DOI: 10.1021/la100384e 9917Langmuir 2010, 26(12), 9917–9926 Published on Web 04/29/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Probing Collagen/Enzyme Mechanochemistry in Native Tissue withDynamic, Enzyme-Induced Creep

Ramin Zareian, Kelli P. Church,† Nima Saeidi, Brendan P. Flynn, John W. Beale, andJeffrey W. Ruberti*

Department of Mechanical and Industrial Engineering, Northeastern University, Boston, Massachusetts 02115.†Currently at Integra Life Sciences Corporation

Received January 27, 2010. Revised Manuscript Received April 9, 2010

Mechanical strain or stretch of collagen has been shown to be protective of fibrils against both thermal andenzymatic degradation. The details of this mechanochemical relationship could change our understanding of load-bearing tissue formation, growth, maintenance, and disease in vertebrate animals. However, extracting aquantitative relationship between strain and the rate of enzymatic degradation is extremely difficult in bulk tissuedue to confounding diffusion effects. In this investigation, we develop a dynamic, enzyme-induced creep assay anddiffusion/reaction rate scaling arguments to extract a lower bound on the relationship between strain and thecutting rate of bacterial collagenase (BC) at low strains. The assay method permits continuous, forced probing ofenzyme-induced strain which is very sensitive to degradation rate differences between specimens at low initialstrain. The results, obtained on uniaxially loaded strips of bovine corneal tissue (0.1, 0.25, or 0.5 N), demonstratethat small differences in strain alter the enzymatic cutting rate of the BC substantially. It was estimated that achange in tissue elongation of only 1.5% (at ∼5% strain) reduces the maximum cutting rate of the enzyme by morethan half. Estimation of the average load per monomer in the tissue strips indicates that this protective “cutoff”occurs when the collagen monomers are transitioning from an entropic to an energetic mechanical regime.The continuous tracking of the enzymatic cleavage rate as a function of strain during the initial creep responseindicates that the decrease in the cleavage rate of the BC is nonlinear (initially steep between 4.5 and 6.5% and thenflattens out from 6.5 to 9.5%). The high sensitivity to strain at low strain implies that even lightly loadedcollagenous tissue may exhibit significant strain protection. The dynamic, enzyme-induced creep assay describedherein has the potential to permit the rapid characterization of collagen/enzyme mechanochemistry in manydifferent tissue types.

Introduction

Collagen is the structural protein and load-bearingmolecule ofchoice in vertebrate animals. It seems that wherever there is adifficult mechanical environment (e.g., joints, tendons, ligaments,etc.), collagen provides the critical link between force andmotion.It is thus reasonable to suggest that mechanical forces or strainsplay a critical role in directing the placement and retention ofcollagen. This idea has been around in various forms since the late1800s.1-7 However, rather than assume that the connective tissuefibroblastic cells direct the removal and placement of individualcollagen monomers, we have suggested that collagen incorpora-tion, removal, and retention in fibrils is controlled directly by thestate of mechanical strain in the tissue.8 Fundamental to this“smart matrix” hypothesis is the idea that collagen molecular

stability is influenced by mechanical strain. Direct strain stabili-zation of collagen could explain why this material appears to“seek out” high tensile forces and how it persists in their path.

Collagen type I can be found in over 80% of collagenousstructures in vertebrate extracellular matrices.9 Collagen mole-cules are adapted to carry tensile loads and are often anisotropi-cally arranged into aligned hierarchical structures (fibrils, fibers,or filaments) which correspond to the direction of tensile load inload-bearing extracellular matrices. In spite of the importance ofthe role of collagen in a number of critical biological events(development, growth, repair of connective tissue), irreversiblepathologies (osteoarthritis, osteoporosis, intervertebral disk de-generation), and difficult tomanage injuries (ACL rupture), therehas been little progress in determining precisely how collagenousload-bearing tissue is initially organized, load-optimized, grown,and maintained.4,5 The manner by which load guides matrixretention and remodeling remains unclear, but the existence of acollagen/enzyme mechanochemical relationship might provideinsight into this important problem.

A series of in vitro experiments on collagen mechanochemistryhave demonstrated that collagen gains stability against thermaldenaturation10,11 and enzymatic cleavage8,12-15 when it is under

(1) Carter, D.; Beaupre, G. Skeletal form and function: Mechanobiology ofskeletal development, aging and regeneration; Press Syndicate of the University ofCambridge: Cambridge, 2001.(2) Carter, D. R.; Van DerMeulen, M. C.; Beaupre, G. S. Mechanical factors in

bone growth and development. Bone 1996, 18 (1 Suppl), 5S-10S.(3) Chalmers, J.; Ray, R. The growth of transplanted foetal bones in different

immunological environments. J. Bone Jt. Surg. 1962, 44B, 149-64.(4) Cowin, S. C. How is a tissue built? J. Biomech. Eng. 2000, 122 (6), 553-569.(5) Cowin, S. C. Tissue growth and remodeling. Annu. Rev. Biomed. Eng. 2004,

6, 77-107.(6) Roux, W. Gesammelte abhandulgen uber entwicklungsmechanics der organis-

men; Wilhem Engelmann: Leipzig, 1895.(7) Wolff, J.DasGesetzDerTransformationDerKnochen; AHirschwald: Berlin, 1891.(8) Bhole, A. P.; Flynn, B. P.; Liles, M.; Saeidi, N.; Dimarzio, C. A.; Ruberti, J.

