exogenous collagen cross-linking recovers tendon functional integrity in an experimental model of...

Exogenous Collagen Cross-Linking Recovers Tendon FunctionalIntegrity in an Experimental Model of Partial Tear

Gion Fessel,1,2 Jeremy Wernli,1 Yufei Li,1,2 Christian Gerber,1 Jess G. Snedeker1,2

1Department of Orthopedics, University Hospital Zurich, Balgrist, Forchstrasse 340, 8008 Zurich, Switzerland, 2Institute for Biomechanics, SwissFederal Institute of Technology (ETH), Wolfgang-Pauli-Str. 16, 8093 Zurich, Switzerland

Received 1 June 2011; accepted 31 October 2011

Published online 18 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22014

ABSTRACT: We investigated the hypothesis that exogenous collagen cross-linking can augment intact regions of tendon to mitigatemechanical propagation of partial tears. We first screened the low toxicity collagen cross-linkers genipin, methylglyoxal and ultra-violet(UV) light for their ability to augment tendon stiffness and failure load in rat tail tendon fascicles (RTTF). We then investigated cross-linking effects in load bearing equine superficial digital flexor tendons (SDFT). Data indicated that all three cross-linking agents aug-mented RTTF mechanical properties but reduced native viscoelasticity. In contrast to effects observed in fascicles, methylglyoxaltreatment of SDFT detrimentally affected tendon mechanical integrity, and in the case of UV did not alter tendon mechanics. As in theRTTF experiments, genipin cross-linking of SDFT resulted in increased stiffness, higher failure loads and reduced viscoelasticity. Basedon this result we assessed the efficacy of genipin in arresting tendon tear propagation in cyclic loading to failure. Genipin cross-linkingsecondary to a mid-substance biopsy-punch significantly reduced tissue strains, increased elastic modulus and increased resistance tofatigue failure. We conclude that genipin cross-linking of injured tendons holds potential for arresting tendon tear progression, andthat implications of the treatment on matrix remodeling in living tendons should now be investigated. � 2011 Orthopaedic ResearchSociety. Published by Wiley Periodicals, Inc. J Orthop Res 30:973–981, 2012

Keywords: acute injury model; genipin; methylglyoxal; therapy; ultraviolet

Tendon injuries are generally associated with a weak-ened collagen structure, and this weakening has beenrelated to mechanical overuse and high tissue strains.1

Tendon tears can start as localized regions of tissuedamage that increase in size as continued mechanicalloading further weakens the tissue. For example rota-tor cuff tears, a common and potentially serious typeof tendon injury with a prevalence of up to 80% inelderly individuals,2 have been reported to measurablyenlarge in approximately 50% of cases after twoyears.3 In the case that a smaller tear progresses to alarge tear, surgical repair of a full rotator cuff tearremains one of the least successful orthopedic inter-ventions, with tear recurrence in up to 94% of cases(as summarized by Ref. 4).

To address this urgent clinical need, we are target-ing a minimally invasive intervention to treat smalltendon lesions, with the goal to stop their enlargementat an early time point. The idea is that together with aminimally invasive diagnosis,5 early repair could pre-vent tendon injuries from becoming clinically unman-ageable. Since tendon tear enlargement is associatedwith progressively weakened collagen tissue struc-tures, we propose an approach using injectable colla-gen cross-linking agents to prevent lesion enlargementby mechanical reinforcement of the surrounding intacttissues. In this sense, cross-linking may potentially

avoid the need for later, more-invasive surgery inpatients.

