mechanical testing is frequently used to characterize tendons , and to describe the effects of interventions such as exercise or medication on their mechanical properties .
force , deformation and stiffness , derived from an ultimate tensile strength test , provide sample specific information about the mechanical behaviour of a tendon sample . from these values ,
stress , strain and elastic modulus are regularly derived in order to provide quantitative data concerning material properties , with the intention of characterizing the material irrespective of sample dimensions . however , when comparing the results of different studies , it is evident that the reported material properties , in particular the strain values , vary hugely , e.g. from 610% ( rigby et al . , 1959 ; tamiwa , 2007 ) to 1720% ( almeida - silveira et al . , 2000
it is well documented that the mechanical properties of tendons are influenced by a range of intrinsic and extrinsic factors including anatomical site ( haraldsson et al . , 2005 ) , age ( nakagawa et al . , 1996 ) , loading history ( simonsen et al . , 1995 ) or hormonal status ( inhofe et al . , 1995 ) .
in addition , results can also be influenced by experimental setup , including environmental conditions and test protocol ( schatzmann et al . , 1998 ;
however , even taking these factors into account , there is a remarkably wide range of reported material properties for tendon , with particularly large strain values frequently reported when analyzing the tendons of small mammals such as rats .
a recent study by tamiwa et al . ( 2006 ) suggested that stress is not able to accurately normalize force data for rat tail tendon fascicles , whilst two brief articles published in the 1980s describe an effect of specimen length on stress 
strain characteristics in rat tail tendon ( haut , 1986 ; sanjeevi et al . , 1982 ) .
a number of reasons for these variations have been hypothesized , including inhomogeneity of samples ( atkinson et al . , 1999 ) and end - effects ( lam et al . , 1988 ) .
the influence of each of these effects is important , as it highlights the difficulties in simply using stress and strain parameters to derive true material properties for non - homogenous soft tissues .
the aims of our study were to highlight the influence of specimen length and csa on apparent material properties , investigate why these occur , and determine if they differ between two functionally different tendons .
we hypothesize that the variations recorded in material properties with specimen dimensions are largely influenced by gripping the specimens .
however , we further suggest that variations in micro - structure between different sized samples may lead to different material properties .
tendon fascicles were dissected according to an established protocol ( screen , 2003 ) from five rat tails and five bovine foot extensors , all from healthy animals sacrificed for other , unrelated reasons . for each fascicle ,
the diameter was determined by a laser micrometer at multiple points along a 1  cm region in the middle of the fascicle .
fascicles were secured in a materials testing machine ( bionix100 , mts , 50n load cell ) by pneumatically driven grips with a serrated surface , exerting a gripping pressure of 3  gpa .
fascicles were loaded to failure at room temperature at a strain rate of 1%/s . during dissection and testing , specimens were kept moist by continually spraying with phosphate buffered saline solution ( pbs , sigma ) .
grip - to - grip length was varied and at each length 34 fascicles per animal were tested : rat tail fascicles were tested at 5 , 10 , 20 , 40 , 60 , 80 and 100  mm ( 103 fascicles in total ) ; bovine extensor fascicles were tested at 5 , 10 , 20 and 40  mm ( 67 fascicles in total ) .
force and deformation were both continuously recorded from the bionix100 at 50  hz and engineering stress and strain calculated using the csa and length of the sample at the start . from the resulting data , the point at which a 0.1  n load was detected was located , and defined as the test start point .
strain data were smoothed with a 5-point moving average filter , before calculating the modulus over every 8 values .
the modulus across the most linear region of the stress  strain curve and the maximum modulus were found ( fig . 1 ) and referred to as the linear and maximum modulus , respectively .
the end - effect was calculated to establish the effective specimen length , which is longer than the daylight length .
compliance ( elongation / linear moduluscsa ) against specimen length was plotted , and the regression line extrapolated to cut the x - axis and indicate the magnitude of the end - effect ( ker , 1981 ) .
material properties were then corrected for end - effects , to determine how they influence the measured data . to determine the strain distribution along the length of the fascicles , markers ( ink )
were drawn every 5  mm along bovine extensor fascicles of 20 and 40  mm lengths .