W. Mechanical strain enhances survivability of collagen micronetworks in thepresence of collagenase: implications for load-bearing matrix growth and stability.Philos. Trans. R. Soc., A 2009, 367 (1902), 3339-62.

(9) Kadler, K. E.; Holmes, D. F.; Trotter, J. A.; Chapman, J. A. Collagen fibrilformation. Biochem. J. 1996, 316 (Pt 1), 1-11.

(10) Bass, E. C.; Wistrom, E. V.; Diederich, C. J.; Nau, W. H.; Pellegrino, R.;Ruberti, J.; Lotz, J. C. Heat-induced changes in porcine annulus fibrosusbiomechanics. J. Biomech. 2004, 37 (2), 233-240.

(11) Miles, C. A.; Ghelashvili, M. Polymer-in-a-box mechanism for the thermalstabilization of collagen molecules in fibers. Biophys. J. 1999, 76 (6), 3243-3252.

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applied mechanical load or strain. In a recent investigation,15

excised rat tail tendon fibers held at constant strain (stretch) andexposed to bacterial collagenase relaxed more slowly at higherstrain. These results suggest that the strain is protective of thecollagen, but there are some experimental concerns including thepossibility that the 4% strain difference between samples in thedense tendon tissue could have affected diffusion rates and the useof a room temperature apparatus. More critically, the initialtransient data were not used in the experiments because of aconcern regarding osmotic loading. As we will demonstrate, theinitial transient is where one can (and should) extract quantitativeenzymatic cleavage rate information before diffusion dominatesthe kinetics.

There has also recently been one in vivo study which hasdemonstrated that the application of tension to an intentionallydamaged intervertebral disk reduces the rate of degradation ofcollagen in annulus fibrosus in a rodent injurymodel.16 The latterstudy suggests a potentially important clinical route to theprevention of tissue loss following injury via the application ofmechanical tensile strain. Such a possibility was predicted in2005.14 However, a connection to strain-altered enzyme/collagencleavage rates was not established.

Taken together, there is a growing evidencewhich suggests thatcollagen may possess mechanochemical stability-enhancementproperties. Strain protection against enzymatic cleavage poten-tially permits collagen to preferentially remain in the path ofapplied loads which could help explain collagen’s dominance asa load-bearing molecule in animals. In this investigation, anattempt is made to extract a lower bound for the relationshipbetween strain and the cutting rate of loaded collagen by bacterialcollagenase at low (near physiological) strains. As will be demon-strated through diffusion/reaction rate scaling arguments appliedat the surface of the sample, this can be done by examiningthe difference in the initial strain rates of native collagenoustissue exposed to a degrading enzyme under constant load(enzymatically induced creep experiments). The enzymatic creepassay is well-suited to examine bulk tissue mechanochemicalbehavior principally because the probe value (strain) is externallydriven. Unlike stress-relaxation experiments in tissue, where theprobe value (load) decays, the energy to induce creep is externallyapplied and under user control. As will be seen, this assaymethodpermits a reasonable estimation of the number of loaded collagenmolecules being cut per second by the enzyme.

Experimental Section

Specimen Preparation. The corneal stroma is a relativelyacellular, hydrated network of fibrils comprising heterotypic typeI/V collagen. In addition to their high collagen content, corneaswere chosen for their transparency and their unique fibril archi-tecture comprising aligned, alternating arrays of monodispersediameter collagen fibrils. Corneaswere obtained from right or lefteyes of 2 week old cows (40-100 pounds) from Research 87 Inc.(Boylston, MA). After cleaning the globes, the corneas were

excised followed by epithelial and endothelial debridementwith a razor blade. The bare stromal tissue was stored at-80 �C until use. Prior to testing, specimens were devitalizedby freezing and thawing (to -80 �C). A custom-made cuttingdie was used to generate accurate and repeatable test stripsfrom the tissue. The central region of each dissected cornea wasused to produce one vertically-oriented (superior-to-inferior)tensile specimen approximately 0.75 ( 0.1 mm thick � 17.5 (2.5 mm length � 6 mm wide. During excision and mountinginto the test chamber, all specimens were kept moistened with37 �C Dulbecco’s Modified Eagle’s Medium (DMEM; Media-tech Inc., Manassas, VA).

Mechanical Loading Apparatus. To investigate the effect ofmechanical forces/strains on the rate of degradation of tissuestrips, an environmentally controlled, miniature uniaxial testingdevice was designed and constructed (see the Acknowledgment,Supporting Information, and thesis of K. P. Church17). Thedevice comprises a load cell (Honeywell Sensotec, model 31;max, 5 lb; resolution, 0.0002 lb; Columbus, OH) and a uniaxialmotor (Zaber Technologies T-LA60; resolution, 16 μm; maxspeed, 4 mm/s; speed resolution, 0.001 mm/s; Vancouver, BC,Canada) which are under the control of a custom LabViewprogram. The combined system load accuracy with PID control-ler is(0.01N.The specimen chamber’s quartz glass allowsopticalaccess for polarization microscopy of the specimen and isequipped with an integrated PID driven temperature controlsystem. Each corneal strip was positioned between two cam gripsinside the chamber, immersed in 37 �C DMEM and uniaxiallystretched. To prevent slippage of the tissue, following clamping,cyanoacrylate glue was applied to the sample grip interface andpermitted to dry (briefly). In addition, the load on the specimenwas monitored for sudden relaxation (an indicator of slip), andmovies were taken during at least two experiments from each loadvalue to verify reported strains (see the Supporting Information).In the experiment, strain, ε, was calculated directly from the tissuegrip displacement, l, using the initial specimen gage length, l0, asthe reference: ε= (l - l0)/l0.Digestion Protocol. Crude bacterial collagenase (BC) from