Collagen cross-linking agents have shown therapeu-tic potential in both experimental models and clinicalapplication. For instance, collagen cross-linking in-duced by irradiation with ultra-violet (UV) light hasbeen reported in augmenting biomechanical propertiesof self-assembled collagen threads.6 A similar proce-dure involving UV stiffening of the sclera has beenclinically applied in patients for treatment of keratoko-nus.7 Genipin (GP), a naturally occurring cross-linkerderived from the gardenia fruit used in traditionalChinese medicine has successfully been applied toimprove intervertebral disc strength and functional in-tegrity in experimental models of disc degeneration.8

GP possesses significantly lower toxicity compared toglutaraldehyde.9 Later studies investigated both GPand methylglyoxal (MG), a naturally occurring metab-olite, for their ability to diffuse, cross-link and alterthe mechanics of the annulus fibrosus as a potentialtreatment for degenerative disc disease.10 Wagneret al.11 previously used MG as a model for glycationinduced cross-linking, related to aging and diabetes,and found an increased stiffness in the annulus fibro-sus after treatment.

Based on this evidence, we hypothesized that exoge-nous collagen cross-linkers could also be candidates foraugmenting the biomechanical properties of tendon—specifically tendon stiffness and failure load. We fur-ther hypothesized that cross-linking could be appliedto arrest mechanical progression of tendon tears. Totest these hypotheses, we first screened three lowtoxicity collagen cross-linkers (GP, MG, UV) for theirability to augment tendon stiffness and resistanceto failure. Here we utilized a highly reproducible

Additional Supporting Information may be found in the onlineversion of this article.Correspondence to: Jess G. Snedeker (T: þ41-44-386-3755;F: þ41-44-386-11-09; E-mail: [email protected]);www.balgrist.ch\research.

� 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2012 973

experimental model for tendon mechanics (rat tailtendon fascicle; RTTF) that has been established inour laboratory.12–14 Next we verified the suitability ofthese cross-linking treatments in application to theequine superficial digital flexor tendon (SDFT), aclinically relevant, large animal, load-bearing tendonmodel.15 Finally, we selected the best candidate cross-linking agent to test the hypothesis that exogenouscollagen cross-linking can arrest tendon tear propaga-tion. To do this, we developed and implemented anovel model of partial-width, full-thickness tendontear propagation under cyclic loading to failure.

METHODSCross-Linking in Rat Tail Tendon FasciclesTails from skeletally mature rats (at least 17 weeks old) wereremoved and frozen (�208C) after sacrifice according to localand federal regulations. After thawing, approximately 6 mmlong rat tail tendon fascicles (RTTF) were carefully extractedfrom the middle part of the tail. They were randomly sepa-rated into three different cross-linker treatment groups andcut into two 3 mm long halves for paired analysis. One-halfof the fascicle pair was incubated for 4.5 h in either genipin(GP, n ¼ 7) or methylglyoxal (MG, n ¼ 7) cross-linking solu-tion, or cross-linked by UV irradiation for 30 min in presenceof a photosensitizer (riboflavin, n ¼ 7) (details in Supplemen-tary Material: S-1). The other half of the fascicle pair wasdesignated as a matched (untreated) control. Samples wereincubated in single baths to minimize contamination of theexperimental batch. Samples were finally tested to failure.In brief, samples were preloaded, preconditioned and rampedto failure at a constant strain-rate. Force-strain curves wereparameterized according to tangential stiffness in the linearpart of the material curve, onset of failure (yield point), fail-ure load (peak force), and failure strain (strain at peak force).Toughness was assessed as strain energy until failure(integral of the force-strain curve from zero strain to failurestrain). Viscoelasticity was quantified as relative energydissipation (hysteresis) during preconditioning and the rela-tive force drop at peak strain between preconditioning cycles(cyclic relaxation). The most promising cross-linker in RTTFwas then further subjected to creep tests in separate experi-ments (UV, n ¼ 6) (for details, see S-2).

