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triamcinolone acetonide ( ta ) injections are often used to treat tendinopathy , but it has substantial adverse effects , such as impaired function of supraspinatus tendon cells and high rate of re - rupture .
previous studies also reported the deleterious effects of ta on histological and biomechanical properties of the injured tendons .
local anesthetics are used for adjunctive pain control in combination with corticosteroids injection in tendinopathies .
however , recent studies suggest that local anesthetics of amide derivatives , such as bupivacaine and lidocaine , have deleterious effects on various cell types [ 611 ] .
it was shown that the cytotoxicity of bupivacaine was dose - dependent in chondrocytes in vitro .
a more recent study showed that lidocaine could significantly decrease positive effects of platelet - rich plasma on human tendon cells .
while local anesthetics are commonly given with corticosteroids injection in treating tendinopathies , the combined effect of steroids and local anesthetics on tenocytes is largely unknown .
based on these studies , we hypothesized that local anesthetics might potentiate the deleterious effects of ta on tenocytes .
the aim of this study was to determine the effects of lidocaine and ta on cultured rat tenocytes and whether there is a synergistic effect .
animal experiments were carried out under the rules and regulations of the animal care and use committee at harbin medical university . sprague - dawley male rats ( weighing 200250 mg ) were used for isolation of tenocytes .
each patellar tendon was cut into small sections in aseptic conditions , and then placed and cultured in 6-well culture plates .
after 5 min of air - drying for better adherence , dulbecco s modified eagle medium ( dmem ) , 10% fetal bovine serum ( fbs ) , and 1% penicillin and streptomycin were supplemented to each well .
the explants were incubated at 37c in a humidified culture chamber supplemented with 5% co2 .
cells began to emerge from the tissue pieces after 34 days in culture and began to attach to the surface of the culture dishes .
after the cells reached 80% confluence , they were passaged by washing with phosphate - buffered saline ( pbs ) and detached with trypsin / ethylene deaminate triacetic acid ( edta ) .
after 24 h of culture , attached tenocytes were treated with ta in culture media ( regular medium with 0.1 mg / ml of ta ) .
samples were then washed with pbs solution , and returned to regular culture media with or without ta at 37c in a humidified 5% co2 incubator . there were 4 groups , including the control group , ta group , lidocaine group , and ta + lidocaine group .
each group was cultured in triplicate , and the culture medium was changed every 3 days .
cell viability , cell morphology , and expression of tenocyte - related genes were assayed at day 7 .
cell viability was determined using the cell - counting kit-8 ( cck-8 ) ( dojindo , kumamoto , japan ) according to the manufacturer s instructions .
tenocytes were seeded in 96-well plates at a density of 510 cells per well for 24 h. cells were exposed to 1% lidocaine for 15 min , followed by removal of each medium and returning to regular culture media with or without ta .
then , tenocytes were allowed to recover in regular medium for 24 h prior to assessing their viability .
for the cell viability assay , each well was supplemented with 10 l cck-8 and incubated at 37c for 2 h. the optical density was spectrophotometrically measured at 450 nm .
scleraxis ( scx ) and tenomodulin ( tnmd ) are closely related to tendon regeneration .
it positively regulates tendon - associated genes such as collagen i and tnmd expression in tendon fibroblasts . as a later differentiation marker of tendon cells ,
therefore , collagen i , tnmd , and scx were evaluated using rt - pcr in this study
. total rna was extracted using the rneasy mini kit ( qiagen , hilden , germany ) according to manufacturer s instructions .
cdna was synthesized using the first - strand kit ( invitrogen , carlsbad , california ) .
the qrt - pcr was carried out with quantitect sybr green rt - pcr kit ( qiagen , hilden , germany ) .
we amplified 2 l total cdna of each sample in a final volume of 50 l reaction mixture .
the cycling conditions were : held at 65c for 5 min , snap cooling at 4c for 1 min , 42c for 50 min , and at 72c for 15 min .
rat - specific primers were used for collagen i , tnmd , scx , and gapdh as follows : 5-ccggactgtgaggttaggat-3 ( forward ) and 5-aacccaaaggacccaaatac-3 ( reverse ) for collagen i ; 5-ccatgctggatgagagaggttac-3 ( forward ) and 5cacagaccctgcggcagta-3 ( reverse ) for tnmd ; 5-aacacggccttcactgcgctg-3 ( forward ) and 5-cagtagcacgttgcccaggtg-3 ( reverse ) for scx ; 5-tgactctacccacggcaagttcaa-3 ( forward ) and 5-acgacatactcagcaccagcatca-3 ( reverse ) for gapdh .
