibroblasts showed that the p expression causing g arrest of bn cells was due to dna damage, 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 is suppressed in normal mammalian cells until the stages of telophase/cytokinesis, by high activity of cyclin-dependent kinase. 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 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; this event results in the recruitment of downstream repair factors into a microscopically visible focus. 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 and numbers of dsbs. phosphorylation of h2ax has also been shown in response to stress-induced chromatin reorganisation; this can occur without dna breaks. the s-phase transition in fibroblasts usually results in reparable dna damage, visible as low numbers of large γh2ax puncta; we only classified cells expressing or more puncta as excessively damaged. in monolayers from the pm group we detected occasional puncta together with micropuncta of γh2ax. micropuncta have previously been shown to arise independently of dna damage, possibly because of the reorganization of chromatin structure that accompanies replication and cell division; only the larger puncta have been associated with dna damage. in contrast, frequent large puncta were noted in fibroblast monolayers from the abattoir group, in 8-25% of both bn and mononuclear cells; the highest percentages were seen in the more sparse monolayers i.e. the amount of dna damage reduced as the fibroblasts became confluent. these levels of γh2ax puncta expression were remarkable, as they have previously only been demonstrated in cells injured using genotoxic agents or ionising radiation. the difference between pm and abattoir groups may have related to the delay of at least 6–7 h prior to placement of specimens into medium for the latter group, or subtle differences in digestion technique. we feel that a tissue digestion effect was unlikely, given that comparable levels of dna damage and binucleation were evident in monolayer and explant cultures derived from the same tissue samples. percentages of dna-damaged cells were % ±  and %± in ~70% confluent monolayer and explant cultures respectively, that were derived from an identical adult abattoir sample. clearly the speed of cell processing post mortem requires further investigation, particularly as it has been considered acceptable to culture fibroblasts up to several days following death from both human and animal subjects. similar numbers of bn cells were detected in abattoir and pm group monolayers. this suggested that bn cell formation was largely due to factors other than dna damage. 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, diffuse or occasionally punctate γh2ax labelling was noted at mitosis at the spindle poles and microtubules. in some interphase cells γh2ax labelling was also noted in small perinuclear foci consistent with localisation to the centrosome; this has been reported previously but is of unknown significance. these distributions of γh2ax are identical to those previously described in other species. in abattoir fibroblast monolayers labelled for p phosphorylated at serine, most of the positive nuclei were seen in bn cells i.e. although the numbers of bn formed were not influenced by dna damage, they were more likely to contain an amount of damage visible in terms of stabilised p enrichment. we did not see prominent p stabilisation in bn cells in monolayers from the pm group. additional positive labelling for p confirmed cell cycle arrest in the p53-positive cells; p mediates the cell cycle arrest, initiated and tightly controlled by stabilised p some researchers have demonstrated the involvement of p in elevation of reactive oxygen species levels in cultured fibroblasts, feeding back to result in further γh2ax accumulation, continuation of cell cycle arrest and activation of cellular senescence. not all of the bn cells in the abattoir monolayers had p53-positive nuclei, and some of them were both p53- and p21-negative. together with the fact that their numbers did not accumulate with time, this indicates survival and eventual re-entry into the cell cycle to complete mitosis. it has previously been demonstrated that if dna damage does not reach a certain level, most bn cells will eventually finish mitosis and repair the dna in the next g phase. dna double-strand breaks in tendon fibroblast cultures are replication-associated but do not result in significant cell death because in the abattoir group the numbers of large γh2ax puncta per nucleus were greatest in subconfluent monolayers, we proposed that dna damage could be occurring in response to replication stress. during dna replication the replication fork forms a y-shaped structure at which dna is unwound and copied. erroneous replication can stall the replication fork complex, and the single-stranded dna generated at that point activates cell cycle arrest. if the stalling is prolonged, dna damage will occur, and if the fork collapses, dsbs are generated that cannot be repaired by simply restarting the fork. however, the precise relationship between the formation of γh2ax puncta and dsbs at blocked replication forks is controversial. some studies have shown that γh2ax puncta form some hours prior to fork breakage, after many hours of cell cycle arrest, i.e. the puncta may not necessarily represent breaks directly, but they do indicate replication stress and possibly the activities of different repair and chromatin modifying complexes. because we needed to determine if the high numbers of γh2ax puncta in abattoir fibroblasts were actually due to dsbs, we double immunolabelled the cells for γh2ax and two markers of replication-induced dna damage, promyelocytic leukaemia protein and rad we previously demonstrated that dsbs juxtapose with nuclear puncta of pml; pml has since been shown to play a prominent role in the homologous recombination of dna during dsb repair. this repair process is favoured uniquely in the replicative fraction. the rad protein searches for homologous sequence to act as a template for repair, having first bound to single stranded dna generated by dna resection at the breakage site. our co-staining experiments revealed the stereotypic juxtaposition of nuclear domains of pml protein with large γh2ax puncta in the abattoir fibroblast monolayers with co-localised foci of rad. the majority of the large γh2ax puncta in abattoir cells expressed co-localised puncta of γh2ax and rad. rarer, large puncta of γh2ax in pm cells also co-localised with rad however, not all γh2ax puncta contained rad protein; these may have represented sites of γh2ax accumulation prior to breakage. alternatively, some researchers have observed continued marking of resealed but poorly repaired dsb sites by γh2ax in order to recruit the more accurate hr