Clostridium histolyticum (Clostridiopeptidase A; Sigma-Aldrich,no. C0130) with a molecular weight range from 68 to 125 kDawas used. In order to activate the solution, each mole of collage-nase requires four moles of calcium [Ca2þ]. DMEM providesenough calcium to support the enzyme at the concentrations usedin our system. Loaded tissue specimenswere “crept-in” for 15minin DMEM. Control experiments without digestion exhibitedslight additional material creep (max 2% over 1.25 h) whichwas much less than the enzymatically induced creep (see Figure 4and the Supporting Information). TheDMEMwas replacedwithsolution containing BC (DMEM and 0.05 mM BC) for theduration of the experiment. The concentration far exceeds theMichaelis-Menten constant,Km, for the collagen/BC interactionand the surfeit of enzyme relative to available monomer shouldensure maximum reaction rate kinetics. It should be statedthat because this is a diffusive, erosion problem, strictMichaelis-Menten kinetics cannot be applied without some modification.Nonetheless, it is possible to estimate the rate of loadedmonomercleavage from this data directly. For consistency, collagenasesolutions were made previously and stored in individual vials at-80 �C until use for each experiment. Before injection into thechamber to initiate the degradation process, collagenase solutionwas preheated in a water bath (37 �C) for 30 min. Collagenaseactivity can vary from batch-to-batch and can decline with time;however, the repeatability of our experimental series indicatedminimal batch-to-batch variation, and the short duration of ourexperiments (

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Polarization Microscopy (Birefringence): Dynamic Ima-ging. One of the reasons the corneal stroma was chosen as a testspecimen is because it comprises a transparent array of alignedcollagen fibrils which alternate in direction.18 Corneal collagenfibrils are also effectively mechanically isolated (with regard totension), as there are only weak proteoglycan interactions bet-ween individual fibrils.19 Thus, uniaxial strain preferentially loadsfibrils oriented in the direction of the load while others remainrelatively unloaded. One of our goals was to use polarization todiscern which fibrils are degrading faster in real time (loaded orunloaded). Polarization lens (43 mm diameter, Prinz, Japan) axeswere set to 45�-135� to select corneal fibrils aligned with the loadaxis (90�) in our setup. To select arrays of unloaded corneal fibrils(i.e., oblique to the loadaxis), the polarization lens axeswere set to0�-90�. The specimens were then digitally imaged (Prosilica,model CV640; black and white; frame rate, 120 fps; resolution,9.9 � 9.9 μm per pixel; Newburyport, MA) every 10 s for up to90min through crossed polarizers during each experiment.Movieswere generated from these pictures tomonitor the degradation rateon the set of illuminated fibrils and permit examination of thespecimen for anomalous behavior (premature tearing or evidenceof grip slippage; see movie in the Supporting Information).

Loading Protocols. All experiments in the protocols wereperformed in load-control mode (fixed load with feedback) whileboth strain and load were recorded. After a specimen was posi-tioned inside the chamber, the samplewas stretched until a loadof0.01 N (our threshold for resolution) was detected and theresulting strain was our reference or “zero strain”. The experi-mental loads of 0.1, 0.25, and 0.5 N were chosen based on anestimate that the initial, per monomer loads in the tensile speci-mens are close to the molecular transition from an entropic to anenergetic mechanical state.20 In load-control, the creep becomesdynamic, permitting the specimens to “sample”multiple strains ina single experiment. The specific experimental loading protocolswere as follows:

Control Test with Load. After preloading 0.01 N, specimens(N = 9) were loaded at 0.1, 0.25 or 0.5 N for 75 min while bothstrain and load were captured from samples. This test wasdesigned to determine the expected normal creep response andto demonstrate that the grip design was adequate at the variousapplied loads.

Digestion Test with Load.After preloading 0.01N, specimens(N= 15) were loaded at 0.1, 0.25, or 0.5 N for 15 min (creep-in).Then the DMEM was replaced by preheated active BC solution(0.05 mM) for an additional 75 min or until failure.

Digestion Test without Load (Probed with Sudden Reload).After preloading 0.01N, specimens (N=5)were loaded at 0.25Nfor 15min (creep-in). Specimenswere thenunloaded, andDMEMreplaced by 0.05 mM preheated, active BC solution for anadditional 35 min. Specimens were then reloaded to probe themechanical integrity of the specimen at 0.25 N loading untilfailure.