one - way anova with the student - newman - kuels test was used for multiple comparisons .
animal experiments were carried out under the rules and regulations of the animal care and use committee at harbin medical university .
sprague - dawley male rats ( weighing 200250 mg ) were used for isolation of tenocytes .
each patellar tendon was cut into small sections in aseptic conditions , and then placed and cultured in 6-well culture plates .
after 5 min of air - drying for better adherence , dulbecco s modified eagle medium ( dmem ) , 10% fetal bovine serum ( fbs ) , and 1% penicillin and streptomycin were supplemented to each well .
the explants were incubated at 37c in a humidified culture chamber supplemented with 5% co2 .
cells began to emerge from the tissue pieces after 34 days in culture and began to attach to the surface of the culture dishes .
after the cells reached 80% confluence , they were passaged by washing with phosphate - buffered saline ( pbs ) and detached with trypsin / ethylene deaminate triacetic acid ( edta ) .
rat patellar tendon - derived tenocytes were plated in culture plates . after 24 h of culture ,
attached tenocytes were treated with ta in culture media ( regular medium with 0.1 mg / ml of ta ) .
samples were then washed with pbs solution , and returned to regular culture media with or without ta at 37c in a humidified 5% co2 incubator . there were 4 groups , including the control group , ta group , lidocaine group , and ta + lidocaine group .
each group was cultured in triplicate , and the culture medium was changed every 3 days .
cell viability , cell morphology , and expression of tenocyte - related genes were assayed at day 7 .
cell viability was determined using the cell - counting kit-8 ( cck-8 ) ( dojindo , kumamoto , japan ) according to the manufacturer s instructions .
tenocytes were seeded in 96-well plates at a density of 510 cells per well for 24 h. cells were exposed to 1% lidocaine for 15 min , followed by removal of each medium and returning to regular culture media with or without ta .
then , tenocytes were allowed to recover in regular medium for 24 h prior to assessing their viability .
for the cell viability assay , each well was supplemented with 10 l cck-8 and incubated at 37c for 2 h. the optical density was spectrophotometrically measured at 450 nm .
scleraxis ( scx ) and tenomodulin ( tnmd ) are closely related to tendon regeneration .
it positively regulates tendon - associated genes such as collagen i and tnmd expression in tendon fibroblasts . as a later differentiation marker of tendon cells ,
therefore , collagen i , tnmd , and scx were evaluated using rt - pcr in this study .
total rna was extracted using the rneasy mini kit ( qiagen , hilden , germany ) according to manufacturer s instructions .
cdna was synthesized using the first - strand kit ( invitrogen , carlsbad , california ) .
the qrt - pcr was carried out with quantitect sybr green rt - pcr kit ( qiagen , hilden , germany ) .
we amplified 2 l total cdna of each sample in a final volume of 50 l reaction mixture .
the cycling conditions were : held at 65c for 5 min , snap cooling at 4c for 1 min , 42c for 50 min , and at 72c for 15 min .
rat - specific primers were used for collagen i , tnmd , scx , and gapdh as follows : 5-ccggactgtgaggttaggat-3 ( forward ) and 5-aacccaaaggacccaaatac-3 ( reverse ) for collagen i ; 5-ccatgctggatgagagaggttac-3 ( forward ) and 5cacagaccctgcggcagta-3 ( reverse ) for tnmd ; 5-aacacggccttcactgcgctg-3 ( forward ) and 5-cagtagcacgttgcccaggtg-3 ( reverse ) for scx ; 5-tgactctacccacggcaagttcaa-3 ( forward ) and 5-acgacatactcagcaccagcatca-3 ( reverse ) for gapdh .
one - way anova with the student - newman - kuels test was used for multiple comparisons .
the cell density was lower for cells cultured with ta or lidocaine compared to control .
the combination of ta and 1% lidocaine induce more obvious morphologic change ( figure 1 ) .
when the tenocytes were exposed to ta or lidocaine , the viability of the tenocytes was lower than that of the control group .
moreover , we found that viability of the tenocytes cultured with ta and 1% lidocaine was significantly lower than with ta or lidocaine alone ( figure 2 ) .