Cross-Linking in Equine Superficial Flexor Tendon StripsEquine superficial flexor tendons (SDFT) were collected onthe day of slaughter at local butcheries. Whole tendons wereharvested from freshly slaughtered horses, wrapped in PBSsoaked gauze and frozen (�208C) until the day of testing.Horses with known pathologic history in the SDFT tendonwere excluded. Tendons with reddish discoloration and localswelling were further excluded after dissection as it indicat-ed potential tendon pathology. After thawing, SDFT tendonswere dissected with three parallel, 1 mm spaced blades. Thetwo paired 1 mm thick strips were then cut with two 3 mmspaced blades to yield an approximate width of 3 mm. Finallythe length was trimmed to 40 mm (Fig. 1A and B). Cross-sectional areas (CSA) were verified with a caliper (accuracy:0.05 mm). Generally, the cutting procedure was very repeat-able with overall mean sample cross-section of 3.04 �0.29 mm2 with no differences between groups. Samples fromone cut were suitable to be analyzed in paired experimentaldesign as they presented very similar mechanical properties

(see details: S-3). Sample pairs were randomly allocated toa cross-linking group: GP (n ¼ 18), MG (n ¼ 15), or UV(n ¼ 10). Each pair was then split into a test (cross-linking)and matched (untreated) control group. MG and GP cross-linking procedures were slightly modified from the RTTFexperiments, due to the size of the samples, with increasedconcentrations (GP: 0.02 M/MG: 0.03 M and incubation pro-longed to 72 h) (Fig. 1B–D). Mechanical tests to failure wereperformed as in the RTTFs with adapted prestress and pre-conditioning strains (details in S-3).

Recovery of Tendon Function After Induced TearSDFT were thawed and dissected with four parallel, 1 mmspaced blades to yield three 1 � 3 � 40 mm3 strips per cut(triplet). CSA of 39 strips from 13 SDFT triplets were mea-sured. Strips from one triplet were randomly allocated toeither a control (intact), injury (partial tear) or a cross-linkedinjury group (complete randomized block design). An injurywas imposed to the samples of the two injury groups usinga 2 mm diameter biopsy punch (verification of damagemodel see S-4). Samples were then incubated for 72 h in0.02 M GP solution (cross-linked injury group) and bufferonly (control and injury group). Following treatment, allsamples were cyclically loaded on an electrodynamic testmachine (ElectroForce 3200, BOSE Corporation, Eden Prai-rie, MN) equipped with a PBS filled climate chamber at roomtemperature. After preconditioning of 10 cycles from 1 to20 MPa, initial length was established at 1 MPa. All tripletswere then cyclically loaded from 5 to 25 MPa at 10 Hz untilfailure (Fig. 2). This high frequency was chosen to reach fail-ure within reasonable time using a physiological stress-range.16 Corresponding strain values (clamp-to-clamp) andnumbers of cycles to failure were recorded. Timed peakand valley strains were used to reduce the data into timedamplitude (eamplitude ¼ epeak � evalley) and mean strain(emean ¼(epeak þ evalley)/2). Constant stress amplitude was

Figure 1. (A) Dissected SDFT were cut into approximately1 mm thick and 3 mm wide strips that provided consistent colla-gen alignment and elastic modulus similar to the whole tendon.Here we show the cutting of three samples with four blades. Inbaseline tests to assess the effects of different cross-linkers weused only three blades to cut pairs of strips. (B) Intact controltendon strip. (C) Injured (biopsy punched) tendon strip. (D) In-jured and cross-linked (GP) tendon strip. (E) Control tendon stripafter failure due to cycling, note cloth was wrapped with cyanoac-rylate at the ends to prevent slippage. (F) Injured tendon stripafter failure. (G) Cross-linked injured tendon strip after failure.A version of the previously reported mechanical clamp assem-bly14 was modified to fit into the climate chamber.

974 FESSEL ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH JUNE 2012

divided by strain amplitude to calculate a secant modulus((speak � svalley)/eamplitude ¼ 20 MPa/eamplitude). Strain-time cur-ves showing an approximately linear region were best fitover timed mean strain as well as timed secant modulususing the least squares method. The slopes of these curveswere used to derive time dependent properties as relativemean strain increase over 1,000 cycles (creep rate) anddecrease of secant modulus over 1,000 cycles (modulusdecrease). Creep-rate and modulus decrease measures wereonly determined for samples that survived more than 60cycles (extracting reliable values from fewer than 60 cycleswas not possible due to a lack of linearity of the correspond-ing strain-cycle plots), but other mechanical parameters werecalculated for these samples and included in the analysis.Finally, numbers of cycles to complete failure (inability tosupport 25 MPa) was recorded.