Mechanical Characterization of Tissue. In general, thecornea is, like most collagenous connective tissue, a nonlinear,viscoelastic material with an increasing equilibrium tensilemodulus as a function of increasing force/strain. Its uniaxialmechanical response is generally more complex than that oftissues which possess uniformly aligned collagen due to thedistributed orientation of collagen lamellae18,21 in the plane ofthe tissue. Control tensile tests on the cornea (see the Support-ing Information) indicate that the tissue has a viscoelastic time

constant which is O(10 min). The initial tensile modulus for thetissue strips for each experiment was determined just prior tothe addition of collagenase. For comparison, the equilibrium(long-time) modulus was also obtained for two of the threeexperimental loads (0.1 and 0.25 N).

Time Scale Ratio Estimate. BC is an aggressive binder andcleaver of collagen, and thus, the time scale for collagen cleavage islikely to be shorter than the time scale for diffusion into the wholetissue strip. Given data obtained from our own assay (see theSupporting Information) for the rate-limiting step in collagenfibril catalysis for our specific enzyme, Kcat (∼0.033 s-1), and anestimated enzyme diffusion coefficient in the cornea, De of ∼1 �10-7 cm2/s (based on the diffusion coefficient of a similar sizedmolecule, albumin22), an initial reaction-diffusion time scale ratiocan be calculated from the following equation:

Ts ¼Cc

CeKcat

� �

h2

4De

ð1Þ

whereCc is the concentration of collagen in the corneal strip,Ce isthe concentration of enzyme in the chamber, and h is the half-width of the tissue strip.Ts calculated in this way is∼0.1. The timescale ratio is a reflection of the balance between the time to digestthe available collagen in the tissue (numerator) and the time forthe enzyme to diffuse to the center of the sample (denominator).The value ofTs less than 1 indicates that, for corneal tissue (h∼375μm), the diffusion delay will dominate the cutting rate. However,at the beginning of the experiment (i.e., for short enzymaticpenetration distances, or small h), the reaction speed of theenzyme will be the limiting factor and will dominate the physics.This is because diffusion is quite fast over short distances.Thus, it is possible to extract good estimates for the reactionspeeds for the enzyme if we examine the initial few minutesof the induced-creep response. Using eq 1, we can calculatethe estimated crossover point, where the reaction-diffusiontime scale ratio is approximately 1.0. The crossover occurs at∼6 min. We use this argument to justify extracting enzymaticcleavage rates during the initial reaction rate limited period (upto 6 min). There is clearly some limitation of the validity of thisassumption due to fibril packing, proteoglycan presence, andthe actual activity of the BC on corneal collagen packed intofibrils. Nonetheless, for awell-designed enzymatic creep experi-ment, there will be an initial time period where the degradationreaction is enzyme activity rate limited. During this period,estimates of enzyme reaction rates on loaded tissue may beextracted.

Estimate of Lower Bound on Enzymatic Cleavage Rate.Figure 1 demonstrates the expected progression of the experi-ment where the outer surface layers of the tissue are cleavedfirst by the diffusing enzyme. During enzyme-induced creep,the enzymatic cleavage rate of loadedmaterial can be obtainedby determining the kinetics of loaded area loss. Area loss maybe estimated with reasonable accuracy by assuming that, as theenzyme removes loaded fibrils from the outside of the tissue,load is transferred to uncut fibrils (deeper in the sample, seeFigure 1). The resulting increase in strain should reflect theamount of loaded area which has been “lost” to the enzymeactivity. Neglecting the viscoelastic contribution (as we do)will result in an underestimate of the area loss in this experi-ment (as viscous dissipation will retard the strain rate). Like-wise, by holding the tensile modulus constant for each force,E 6¼ f(t), the area lost will be further underestimated. This isdue to the fact that there is a nonlinear relationship betweenstress and strain in the corneal tissue in the regime examined.

(18) Meek, K. M.; Leonard, D. W. Ultrastructure of the corneal stroma: acomparative study. Biophys. J. 1993, 64 (1), 273-280.(19) Scott, J. E. Proteoglycan: collagen interactions and corneal ultrastructure.

Biochem. Soc. Trans. 1991, 19, (4), 877-881.(20) Sun, Y. L.; Luo, Z. P.; Fertala, A.; An, K. N. Direct quantification of the

flexibility of type I collagen monomer. Biochem. Biophys. Res. Commun. 2002, 295(2), 382-386.(21) Meek, K.M.; Boote, C. The organization of collagen in the corneal stroma.

Exp. Eye Res. 2004, 78 (3), 503-512.(22) Maurice, D. M.; Watson, P. G.,The distribution and movement of serum

albumin in the cornea. Exp. Eye Res. 1965, 4 (4), 355-363.

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Our simplified equation for calculating the instantaneousloaded area, A(t), is

AðtÞ ¼ FEEðtÞ ð2Þ

where F is the force (known and fixed), E is the initial modulusof the tissue (specific and fixed for each applied load), and ε(t)is the instantaneous strain (measured). Given the argumentsabove, this estimate of the load-bearing area “consumed” bythe enzyme will be a lower bound. Samples which undergolarger strains or strain rates will be more affected by theunderestimate created by eq 2. This is an important point.

To obtain the rate of load bearing area loss (which is reflectiveof the enzymatic activity on the load-bearingmonomers), the firstderivative of eq 2 can be taken. To do this, a five point centereddifference was used (Figure 6 data).