repair machinery. having confirmed a strong association between γh2ax puncta and multiple dna damage markers in these tendon fibroblasts, we then investigated the link with replication status. double immunolabelling was performed for γh2ax and tpx2/repp or centromere protein f antigens respectively; both of these markers are expressed diffusely in the nucleus during the s and g phases of the cell cycle, with relocation to the spindle during mitosis. there was almost exact matching of nuclei expressing increased numbers of γh2ax puncta with positive labelling for each of the replication markers, with the exception of bn cells. other evidence of replication stress was noted morphologically, including the generation of nuclear blebs, microbodies, and aberrant multipolar mitoses. there was one exception to this rule, found by comparing replicative and damaged fractions in coverslips from each of the abattoir horses at different levels of confluence on collagen or fibronectin surfaces. an effect of age of the donor was not shown, but for all horses, in the monolayers of the highest confluence level, the damage levels on fibronectin remained high i.e. there was damage-replication uncoupling on that matrix substrate. confluence was not noted to be reached more rapidly on fibronectin versus collagen. interestingly, recent data have indicated that fibronectin promotes dna damage recognition in an α integrin-dependent fashion. this requires further investigation in three-dimensional cell culture systems and in tissue, as it has relevance for cellular therapies: fibronectin expression is markedly upregulated in damaged tendon tissue, where it occurs pericellularly. 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. we routinely screen our cells for mycoplasma infection and, additionally, we sent cells to a microbiology laboratory for testing. all isolates were negative. despite this, we additionally ‘treated’ our cultures with a bacteriocidal agent that targets mycoplasma protein synthesis and dna replication, to see whether levels of dna damage would be reduced. following several weeks of treatment 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 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, 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, 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 gb i.e. it is slightly smaller than the human genome at gb. as comet analysis is capable of measuring to breaks per daltons, the lowest level of sensitivity would be approximately breaks per equine genome; this is more than adequate to measure levels of background dna damage in control cells. we used hydrogen peroxide 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. significant damage was recorded for both abattoir and pm group cells following h2o treatment, but the majority of comet-tails were lost after 1 h recovery i.e. these data showed robust repair activity for both groups. we were particularly interested to see how subtle variation in initial dna damage levels could alter damage and repair following the h2o treatment; to achieve this we reduced the biologic noise in our assay by comparing cells derived from the same animal. we used pm cells as they carried a lower amount of endogenous damage. our assay involved two sets of cells; one early passage and the second, derived after several weeks in continuous culture. to more accurately identify changes in repair capacity we used the cometscore software that allows analysis of individual comets with definition of their dna distributions. the cometscore software generates histograms of fluorescence intensity across each comet which can then be quantified. cometscore analyses from our two groups of cells are shown in the box-whisker charts in figure 7c. the upper chart shows ep cells; the lower, lp. the median values for control levels of dna damage in ep and lp cells were % and % respectively on collagen-coated surfaces and % and % on fibronectin. following h2o treatment, the median values of these two groups diverged by more than 20% i.e. % for ep versus % for lp on collagen, with similar differentials when grown on fibronectin. this difference was maintained following the 1 h recovery period. we could speculate that the increased injury generated by peroxide treatment of the lp cells delayed repair; the contribution of a minor increase in basal dna injury prior to peroxide treatment remains to be established. however, if small differences in basal dna damage can alter the injury thresholds for tendon fibroblasts, researchers will have to be wary of this possibility and carefully control for genotoxic stress in their experiments. interestingly, pairwise analyses to compare cells on either matrix surface within each of the ep and lp groups 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 % in the recovery group on fibronectin, versus % for collagen. 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 require further investigation, given the high levels of this matrix glycoprotein in injury sites. 
enrichment for injury detection and cell cycle arrest factors 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. low density cell culture should be avoided as it enriches for both dna damage and mitotic defects. 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. superficial digital flexor tendontendonhorsescell culturepolyploidydna damagedna repairreplicative stressgammah2ax proteincomet assay however, significant levels of cell death were not measured, suggesting that dna repair was occurring. 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. comet assays showed that dna repair was delayed in proportion to levels of genotoxic stress. 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. we found significant evidence of replicative stress, initially observing consistently large numbers of binucleate cells. superficial digital flexor tendon injuries of horses usually follow cumulative matrix microdamage; it is not known why the reparative abilities of tendon fibroblasts are overwhelmed or subverted. both dsbs and bn cells were greatest in subconfluent monolayers.
 superficial digital flexor tendon 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 cells. a more variable but prominent feature was the presence of numerous gammah2ax puncta in nuclei, this being a histone protein that is phosphorylated in response to dna double-stranded breaks. enrichment for injury detection and cell cycle arrest factors 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 monolayers. the dna-damaged cells co-expressed the replication markers tpx2/repp 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. 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. superficial digital flexor tendontendonhorsescell culturepolyploidydna damagedna repairreplicative stressgammah2ax proteincomet assay 