Statistical AnalysisA one-sample t-test was used to assess the relative effects ofcross-linking in the quasi-static ramp to failure tests. ExactWilcoxon signed-rank test was used to assess parametersfrom creep tests (time limited experiments of 30 min did notyield approximately normally distributed data). Effects of in-jury/tear on local strains were also assessed with Wilcoxonsigned-rank tests. Fatigue experiments were assessed by

two-way ANOVAs analyzing main effects (random factor:triplets; fixed factor: treatment). Since cycles to failure, creeprate and modulus decrease were positively skewed, datawere log10 transformed before analysis to meet modelassumptions of normality, independence of errors, and sphe-ricity (equality of variances and covariances were assessedby Mauchley’s test). Post hoc pairwise comparisons of treat-ment effects were then made with Tukey Honestly Signifi-cant Difference Test for multiple comparisons. All pairwisetests were two-sided. Differences were deemed significant forp-values less than 0.05. Results are reported as means withstandard deviations if not stated otherwise. All statisticswere analyzed using SPSS v18.0 (IMB Corporation, NewYork, NY).

RESULTS

General Remarks and Quasi-Static ExperimentsCross-linking changed color of the samples to darkblue (GP) and dark yellow (MG) or remained clear(UV) after riboflavin was washed out. Material testcurves for all samples exhibited characteristics typicalof collagenous tissue mechanical behavior, with a non-linear toe and heel region, followed by approximatelylinear stiffness, followed by sample failure. In tests to

Figure 2. (A) Schematic of the loading protocol with 10 cycles of preconditioning, initial length (L0) measurement at 1 MPa andfatigue to failure cycling. (B) Schematic of the strain response during fatigue cycling (not to scale; number of cycles not representative)with two straight lines that mark the peak and valleys strains in the approximate linear part of the strain-time curve. (C) Plot of cyclesversus mean strain of a typical triplet. In this case the fatigue life of the GP cross-linked/injured sample exceeds that of its intactcontrol. (D) Peak and valley strain recorded from the machine with calculated mean strain of one sample. A straight line was fit to thelinear part of the mean strain cycle curve. The slope of that curve (0.0047�) was multiplied by 1,000 cycles to estimate a creep rate per1,000 cycles (here 4.7%/1k cycles). E: Secant modulus calculated from timed amplitudes (peak to valley strain) plotted against cyclecount. Over the straight line, a linear curve was fitted. The slope (�0.0985�) was used to estimate a modulus decrease over 1,000 cycles(here 98.5 MPa/1k cycles). R2 values indicate good approximation by the fit.

TENDON CROSS-LINKING 975


failure, nearly all untreated tendon samples (RTTFand SDFT) ruptured in the mid-substance, indicatingthat end effects of mechanical clamping were generallynegligible. Approximately 50% of GP cross-linked ten-don samples clearly failed at the clamps.

Force-strain curves of cross-linked RTTFs showedlittle ‘‘post-yield’’ deformation (onset and accumulationof damage) with a sudden and abrupt decrease in forceafter reaching maximum force (Fig. 3). While thesesamples exhibited a more abrupt failure mode, tough-ness (strain energy to failure) nonetheless increased(GP: p ¼ 0.001, MG: p ¼ 0.01, UV: p ¼ 0.02). Stiffnessincreased in the GP (p ¼ 0.003) and UV (p ¼ 0.01) butviscoelasticity measured as cyclic relaxation (GP:p ¼ 0.0001, UV: p ¼ 0.002,) and hysteresis (GP:p ¼ 0.0001, MG: p ¼ 0.0001, UV: 0.004) decreased. Incontrast, stiffness (p-value ¼ 0.28) and cyclic relaxa-tion (p ¼ 0.84) did not change in MG cross-linkedgroup (for all relative changes see Fig. 4).