To estimate the modulus of the loaded fibrils in the tissue, thecutting rate of the enzyme, and the average per monomer load, itwas necessary to determine how many collagen monomers in acorneal strip cross section are carrying the load. The initial load-bearing area in a representative tissue strip was calculated usingthe assumption that approximately 18%of the fibrils in the cross-sectional areaof the tissue strip are oriented such that theydirectlysupport the load (i.e., run from grip-to-grip). This estimate is

based on calculations performed on X-ray data on bovinecorneas from Prof. Keith Meek’s laboratory (refs 18 and 21,and personal communication with Prof. Meek). Calculationsbased on the hydration of the normal cornea, the tissue cross-sectional area, themolecular packing of the collagenmonomersinto fibrils, and the number of fibrils aligned with the loadsuggest that there are approximately 9.0 � 1010 monomers in agiven cross section which are carrying the applied load. If theapplied forces are divided by the number of monomers in crosssection, an average initial load of approximately 1.1 pN permonomer in the 0.1 N test, 2.75 pN per monomer in the 0.25 Ntest, and 5.5 pN per monomer in the 0.5 N test is found. Thisload range occurs near the entropic to energetic transition inmechanical properties for type I collagen.20

Statistical Analysis. The single controlled-variable in theexperiments was the applied load. Comparisons of the dependentparameters of interest (time to tissue failure, fraction of arearemaining, and enzyme cutting ratemaximum) weremade using atwo-tailed Student’s t test for unpaired experiments. These statis-tics were designed to reject our null hypothesis: Enzyme cuttingrate is independent of applied load.

Results

Polarization Imaging. The dynamic polarization imagesconfirmed the amount of creep in the samples and indicated thatthe grips held the tissue throughout the experiments. While thereis significant birefringence signal change in the loaded/degradedsamples, the rapidity of the experiments and the dynamic stretch-ing limited our ability to extract a distinct preferential attackon the unloaded fibrils (which was the original goal). Figure 2displays the polarization images captured from specimens at0�-90� and 45�-135� relative to load direction in control anddegradation tests. Figure 2A (control test with load 0.25 N)demonstrates that the polarization signal does not change appre-ciably with time. Figure 2B (digestion test with 0.25 N load)demonstrates that the loaded specimens lose birefringence inten-sity in general over the course of the experiment but that the signalfrom off-axis aligned structures is nearly extinguished. Figure 2Cdemonstrates that when samples were unloaded, the degradationdid not change the birefringence signal appreciably. However,upon reloading, the sample rapidly loses birefringence for fibrilsoff the load axis. This is likely because fibrils are degraded, butremain in place until loaded. In general, in these rapid degrada-tion tests, the birefringence images do not provide clear andconclusive evidence of preferential degradation of unloadedfibrils. Nonetheless, the images are quite useful in support ofthe instrument readings bypermittingdirect examination of strainrates and load-bearing tissue remaining. Examination of the lastframes in Figure 2B, S4 and 2C corroborates the strain datareadings and shows that the 0.25 N continuously loaded sample(2B; last frame) clearly has more loaded material left than eitherthe 0.1 N continuously loaded sample (S4; last frame) or the0.25 N load/unloaded sample (2C; last frame) even after a longerexposure time (75 vs 65 min). See Supporting Information moviefor dynamic view of experiment.General Strain versus Time Response of Samples. Mecha-

nical Response of the Tissue. The initial modulus obtained fromthe mechanical characterization of the tissue strips was 3.0 MPafor the 0.1 N load, 5.9 MPa for the 0.25 N load, and 6.3 MPa forthe 0.5 N load. This modulus is calculated by using the instanta-neous values of stress and strain at the 15 min time point. Theequilibrium strain is

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indicates that, for the corneal strip in this experiment, the tissue istransitioning to a stiffer mechanical regime.

Controls. The strain in the 0.1 N control is relatively stableafter 15min, but the strain in the 0.25N and 0.5N controls creepssteadily for 1 h after the 15 min initial creep-in period. The totalcreep during the post creep-in hour is below 2% in the 0.1, 0.25,and 0.5N tests. Given the large rates of creep induced by exposureto enzyme in the experimental series, this small control creep rateis acceptable. In addition, the slightly increased steady creep in themore highly loaded samples makes the final conclusions of thisstudy even more conservative (a higher creep/stretch for lowerloaded sample).

Experimental Series: Continuous Load.Figure 3A-C showsthe tissue response in the experimental series where the appliedload is held continuously until specimen failure in the presence ofcollagenase. Failure is defined as the point where the load can nolonger be sustained by the tissue at the maximum rate of strain

(which is limited by the linear actuator maximum speed). Whatcan be observed in the enzymatic creep response of the specimensis that degradation begins to weaken the tissue soon afterintroduction of the enzyme to the chamber. There is an initialincrease in the strain for all of the continuously loaded specimens.Following the initial relatively linear strain response (after about30 min of exposure), there is a rapid and nonlinear increase in thestrain for all samples which culminates in failure. This phase ofthe creep response is more variable (see error bars) and likelyindicates amore chaotic period where there is both enzymatic andoverload-induced mechanical fibril failure.