the quasi - static tests to failure were filmed ( olympus c-740 , 15  hz ) and the strain distribution determined at the beginning of the test , 50% of strain to failure , and in the last few frames prior to failure .
the grip - section was defined as the distance of the second highest marker to the upper grip and the second lowest marker to the lower grip , resulting in a constant grip - section length of 15  mm for both the 20 and 40  mm long samples .
the remaining distance was defined as the mid - section , which was 5  mm long in the 20  mm sample and 25  mm long in the 40  mm sample .
distances between the markers and the grips were measured using an image processing program ( imagej , national institutes of health , usa ) and local strains in each section were determined and expressed as a contribution to total sample strain . a one - way - anova ( post - hoc test : tukey ) was used to determine the effect of specimen length on mechanical parameters . to describe relationships between mechanical parameters and either csa or specimen length , pearsons correlation coefficient was used .
bovine extensor and rat tail fascicles at a specific specimen length were compared by an unpaired t - test , as were mid - section and grip - section strains in short and long samples .
mid - section and grip - section strains within a group of samples of the same length were compared by a paired t - test .
gripping samples during mechanical testing will always lead to stress concentrations at the grips , hence the reported failure properties should be considered a minumum . however , using identical testing methods for all specimens , the values can be compared .
failure stress , strain and modulus , derived directly from the bionix100 force and extension data , are compared for different sample lengths or diameters in figs . 2 and 3 , respectively .
strain to failure was significantly influenced by specimen length and consistently reduced as the sample length increased . by contrast ,
specimen length did not influence failure stress in rat tail fascicles , although in bovine fascicles , it was significantly lower in 40  mm than 5 or 10  mm long specimens .
the linear modulus increased with increasing specimen length in both sample types . whilst the effect of specimen length on mechanical parameters was similar in rat tail and bovine extensor fascicles , failure strains were consistently significantly lower in rat tail than bovine extensor fascicles , resulting in a significantly higher linear modulus in rat tail fascicles ( fig .
2 ) . strain measurements at longer specimen lengths were very consistent as evident from the particularly small standard deviations .
failure strain , stress and linear modulus all correlated significantly with csa at certain specimen lengths . in bovine extensor fascicles , correlations were only apparent in short specimen lengths ( 5 and 10  mm lengths ) , where csa positively correlated with strain and negatively correlated with stress and linear modulus . in rat tail fascicles , failure strain correlated positively with csa at specimen lengths of 5 and 20  mm , whilst stress and linear modulus correlated negatively with csa at the majority of sample lengths ( 1060 and 100  mm and 560 and 100  mm , respectively ) ( fig .
the linear regression lines , indicating that compliance is proportional to length , crossed the x - axis at 5.86  mm ( bovine extensor ) and 7.50  mm ( rat tail ) ( fig .
the end - effect at each grip can thus be estimated at 2.93  mm in bovine extensor and 3.75  mm in rat tail samples .
these data highlight that , having accounted for end - effects , a single linear modulus value can be used to describe each tendon type , found from the reciprocal of the regression lines to be 1000165  mpa for rat rail tendons and 714120  mpa for bovine extensors . correcting failure strains for end - effects reduced differences between short and long sample lengths ; however , significant reductions in failure strain with increase in sample length were still evident ( fig .
strain curves was consistently different for bovine extensor and rat tail fascicles , and in the case of the rat tail fascicles it also changed with specimen length .
the continuous modulus curves showed a characteristic two - peak course in rat tail fascicles in contrast to one peak in bovine extensor fascicles ( fig .
1b ) . in rat tail fascicles , the height of the first peak , rising to the maximum modulus , increased with increasing specimen length ( r=0.592 ; p<0.01 ) , enlarging the difference between the maximum and linear modulus .
by contrast , this difference remained constant in bovine extensor fascicles ( r=0.107 ; p=0.387 ) ( fig .
the location of the maximum modulus on the stress  strain curve , relative to the strain at failure , appeared to be constant and independent of specimen length in both types of tendon fascicles .
however , it occurred significantly earlier in rat tail fascicles at 217% of the strain at failure compared to 3613% of failure strain in bovine extensor fascicles ( fig .