SDFT cross-linking augmented mechanical proper-ties only the GP group with 17% increased stiffness,15% higher failure stress and 35% increased toughness(for absolute values and p-values see Table 1). Onsetof failure (yield) was not significantly increased bycross-linking. Similar to observed cross-linking effectsin RTTF, viscoelasticity was strongly affected by GPtreatment with 54% reduced cyclic relaxation and 28%reduced hysteresis. MG samples showed a 32% reduc-tion in elastic modulus and a 21% reduction in failurestress. UV samples remained unaffected in all meas-ures (data not shown). Based on these results GP wasselected for further investigation in the injury model.

Recovery of Tendon Function after Induced TearEleven of 39 samples (from 13 triplets) failed withinthe first 60 cycles and could not be used to measurecreep rate and modulus decrease (although othermeasures were extracted). Among these specimens, allsamples from one triplet failed completely within thefirst 60 cycles suggesting that the initial cut had dam-aged sufficient collagen fibrils/fascicles such that theentire triplet was compromised. Two other triplets(one from each the injury and cross-linked injurygroups) also indicated compromised integrity with thebiopsy punched samples of these triplets failing before60 cycles. The remaining premature failures occurredin single samples of triplets (one sample in the injury/cross-linked group and three samples in the injurygroup). Collectively, analysis of premature sample fail-ure indicated that the test protocol was generally re-peatable but that sample formation (cutting andbiopsy punching) was critical with respect to the me-chanical integrity of the samples. Further, contrary tofailure tests on intact samples, fatigue testing failurein all groups occurred mid-substance (Fig. 1E and F).

No differences in overall effect were observed afterrunning the two-way ANOVA both excluding tripletswith missing values due to premature cyclic failure aswell as including these triplets (number of cycles tofailure was the only parameter with no missing data).Data are therefore presented based on all samples.

The two-way ANOVA indicated a highly significantoverall effect of treatment on all measured variablesduring fatigue testing: Initial modulus (p-value ¼0.0001), initial mean strain (p-value ¼ 0.003), number

Figure 3. Force-strain curves of genipin (GP), methylglyoxal (MG), and ultra-violet/riboflavin (UV) cross-linked RTTFs and theircontrols. Samples were selected to show variability of different animals and fascicles that can be seen when comparing control curves.

Figure 4. Relative changes in mechanical properties induced by cross-linking. The baseline group shows the similarity of untreated(non-incubated) paired samples that was used to calculate design effects and statistical power of the experimental measures. Hysteresisand cyclic relaxation are only reported of the 2nd and 2nd to 3rd cycle of pre-conditioning, respectively. Means are reported withstandard deviations and stars denote significant differences at a p < 0.05.

976 FESSEL ET AL.


of cycles to failure (p-value ¼ 0.001, log10 trans-formed), creep rate (p-value ¼ 0.004, log10 trans-formed) and modulus decrease (p-value ¼ 0.004, log10transformed). Creep rate and modulus decrease washighly correlated (R2 ¼ 0.985), so only post hoc analy-sis for creep rate is reported. The effect of blocking ofsample triplets was only significant with respect tonumber of cycles to failure (p-value ¼ 0.001) comparedto the other measures (p-values ranging from 0.13 to0.30). Post hoc tests (Fig. 6) revealed that initial meanstrain was increased due to injury (injured: 5.2 � 1.1%vs. intact 3.6 � 0. 7%, p < 0.001) and cross-linkingpartly mitigated this effect (injured: 5.2 � 1.1% vs.cross-linked/injured: 4.2 � 0.6%, p ¼ 0.038). Whilestrains were higher in the injured/cross-linked sam-ples compared to intact samples, these differenceswere not significant different (cross-linked/injured:4.2 � 0.6% vs. intact 3.6 � 0.7%, p ¼ 0.191). Initialmodulus followed a similar but inverse trend withnon-significantly higher modulus of cross-linked/injured samples compared to injured (injured: 411 �97 MPa vs. cross-linked/injured: 481 � 63 MPa, p ¼0.224) and lower modulus of cross-linking/injuredsamples compared to intact controls (intact: 623 �105 MPa vs. cross-linked/ injured: 481 � 63 MPa,p ¼ 0.003). Number of cycles to failure was lower ininjured compared to intact samples (injured: 72 vs.intact: 14,842, p < 0.001), an effect that was partlyrecovered by cross-linking (injured: 72, cross-linked/injured: 3,307, p ¼ 0.012), however cross-linking of in-jured samples did not reach values of intact samples(p ¼ 0.01). Creep rate per 1,000 cycles of injuredsamples (4.4% strain/1k cycles) was higher comparedto cross-linked/injured (0.032% strain/1k cycles, p ¼0.014) and controls (0.062% strain/1k cycles p ¼0.028). Cross-linked/injured creep rates were quitesimilar to intact samples (p ¼ 0.871).