Experimental Series: Load-Unload-Reload. This experi-ment was conducted because there was no simple way to con-tinuously probe the tensile mechanical behavior of the entiresample at zero load. Instead, the tissue was exposed to enzymeunder zero load conditions for a set period of time, then reloaded(at a judicious time point) to probe the mechanical state of the

Figure 2. Polarization imaging of tissue strips before collagenase exposure and after sample failure. Polarization axes were oriented at either0�-90� or 45�-135� (see first frames of (A) for polarization axis orientation example indicated by dashed lines). (A) Control test with 0.25Nload, no degradation; (B) digestion test with 0.25 N load; (C) digestion test without load. Figure S4 in the Supporting Information showsdigestion test with 0.1 N load.

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sample. In Figure 3D, it can be observed that specimens whichwere unloaded and exposed to 0.05 mM BC at 37 �C for 35 min,then reloaded, experienced a sharp rise in strain and could notsustain the load (0.25N) formore than 5-10min upon reloading.Comparison of Strain versus Time Response of Samples.

In Figure 4A, the 0.25 N control and experimental runs areplotted on the same axes (strain vs time) with error bars. The datain Figure 4A indicate that completely unloading the tissue duringexposure to collagenase results in a significant decrease in the timeto failure (2550 ( 88 s vs 3730 ( 137 s; p , 0.05). In Figure 4B,the three continuously loaded samples (0.1, 0.25, and 0.5N) areplotted on the same axes (strain vs time). It can be seen thatthe 0.25 and 0.1 N curves appear to be different only at the verybeginning of degradation and at the end. Further, the 0.1 N loadfails before the 0.25 N loaded sample (2822 ( 384 s vs 3730 (137 s; p , 0.05). It is important to note that if the degradation

rates were equal, the strain should be different at all time pointsfor all samples. The convergence of the strains for the 0.1 and0.25N samples (early) and the convergence with the 0.1 and 0.5Nsamples (late) are consistent with the hypothesis that strain (asopposed to load) is the relevant parameter for mechanochemicalinfluence on enzymatic activity.Area Loss Dynamics. In this investigation, we are trying to

reject the null hypothesis which states that the degradation rate ofthe tissue is unaffected by small strain differences. Another goal isto determine themagnitude of the strain protection in terms of theenzymatic activity.

Effective Load-Bearing Area Loss. Figure 5 shows the decayin the effective load-bearing area for the entire time coursecalculated with eq 2. In spite of the conservative assumption ofconstant modulus, it can be readily observed that during this timethere is a significant increase in the loss of loaded area from the

Figure 3. Strain and load versus time for the four loading protocols. (A) Load controlled to 0.1 N, (B) load controlled to 0.25 N, (C) Load-controlled to 0.5N. (D)Load initially controlled to 0.25Nand then unloaded at time of addition of collagenase; load reapplied after 35min toprobe degradation of structural components. Load was the controlled variable, and data are representative of one experiment. Arrowsindicate time of addition of collagenase and point of failure of tissue (load can no longer be maintained).

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sample which is under the 0.1N load (relative to both the 0.25 and0.5 N loaded samples). Within the first 10 min of enzymeexposure, the corneal strips loaded with 0.1 N have lost morethan 50% of their load bearing fibrils compared to∼30% for the0.25 and 0.5 N loaded specimens.

Rate of Load-Bearing Area Loss. Figure 5 shows that, at thevery beginning of the experiment, just after enzyme addition to thechamber, there appears to be a substantial difference in the loaded-tissue area loss rate between the 0.1 and 0.25/0.5 N samples. Takingthe first time derivative of the load-bearing area and plotting itagainst time, the strain produces a set of curves which suggest thecomposition of multiple physical processes (Figure 6 series). InFigure 6A and B, the enzymatic cutting rate is plotted against time.The curves showan initial rise in cutting rate to amaximumfollowedby a decline. The initial rise represents the rate of cutting as theenzyme arrives at the specimen surface and begins cutting load-bearing tissue. The peaks of the curves represents the maximumcutting rate obtained.Thedecline in the cutting rates are likely due to

diffusion limitations at longer times, but to strain effects at shortertimes (before diffusion limitations slow the reaction rate, i.e., up to6 min). Examination of the data shows that the maximum rate ofarea loss (which is directly reflective of the enzymatic cleavage rateon loaded tissue) ismore than 2 times larger (p

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from the 0.25 N sample. The ratio of the 0.1 N to the 0.25 N(or 0.5 N) cutting rates is ∼2.6. Figure 6C and 6D show thecollagen cleavage rate against strain. These data demonstrate thatthe sharp decline in the enzyme activity occurs over a very smallstrain difference (∼Δ1.5%). Further, the cutting rates convergerapidly as the strain values for the two samples converge.A linear regression of the data for the 0.1 N loaded sampleindicates that the relationship between strain and enzyme activityon loaded fibrils is linear from 4.5 to 6.5% strain and declines at arate of ∼30%/1%. The maximum cutting rate for the 0.5 Nsample suggests that the strain protection effect does not continueto increase with strain after 6.5%, but rather the relationshipbetween strain and cutting rate flattens out as the strain increases(see Figure 6D). Thus, protection appears to occur quickly andremain in effect up to at least 9.5% strain.Note onDiffusion andMechanical Failure Effects. Diffu-