rt - pcr analysis on the cultures exposed to ta and lidocaine was performed as above .
we found that the expressions of tenocyte - related genes , collagen i and scx , were decreased by the treatment of ta or 1% lidocaine ( figure 3 ) , whereas no significant difference has been detected for the expression of tnmd .
when the tenocytes were incubated with the combination of ta and lidocaine , the expressions of tenocyte - related genes were significantly decreased when compared with ta or 1% lidocaine administration alone .
the cell density was lower for cells cultured with ta or lidocaine compared to control .
the combination of ta and 1% lidocaine induce more obvious morphologic change ( figure 1 ) .
when the tenocytes were exposed to ta or lidocaine , the viability of the tenocytes was lower than that of the control group .
moreover , we found that viability of the tenocytes cultured with ta and 1% lidocaine was significantly lower than with ta or lidocaine alone ( figure 2 ) .
rt - pcr analysis on the cultures exposed to ta and lidocaine was performed as above .
we found that the expressions of tenocyte - related genes , collagen i and scx , were decreased by the treatment of ta or 1% lidocaine ( figure 3 ) , whereas no significant difference has been detected for the expression of tnmd .
when the tenocytes were incubated with the combination of ta and lidocaine , the expressions of tenocyte - related genes were significantly decreased when compared with ta or 1% lidocaine administration alone .
current literature reports indicate that either ta or local anesthetics can have significant detrimental effects on tendon cells .
this study shows that ta has cytotoxic effect on tenocytes in vitro , which is synergistically increased when used in conjunction with 1% lidocaine .
many studies have been performed to investigate the effects of glucocorticoids on cultured tendon cells .
they showed that ta could decrease cell viability , induce apoptosis , block cell migration , and reduce collagen secretion of tendon cells .
tenocytes showed significantly lower mrna levels of collagen i and scx in the ta - treated group than in the control group .
. these results might account for the poor healing in tendons after injection of ta .
ta is commonly used to treat a wide range of tendon tissue disorders ; therefore , we selected it for use in our study .
several other investigators have studied the effects of local anesthetics on tendon cells in vitro and in vivo .
lehner et al . discovered a severe , reactive oxygen species - mediated effect of bupivacaine on tendon cells .
moreover , functional damage of tendon tissue was also elicited by bupivacaine . because local anesthetics are commonly used with corticosteroids injection , our study attempted to show the combined effect of lidocaine and ta on tenocytes .
this study showed that lidocaine affects not only morphology , but also functional activities of tenocytes .
we showed that the deleterious effects of ta were enhanced by the administration of lidocaine .
therefore , it we suggest that the combination of ta and lidocaine has greater adverse effects on tendon tissues than ta alone . these results are consistent with previous reports describing the toxicity of lidocaine in other cell types .
future in vivo studies are necessary to determine whether the combination of ta and lidocaine could induce the increased risk for tendon pathology .
ta injection in combination with lidocaine should be used with caution due to the deleterious effects .
one limitation of our study is that the underlying molecular mechanisms that are responsible for the deleterious effects of ta and lidocaine in this study are yet to be determined and need to be elucidated in further investigations .
our findings indicate that ta affects the properties of tenocyte by blocking the migration , decreasing the cell viability , and inhibiting the functional activities of the tenocytes .
this in vitro study provides information on the detrimental effects of these drugs on tendon tissues .
the use of ta with lidocaine in humans should be done with caution given the outcomes of this study .
backgroundlocal anesthetics are commonly used for the treatment of a variety of tendinopathies in combination with corticosteroids injection . 
 the goal of this study was to evaluate the effects of lidocaine and triamcinolone acetonide ( ta ) on cultured rat tenocytes and to determine whether there is a synergistic effect.material/methodsrat patellar tendon - derived tenocytes were cultured with or without ta and lidocaine , and the culture without any additive served as the control . 
 cell morphology and cell viability were evaluated . 
 expressions of tenocyte - related genes were measured by qrt-pcr.resultsta , when exposed to tenocytes in vitro , significantly decreased cell viability . 
 the cells cultured with ta had a flattened shape . 
 moreover , the expressions of tenocyte - related genes in tenocytes were markedly decreased in the ta - treated group . 
 we found that 1% lidocaine synergistically increased the deleterious effects of ta.conclusionsour data provide evidence of the detrimental effects of these drugs on tendon tissues . 
 injection of ta in combination with 1% lidocaine should be used with caution .