DISCUSSIONWe hypothesized that exogenous collagen cross-linkingmay have potential to augment the mechanical proper-ties of tendon, and could be used to mitigate mechani-cal tear propagation. The present study tested thesehypotheses using three cross-linking agents, genipin(GP), methylglyoxal (MG), and ultra-violet irradiationwith photosensitizer riboflavin (UV). These agentswere chosen due to their demonstrated potential tocross-link other collagenous tissues,6–8,10,11 theirpotential to diffuse into tissue,10 and their reportedlow toxicity.9

Our experiments confirmed that GP, MG, and UVaugment tissue mechanics on the fascicle level in a rattail tendon model (RTTF). However in contrast toobserved effects in tail fascicles, MG cross-linkingtreatment detrimentally affected tendon mechanicalintegrity in equine superficial digital flexor tendons(SDFT), and UV cross-linking had no apparent effecton SDFT mechanics. Genipin cross-linking of SDFTresulted in similar but smaller mechanical changesT

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(e.g., stiffness and failure stress increased maximally17%) compared to those observed in rat tail tendon(increases of at least 37% in RTTF according to thesetwo measures).

The observed discrepancies between MG cross-link-ing effects in RTTFs and strips of equine SDFT mayindicate a potentially important dependency on thehierarchical tissue level at which the cross-links areformed and/or the type of tendon. MG cross-links prob-ably bridge only between and within collagen mole-cules,17 in contrast to GP which can form polymersthat may also bridge between collagen micro-fibrils.9

This may partly explain why reinforcing MG cross-linking effects found in fascicles did not translate tothe more complex structure of the load bearing SDFT,while GP similarly affected both tendons. It is alsopossible that the two cross-linking agents differentlyaffected collagen fiber kinematics with correspondingimplications to failure mode. We suspect that the ob-served large reductions in viscoelasticity (hysteresis,cyclic relaxation, and creep) point to profound alter-ation of matrix mechanics, such as altered collagenfibril sliding behavior and not just increased collagenfibril stiffness. Such profound alterations could also beindicated by the observed changes in failure mode(Fig. 1G) with more abrupt force decrease of thestress-strain curves in cross-linked samples (Figs. 3and 5) and a tendency to rather fail at the clamp incross-linked samples. What implications these altera-tions may have in even more complex tendon struc-tures (such as the rotator cuff tendons) remains to beevaluated. In any case, MG cross-linking mimics theadvanced glycation end products related cross-linkingbetween and within collagen fibrils (or higher architec-tural levels of collagen organization) that is generallyaccepted to increase mechanical stiffness of collage-nous tissue.17 It is possible that the conflicting trendswe observed in these two tendon models may be usefulto investigate how increased age-related cross-linkingmay detrimentally affect mechanical properties of loadbearing tendons.18

With regard to between-model discrepancies ob-served in the UV treatment, we suspect that lack ofeffect in SDFT could have been attributed to

inadequate tissue penetration by the UV in the denserand larger equine tissue, although this remains to beconfirmed. It is also possible that incomplete penetra-tion by GP in the SDFT may have led to the reducedmechanical effects compared to RTTF, although themacroscopic color changes associated with GP penetra-tion were observed throughout the sample volume. Itis further possible that the differences in the mechani-cal testing protocols (e.g., level of preconditioning)could explain differences in the observed effects.