sion. Enzymatic erosion tests of differentially loaded tissuesamples are generally subject to diffusion differences caused bymatrix compaction/extension. At the beginning of the load-control tests in this investigation (where the largest differentialin enzymatic activity was found), the difference in the strainbetween the 0.1 N sample and the 0.25 (or 0.5 N) which show thegreatest strain effect is only ∼1.5%. The sample strains thenrapidly converge. The initial strain difference is so small that it ishighly unlikely to retard diffusion enough to reduce enzymeactivity appreciably. The hydraulic diameter of the network in anormal cornea is approximately 18 nm.23 Assuming a Poisson

ratio of 0.4-0.5 for the corneal stroma, the maximum differencein the hydraulic diameter between the two samples will be lessthan ∼0.2 nm for the strain difference. Thus, with regard todiffusion of the enzyme, the 0.1 N and 0.25 N-loaded specimensare virtually identical through the first 30 min. In more densetissue (e.g. rabbit tendon), with a higher strain difference, Nabe-shima et al. found little influence of strain on the ability of theenzyme to diffuse through and bind to the matrix.13

Mechanical Failure. The mechanical failure of the tissuewill proceed with increasing probability as the strain on thematrix increases. Thus, the effective cutting rate at larger valuesof strain should include both enzymatic cleavage and mechanicalbreaking/separation ofmonomers. The effective cutting rates as afunction of strain for the samples are similar from their conver-gence at 20% to∼35% strain after which the lower loaded samplebegins to fail rapidly. This is difficult to interpret, but it indicatesthat as themechanical failure rate increases, the enzymatic cuttingrate may decrease further. The decrease could be mechanochem-ical or diffusion-related at higher strains. These effects cannot bereadily separated without further investigation.

Discussion

Toprevent dissipation of their structure,metazoans necessarilyplace load-bearing material in the path of forces, where it mustpersist, even in the presence of catabolic enzymes. In vertebrates,this material is often collagen. Simple free-energy argumentswould appear to favor an increase in the rate of collagen fibrilcatabolismwith increasing strain energy density (to reduce storedenergy in strained monomers). However, there is a mountingbody of evidence indicating that collagen fibrils are generally

Figure 5. Effective load-bearing area versus time. Sample loaded area plotted as a function of time for all four experimental protocols. Thereis a rapid loss in the area after the addition of collagenase for all three continuously loaded samples.No loss is shown for the unloaded sample,as it was not possible to probe the sample without load. However, upon reloading, the fraction of area remaining converged on the data fromthe 0.1 N sample. Vertical bars are standard deviations. Differences in remaining area were statistically significant for loading conditions:0.1 versus 0.25 N and 0.1 versus 0.5 N. There were no statistically significant differences between the 0.25 and 0.5 N runs.

(23) Overby, D.; Ruberti, J.; Gong, H.; Freddo, T. F.; Johnson, M. Specifichydraulic conductivity of corneal stroma as seen by quick-freeze/deep-etch. J.Biomech. Eng. 2001, 123 (2), 154-161.

DOI: 10.1021/la100384e 9925Langmuir 2010, 26(12), 9917–9926

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stabilized by mechanical load, which is nonintuitive, but whichmakes practical sense given the load-bearing role of collagen as amaterial. Further, if collagen cleavage rates were to increase withapplied strain, then the presence of catabolic enzyme in loadedtissue would constitute the dangerous and unstable situation,where increased cleavage of load-bearing structure could lead toincreased strain on remaining tissue and still faster cleavage. Theidea that collagen fibrils aremademore stable bymechanical loadappears to be consistent with what can be generally observed invertebrate animals: applied nominal strains appear to cause theretention and organization rather than the removal of load-

bearing structure.1,3-5,7,24 In the current investigation, we havefound that increasing the applied load increases the time-to-failureof corneal collagen strips. This is definitive and likely to be un-related to diffusion effects as discussed above. We further deter-mined that there appears to be a precipitous reduction in themaximum rate of load-bearing area loss (enzymatic cleavage rate)as a function of strain over a very small initial strain differencebetween the two samples (Δ of∼1.5%). Themagnitude of change

Figure 6. Dynamics of enzyme induced creep experiment. Rate of loss of load-bearing area versus time and strain (i.e., proportional toenzyme cutting rate). For all graphs, the ordinate axes represent the normalized rate of load bearing area loss which may be interpreted as ameasure of enzymatic activity on load-bearing tissue components (at low strain). The first derivative was calculated using a 5 point centereddifference. (A)Area loss rate versus time for the whole experiment (smooth line is a 50 pointmoving average trendline). The general temporaltrend in the lossof loadedmaterial beginswithan initial climb toamaximumdegradation rate, followedbyadecline in the rateofmaterial lossduring the remainder of the experiment. (B) Area loss versus time for the initial 1000 s after enzymatic exposure (trendline). In this plot, thetemporal dynamics of the initial enzyme-induced creep is shown. Given our scaling argument, we assume that during the initial 300 s (after ashort transient indicating enzyme arrival and binding), the enzymatic reaction is not limited bydiffusion.During this time (dotted box), for allthree experiments, a peak in the cutting rate is reached. As the reaction proceeds, diffusion is likely to begin to dominate the reaction rate(dotted box). (C) Rate of load-bearing area loss as a function of strain (trendlines shown only). The effect of strain on the cutting rate can bedirectly extracted. Remarkably, at the beginning of the experiment, it can be seen that∼1.5% increase in applied strain decreases the cuttingrate by a factor of∼2.6. The difference inmaximum cutting rates (peaks) was statistically significant between the 0.1N and the 0.25 and 0.5Nruns (p