6d ) in the grip - section were consistent for both sample lengths , mid - section strains were significantly smaller in larger specimens , highlighting that the variable strain distribution extended well beyond the gripping regions ( fig .
whilst similar trends were unsurprisingly seen at 50% of failure , local strains in the mid - section of short samples were significantly smaller than in the grip - section at this time point only ( fig . 6a ) , highlighting that strains did not increase homogenously throughout the test either , but increased more rapidly in the grip - section for both sample lengths ( fig . 6b and e ) .
we have highlighted how the material properties of tendon fascicles appear to be influenced by both specimen length and csa .
measurement of grip - to - grip failure strains yielded significantly larger values in shorter specimens , with a subsequent reduction in modulus in these samples , while fig .
3 also highlighted how failure stress and modulus both decreased with increasing sample diameter . increased total strains at short specimen lengths
further studies have suggested that this might be due to the end - effect , where extra unseen sample length within the grips contributes heavily to overall strain measurements in short specimens , whereas the response of longer samples more closely reflects true material properties ( bennett et al . , 1986 ; lam et al . , 1988 ) . a correction for end - effects in the current data notably reduced the magnitude of differences in strain between short and long samples , and also enabled us to find a single modulus value for each tendon type , indicating that the apparent changes in sample properties with length are likely to be almost entirely artifactual .
one method to avoid this is to use an extensometer , which provides a method of measuring sample length without including the grip -region , and thus eliminating end - effect artifacts .
3 indicates that material properties are also influenced by csa . while this is at odds with fig .
4 , in which a single modulus value was appropriately found for each tendon type , it may explain the large standard deviations for each sample length in this data , if modulus is additionally influenced by csa . a correlation between csa and failure stress
it is possible that some of the variation is still related to gripping , as collagen fibers in the centre of a larger fascicle experience less gripping pressure and are therefore more likely to shear and add to the end - effect , thereby increasing strain .
however , in rat tail specimens of 10 , 40 , 60 and 100  mm length , no correlation between csa and strain was observed , but csa did correlate with stress and modulus .
the smaller stress values of larger diameter samples may also be related to structural differences in the internal organization of samples .
this may encompass fiber organization , interactions between subfascicles connected by connective tissue sheaths , or the relative contribution of collagen fascicles and connective tissue sheaths .
it has been suggested that the decrease in the amount of areolar connective tissue in smaller samples might lead to an increase in modulus ( danylchuk et al . , 1978 ) .
alternatively , the proportion of fiber bundles running parallel to the axis of force might be larger in smaller specimens ( butler et al . , 1986 ) .
indeed , on a macro - scale , a study of human patella tendon has shown that small ( 1  mm ) and large ( 20  mm ) sections of tendon do not exhibit the same mechanical properties with larger modulus values in small specimens ( atkinson et al . , 1999 ) .
this has been attributed to structures other than the subfascicle , such as the epitenon or other connective tissue components .
average fascicle csa has been shown to be negatively correlated with tendon modulus , but positively correlated to the total tendon csa in equine superficial digital flexor tendon , also indicating an effect of structural organization on mechanical properties ( gillis et al . , 1997 ) .
indeed , differences in composition and structure of functionally different tendons , such as the rat tail and bovine tendons investigated in the current study , may explain many of the differences seen in their mechanical properties .
although failure strain , stress and modulus displayed similar trends with specimen length in both tendons , the shape of the stress  strain curve at different specimen lengths changed in rat tail fascicles only , and the effect of csa on mechanical properties was more pronounced in rat tail fascicles .
whilst end - effects explain the variation in failure strain with sample length , fig .
it has been reported previously that the strains close to the gripping points are higher than in the mid - section of specimens ( butler et al . , 1984 ; devkota and weinhold , 2003 ; wu et al . , 2004 ) .
this has been attributed to sample slippage in the grips , or premature failure , resulting from local stress concentrations at the grips ( butler et al .