Given the demonstrated potential of GP to improvefailure resistance of SDFTs, we implemented an acuteinjury model to evaluate the effects of GP in mitigat-ing tear propagation during high cycle fatigue tests.Promisingly, GP cross-linking was able to partly recov-er mechanical integrity in biopsy punched samples,with reduced tissue strains, increased modulus andincreased resistance to fatigue failure compared to in-jured controls. We conclude from these findings thatGP cross-linking of injured tendons seems therefore tohold potential for arresting partial tendon tears oraugmenting injured tendons.

We attempted to predict experimental outcomesfrom the high cycle fatigue testing (sample failure,creep-rate, and modulus decrease) based upon the ini-tial mechanical properties at the onset of the fatiguetest. Linear regression models with initial modulusand mean strain as predictors (not described in theMethods and Results Section) best fitted but still wereunable to predict cyclic loading outcome parameters(R2 ranging from 0.31 to 0.46) in cross-linked samplescompared to injured and intact controls (R2 rangingfrom 0.63 to 0.73). However, creep rate and modulusdecrease correlated highly (R2 ranging from 0.96 to0.98) with cycles to failure in all groups. This suggeststhat failure in long-term cyclical loading cannot simplybe estimated by characterizing initial values or from anon-destructive analysis of the material curve, andthat high cycle testing is in fact necessary to elucidatecertain cross-linking effects.

The present study only assessed the passive me-chanical response (damage accumulation) of acellulartendon matrix to collagen cross-linking agents, andassessing biological impact in cell culture and living

Figure 5. Representative stress-strain curves from genipin (GP) and methylglyoxal (MG) cross-linked sample pairs along with theircontrols.

978 FESSEL ET AL.


tendons is a next critical step. GP has been reported tohave low toxicity,9 but has also been shown to altercell biology for instance by promoting neurite out-growth and differentiation, reversing B-cell dysfunc-tion in isolated pancreatic islets and inhibitingendothelial exocytosis in thrombosis and inflamma-tion. GP has been described as ‘‘cell tolerated’’ or mito-genic, as summarized by Wang et al.19 However, basedon their own findings, Wang and his colleagues pointout that GP has distinct effects on different cell typesand provide evidence of dose dependent and transienttoxicity in chondrocyte and osteoblast culture alreadyat much lower concentrations than those used in thepresent study. At the tissue level, it has been shownthat GP cross-linked artificial blood vessels yieldhigher initial stiffness, but that GP can affect invasionof smooth muscle cells and their ability to contract.20

These studies highlight the potential complexities ofintroducing an exogenous tissue cross-linking agentand the need to balance purely mechanical consider-ations against biological function. Future studies arerequired to determine whether specific concentrationsof GP can offer acceptably low cytotoxicity but stillmaintain the potential to augment tendon mechanicalproperties. It then would remain to be determined howthis critical concentration could be delivered in aclinically relevant therapy. Thus verifying the

potential to arrest tear progression in living tendons,better defining cell–matrix interactions and implica-tions to long-term tendon homeostasis, and the devel-opment of a viable delivery method all representessential future steps.