9926 DOI: 10.1021/la100384e Langmuir 2010, 26(12), 9917–9926

Article Zareian et al.

in the enzyme activity rate (more than 2-fold) is remarkable,since it appears to occur over such a small change in tissuelength. This large difference in the rate of enzymatic activityresults in the strain of the 0.1 N sample “catching up to” the0.25 N sample in the first few minutes of the experiment andthen catching up to the 0.5 N sample at the end of theexperiment. The extraction of the decreasing linear relation-ship between strain and enzymatic activity (which was ex-tracted from the 0.1 N loaded sample) depends on the scalingcalculation to provide a reasonable estimate of when diffusionwill begin to limit the reaction rate. If the diffusion limitationoccurs much earlier than our calculation suggests, there may besome error in the data extracted from the linear regression.However, the fact that the more highly loaded sample does notexhibit a similar decline during the same period supports ourassertion that it is strain, not diffusion which is slowing theenzymatic activity (at least initially).

Conclusion

In this study, we use a dynamic, enzyme-induced creep experi-ment to demonstrate that small mechanical strain differencesinduce loaded specimens to degrade at substantially differentrates (higher strain, lower rate of degradation). Further, weextract a continuous relationship between mechanical strainand the enzymatic cleavage rate of collagenous tissue at lowstrains. The experiment differs fromother experiments in that it isperformed at physiological temperature and it takes advantage ofthe physics of the diffusion/reaction time scale to probe theenzyme activity when diffusion is not expected to interfere withthe results. The data provide a clear demonstration that theenzymatic cleavage rate depends on the mechanical state of thetissue independent of diffusion effects. By all measures, thesamples under lower load (unloaded/reloaded and 0.1 N load)fail faster than the more highly loaded samples (0.25 and 0.5 N).Perhaps the most striking finding is the fact that the maximumenzymatic activity rate differs by more than 50% over straindifference of only ∼1.5%. This highly sensitive drop-off in thecleavage rate occurs where the tissue exhibits stiffening which islikely due to the transition from an entropic loading regime to anenergetic one.20 In addition to demonstrating that strain isprotective, the data also indicate that the protection is notabsolute. All specimens will degrade to failure (at this concentra-tion of enzyme, 0.05 mM) in spite of the fact that the strains are

continually increasing throughout the experiment. Crude BC is anonspecific and aggressive enzyme capable of degrading collagenin addition to many other molecules. This enzyme attacks thecollagen molecule at multiple binding sites and is not a particu-larly relevant probe for in vivo collagenase activity. However, thefact that mechanical strain strongly depresses BC activity is veryencouraging for the strain-stabilization hypothesis in vertebrateanimals. There are very few native enzymes (matrix metallopro-teinase (MMP) family and cathepsins) and non-native enzymes(BC) capable of degrading the collagen triple helix directly. Ingeneral, these enzymes must first bind to and then cleave thecollagen monomer. In the case of MMPs, it has been suggestedthat the enzyme must first orient and then unwind the triple helixto perform an effective cleavage event.25,26 The manipulationtakes place near the thermally labile domain which is devoid ofstabilizing hydroxyproline27 and is likely to preferentially strainrelative to the remaining collagen monomer. Thus, MMPs arepotentially even more susceptible to strain-induced stability. Themethod and bioreactor developed in this paper for probingcollagen/enzyme mechanochemistry (dynamic, enzyme-inducedcreep) should apply quite well to the investigation of collagen/MMP mechanochemistry.

Acknowledgment. This work was partially supported by NEIR01EY015500 and NIAMS R21AR057056. The authors wouldlike to acknowledge the Northeastern UniversityMechanical andIndustrial Engineering Undergraduate Capstone course which isresponsible for supporting and providing the initial design of thebioreactor. We would like to thank Professor Keith Meek andSally Hayes for their efforts in helping us estimate how manyfibrils are oriented in the direction of the applied load in bovinecorneas.

Supporting Information Available: Additional figures andmovie; details of collagenase activity assay. This material isavailable free of charge via the Internet at http://pubs.acs.org.

(25) Chung, L.; Dinakarpandian, D.; Yoshida, N.; Lauer-Fields, J. L.; Fields,G. B.; Visse, R.; Nagase, H. Collagenase unwinds triple-helical collagen prior topeptide bond hydrolysis. EMBO J. 2004, 23 (15), 3020-3030.

(26) Lauer-Fields, J. L.; Juska, D.; Fields, G. B. Matrix metalloproteinases andcollagen catabolism. Biopolymers 2002, 66 (1), 19-32.

(27) Miles, C. A.; Burjanadze, T. V.; Bailey, A. J. The kinetics of the thermaldenaturation of collagen in unrestrained rat tail tendon determined by differentialscanning calorimetry. J. Mol. Biol. 1995, 245 (4), 437-446.