, 1984 ; wu et al . , 2004 ) . while gripping may reduce the sample diameter at these points and lead to greater stress and more extension in these regions , failure occurred throughout the length of the samples , opposing the suggestion of localized damage near the grips , although we can not exclude grip - related damage triggering the rupturing process .
furthermore , two studies investigating the failure mechanics of tendons have found no reduction in the ultimate tensile strength of tendons failing at the grips compared with those failing in the mid - section ( ng et al . , 2005 ; smith et al . ,
the act of gripping is likely to alter the stiffness of the ends of the sample close to the grips .
however , the small diameter of the samples would suggest that this effect should only extend a minimal distance into the sample .
it does not readily explain a strain distribution along the sample length , evidently extending in excess of 5  mm from the gripping points .
considering these findings from a micro - structural perspective , they may indicate discontinuity of fibrils .
the applied strain is evidently transferred to those fibrils held within the grips , but our data imply that it may not be fully transferred to the adjacent fibrils near the centre of the sample , resulting in reduced strains in this area .
since the grip - section in our experiment was between 5 and 10  mm long at each end ( in sum constantly 15  mm ) , and the mid - section strains were notably higher in the short samples ( 20  mm ) than in the long samples , we conclude that this subunit is on average more than 5  mm and less than 20  mm long .
while tendon fascicles are thought to span the entire length of a tendon ( basso et al .
, 2001 ) and to be mechanically and functionally independent ( haraldsson et al . , 2008 ) , controversy exists regarding the length and continuity of collagen fibrils in skeletally mature tendons and ligaments ( provenzano and vanderby , 2006 ) .
fibrils are clearly discontinuous during tendon development ( birk et al . , 1997 ; provenzano and vanderby , 2006 ) and the variable csa along the length of some tendons , such as the human achilles tendon ( kongsgaard et al . , 2005 ) , suggests that discontinuous subunits are necessary to allow for this change in csa . however ,
in mature tendon only two fibril ends have been spotted using transmission electron microscopy to analyze 5639 fibrils ( trotter and wolfsky , 1989 ) , and no ends have been found in a study analyzing 7275 fibrils ( provenzano and vanderby , 2006 ) .
the extremely high aspect ratio and interweaving of collagen fibrils make imaging complex ( craig et al . , 1989 ) , and with a fibril length in excess of 5  mm , the likelihood of seeing an end is low . since the large aspect ratio of collagen fibrils complicates the experimental assessment of fibril length ,
theoretical approaches have also been used , estimating a mean collagen fibril length in mature rat tail tendon of 6.412.7  mm ( craig et al . , 1989 ) .
this corresponds positively with our estimation of > 5 and < 20  mm length of a discontinuous subunit .
our findings have implications for the mechanical testing of tendon tissue : while it is not always possible to control for fascicle length and/or csa , these parameters have to be taken into account when comparing samples of different dimensions .
it seems advisable to use longer specimens whenever possible to reduce the variability within a given subgroup of a defined fascicle length , and to avoid end - effects by using an extensometer in a portion of the specimen well clear of the clamps .
 stress , strain and modulus are regularly used to characterize material properties of tissue samples . however , when comparing results from different studies it is evident the reported material properties , particularly failure strains , vary hugely . 
 the aim of our study was to characterize how and why specimen length and cross - sectional area ( csa ) appear to influence failure stress , strain and modulus in fascicles from two functionally different tendons . 
 fascicles were dissected from five rat tails and five bovine foot extensors , their diameters determined by a laser micrometer , and loaded to failure at a range of grip - to - grip lengths . 
 strain to failure significantly decreased with increasing in specimen length in both rat and bovine fascicles , while modulus increased . 
 specimen length did not influence failure stress in rat tail fascicles , although in bovine fascicles it was significantly lower in the longer 40  mm specimens compared to 5 and 10  mm specimens . 
 the variations in failure strain and modulus with sample length could be predominantly explained by end - effects . 
 however , it was also evident that strain fields along the sample length were highly variable and notably larger towards the ends of the sample than the mid - section even at distances in excess of 5  mm from the gripping points . failure strain , stress and modulus correlated significantly with csa at certain specimen lengths . 
 our findings have implications for the mechanical testing of tendon tissue : while it is not always possible to control for fascicle length and/or csa , these parameters have to be taken into account when comparing samples of different dimensions . 
Introduction
Methods
Results
Discussion