effects of enzymatic treatments on the...

Effects of Enzymatic Treatments on the Depth-Dependent ViscoelasticShear Properties of Articular Cartilage

Darvin J. Griffin,1 Josh Vicari,1 Mark R. Buckley,2,3 Jesse L. Silverberg,3 Itai Cohen,3 Lawrence J. Bonassar1,4

1Department of Biomedical Engineering, Cornell University, Ithaca, New York, 2University of Rochester, Biomedical Engineering, Rochester,New York, 3Department of Physics, Cornell University, Ithaca, New York, 4Sibley School of Mechanical & Aerospace Engineering, CornellUniversity, Ithaca, New York

Received 15 April 2014; accepted 14 July 2014

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jor.22713

ABSTRACT: Osteoarthritis (OA) is a disease that involves the erosion and structural weakening of articular cartilage. OA ischaracterized by the degradation of collagen and proteoglycans in the extracellular matrix (ECM), particularly at the articularsurface by proteinases including matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondinmotifs (ADAMTSs).1 Degradation of collagen and proteoglycans is known to alter shear mechanical properties of cartilage, but studyof this phenomenon has been focused on bulk tissue properties. The purpose of this study was to assess microscale cartilage damageinduced by trypsin or collagenase using a technique to measure the local shear viscoelastic properties. Safranin-O histology revealeda decrease in proteoglycans near the articular surface after collagenase and trypsin digestions, with proteoglycan depletionincreasing in time. Similarly, confocal reflectance micrographs showed increasing collagen degradation in collagenase treatedsamples, although the collagen network remained intact after trypsin treatment. Both treatments induced changes in shear modulusthat were confined to a narrow range (�400mm) near tissue surface. In addition, collagenase altered the total energy dissipationdistribution by up to a factor of 100, with longer digestion times corresponding to higher energy dissipation. The ability to detectlocal mechanical signatures in tissue composition and mechanics is an important tool for understanding the spatially non-uniformchanges that occur in articular cartilage diseases such as OA. � 2014 Orthopaedic Research Society. Published by Wiley Periodicals,Inc. J Orthop Res

Keywords: confocal strain mapping; viscoelasticity; cartilage; collagen; proteoglycan

Currently affecting more than 20 million Americans,osteoarthritis (OA) is the second leading cause ofphysical disability among adults in the UnitedStates.1 OA involves the deterioration of cartilage indiarthroidal joints that can involve both enzymaticactivity and mechanical factors. Diarthroidal jointsare inevitably subjected to repeated stresses thatleave disease cartilage susceptible to mechanicalfailure; thus, characterizing the changes in diseasedcartilaginous material properties that occur in OA isintegral to understanding the progression of thisprominent disease. The compromised functionalityof cartilaginous tissues can arise from very subtlechanges in the organization of their structure.Furthermore, it has been reported that in OAstructural damage to the collagen and proteoglycannetwork begins near the cartilage surface beforeprogressing deeper into the tissue.1,2 Degradation ofthe ECM by proteinases is known to alter themechanical behavior of bulk cartilage samples,2 butlittle is known about the specific changes in materi-al properties near the tissue surface. The lack ofknowledge of these changes is in part due to the factthat a reliable method for detecting microscalechanges in early OA has not yet been established.As such, observing the effects of damage on the local

mechanical properties of cartilage can provide infor-mation that may be obscured by measurements atthe bulk level.

Previous studies have used degradative enzymes,such as matrix metalloproteinase-1 (MMP-1),3,4,5

MMP-36,7 or inflammatory cytokines, including both aand b forms of interleukin-1 (IL-1)5,6 and TNF-a6,7 toselectively examine the effect of proteoglycan andcollagen degradation on the mechanical properties ofcartilage explants. While these studies demonstrateddecreases in tensile,8,9 compressive,10,11 and shearstiffness,12 the analyses were limited to bulk mechani-cal behavior. In contrast little is known about localmechanical changes (on the scale of <100mm) thatoccur in cartilage that has been degraded. Recently,our lab has developed a novel system for measuringthe local dynamic shear properties of articular carti-lage on the length scale of 20mm.13 This techniquerevealed that a narrow region of the tissue 100–200mm below the articular surface has remarkablydifferent properties than the rest of the tissue.13,14

This surface region is also damaged or degraded earlyin the process of arthritis.15 However the consequencesof damage to this region on shear mechanical behaviorare not well understood. The purpose of this study wasto assess the effect of progressive proteoglycan andcollagen degradation induced by trypsin and collage-nase on the spatially localized shear properties ofcartilage. Understanding the degradation of cartilagelocally will provide a more comprehensive picture ofthe intricate relationship between tissue structure andproperties as well as providing insight into the me-chanical changes that occur very early upon surfacedamage.