This study has some further limitations. We did notassess any changes of structure and composition (e.g.,histology and wet weight) due to long-term incubationin either control or treatment buffer, such as watercontent or collagen breakdown. Potentially, controlbuffer incubation may have increased viscoelasticityand could possibly explain the large reduced viscoelas-ticity we otherwise attributed to cross-linking. Long-term incubation without protease inhibitors couldhave weakened the matrix in our controls due to ma-trix metalloproteinase activity, although our previouswork indicates that these effects, together with struc-tural and compositional changes, should be negligiblein freeze-stored tendons.13 Despite these potentiallimitations to the sample conditioning protocols, weare confident that the likelihood of confounding experi-mental artifacts is small. With regard to our conjec-ture that mechanical differences in cross-linkingeffects on RTTF and SDFT could be related to tendonstructural hierarchy, these difference may also be dueto innate differences between species or differencesinherent between positional (RTTF) and load-bearing

Figure 6. Post hoc pairwise comparison of treatment effects from cyclic loading at 10 Hz from 5 to 25 MPa to failure. Means arereported with standard deviations. Horizontal bars indicate significant differences (p < 0.05).



(SDFT) tendons. However, the biochemical compositionand ultra-structure of both tendons is similar.12 A fur-ther limitation is that we did not assess collagen cross-linking density, however the clear color changes due tocross-linking indicated successful treatment (and tis-sue penetration) and the employed protocols are welldescribed in the literature.10 Finally, with regard totendon strain measurements, except for the character-ization of altered strains in the injury model, strainswere based on grip-to-grip separation. The use of ma-chine strains was necessary since the use of opticalstrain measurements in long-term, high-frequencytesting is impractical. While the effects of clamp slip-page cannot be precluded, we employed establishedsample mounting protocols that have shown slippageto be minimal.14

Concerning the plausibility of our tendon tear mod-el, the altered strain patterns around the injury well-matched our own theoretical predictions (Fig. 7) aswell as predictions of torn tendon strain patterns de-scribed by others.21 We thus believe that the injurymodel we present is suitable to assess the therapeuticpotential of exogenous cross-linking in partial tendontear. Application of the full-thickness, partial-width in-jury strongly altered the mechanical properties of thesample in fatigue to failure tests as some did not out-last 60 cycles. These samples were not used to mea-sure modulus decrease and creep-rate, as no linearbehavior could be observed. In the statistical analysiswe addressed the problem of missing data values byperforming the two-way ANOVA both excluding trip-lets with missing values as well as including thesetriplets. The relative inclusion or exclusion of samples

had no influence on statistical outcome, or the conclu-sions we were able to draw.

In conclusion, we demonstrated that GP has largepotential to mechanically augment tendon behavior ina wide range of loading conditions, and warrantsfurther (biological) investigation as a treatment fortendon injury. We found conflicting effects of MGcross-linking in a model using isolated fascicles com-pared to a more structurally complex load-bearing ten-don. These differences could be potentially interestingwhen trying to understand age-related alterations dueto endogenous cross-linking in tendon. We view this studyas an important first step for future investigations ofcross-linking in tendon—both patho-physiologicallyand therapeutically.

ACKNOWLEDGMENTSThe authors thank Mr. Hansrudolf Sommer for his expertiseand contribution to the mechanical testing. This study waspartly funded by the Swiss National Science Foundation,grant number 205321–118036 and the Balgrist Stiftung.

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Figure 7. (A) An example of a tendon strip used for analysis of surface strains in four regions of interest (rectangles) around theeventual biopsy punch site. Strips were marked with graphite surface markers to facilitate strain measurements. (B) Measured localsurface strains along the loading axis (y-strains) overlaid with the regions of interest. Average y-strains in each region were deter-mined. (C and D) Same sample as panels A and B with identical regions of interest and overlaid y-strains. Equivalent loads wereapplied to both intact and injured tendons. Injured samples showed lower strains at regions above and below the injury site and higherstrains at both sides; whereas intact tendon samples had more uniform strain fields. Statistical analysis showed significantly lowerstrains (�1.9%, p-value ¼ 0.002) in injured samples in the top and bottom regions compared to intact samples and significantly higherstrains (0.3%, p-value ¼ 0.008) in the left and right regions. This indicates load shunting from injured regions to intact regions, consis-tent with theoretical model predictions presented by others21 (method in S-4). [Color figure can be seen in the online version of thisarticle, available at http://wileyonlinelibrary.com/journal/jor]

980 FESSEL ET AL.


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