Grant sponsor: Cornell University, NIH; Grant numbers:R21-AR054867 and R21-AR062677; Grant sponsor: NIH NIAMS;Grant number: 3R01AR053571-0351.Correspondence to: Lawrence Bonassar (T: þ607-255-9381;F: þ607-255-7330; E-mail: [email protected])

# 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2014 1

MATERIALS AND METHODSTissue Sample PreparationFifty-six full thickness, 6mm diameter explants were har-vested sterilely from the patellofemoral groove of seven 1–3day old calves (Gold Medal Packing, Oriskany, NY) withsamples from each animal randomly assigned betweencontrol and experimental groups (Fig. 1A). The harvestingprocedure produces cylinders with an undamaged articularsurface.16 After dissection, 56 samples were soaked inphosphate-buffered saline (PBS) supplemented with 100U/ml penicillin and 100mg/ml streptomycin for 30min. Eachcylinder was then cut along its long axis into two hemi-cylinders yielding a total of 112 hemi-cylinders (Fig. 1B).A small section (1–3mm) of the deep region of each hemi-cylinder was removed with a razor blade to flatten the facetopposing the articular surface.

Enzymatic Digestion and Confocal Reflectance (CR) MicroscopyCartilage samples designated for enzymatic digestion andCR imaging of collagen were coated with epoxy resin(Devcon, Danvers, MA) to ensure that enzyme exposureoccurred only at the articular surface (Fig. 1C). CR microsco-py was used to obtain real-time images of cartilage sampleswithout the need for histologic stains,17 where the fluore-scence signal intensity is proportional to collagen density. Atotal of eight samples were imaged on a confocal reflectancemicroscope (Zeiss 710 Confocal, Germany) at 25� magnifica-tion in real-time at a frame rate of four frames/min. Sampleswere digested for 15, 30, and 90min using two differentenzymes, 2mg/ml of bacterial collagenase to remove bothcollagen and proteoglycans (Worthington, type II collagenase,Lakewood, NJ) or 50mg/ml of trypsin (Cellgro, 0.25% trypsinEDTA, Manassas, VA), which selectively cleaves the coreproteins in proteoglycans without affecting the collagennetwork.4 During degradation samples were imaged seriallyat 0, 15, 30, and 90min with four samples observed forcollagenase or trypsin exposure. After degradation, solutionswere removed by serial washing with protease inhibitors inPBS. Additionally, 40 samples were fixed, embedded, sec-tioned, and stained with Safranin-O to observe proteoglycandistribution after 0, 15, 30, and 90min of exposure tocollagenase (n¼ 6 at each time point) or trypsin (n¼ 4 ateach time point). In parallel, 44 samples were used forconfocal strain mapping studies, with n¼ 7 samples at eachtime point of collagenase treatment and n¼ 6 samples ateach time point of trypsin treatment.

Confocal Strain MappingThe local shear modulus of samples was measured using gridresolution automated tissue elastography (GRATE) as describedpreviously.16 Samples were placed in PBS with 200mL/ml

5-dichlorotriazinyl aminofluorescein (5-DTAF) (MolecularProbes1, Grand Island, NY) for two hours (Fig. 2A).18,19

5-DTAF modifies amines in proteins and fully stains theextracellular matrix (ECM). The deep zone of the hemi-cylinders were glued to a tissue deformation imaging stage(TDIS) and compressed to 10% axial strain. The TDIS wasmounted on an inverted Zeiss LSM 5 LIVE confocal microscopeand imaged using a 488nm laser (Fig. 2B). To directly imageapplied strain, a line perpendicular to the articular surface wasphotobleached with the confocal laser. Sinusoidal shear dis-placements were applied to the articular surface by the TDISat a frequency of 0.1Hz and amplitude of 16mm, and theresultant forces were measured with a load cell (Fig. 2C).

Custom software written in Matlab (The Mathworks, Inc.,Natick, MA) was used to track the motion of the photo-bleached line as a function of depth.13 These local displace-ments were fit to a sinusoidal function and differentiatedwith respect to z to calculate the strain amplitude g0(z) andphase angle relative to the resultant stress d(z). The magni-tude of the dynamic shear modulus |G�(z)| was calculatedas the ratio of stress amplitude t0 to strain amplitude g0,where t0 is the total applied stress required to deform thetissue from zero strain to g0. The rate of energy dissipation

Figure 1. (A) 6mm cylinders are bisected into hemi-cylinders(B) coated with epoxy resin (C) 50mg/ml trypsin or 2mg/mlcollagenase are added to the tissue surface for 15, 30, and90min.

Figure 2. (A) Samples were placed in a 200mL of 5-DTAFsolution to fluorescently stain the ECM, (B) Schematic of thetissue deformation imaging stage; (C) A line was photobleachedperpendicular to articular surface and subjected to 0.32 shearstrain before and after digestion. The photobleached line for thedegraded specimen exhibited a steeper slope from strain localiza-tion, implying inferior surface properties.

2 GRIFFIN ET AL.

JOURNAL OF ORTHOPAEDIC RESEARCH MONTH 2014

per unit volume (Dv) at a depth z was calculated by thefollowing equation:

DE

Dv¼ pT2

0jG�ðzÞj�1sind

Statistical AnalysisTwo-way ANOVA was used to evaluate the statistical signifi-cance of enzymatic treatments on the depth-dependentviscoelastic shear properties of articular cartilage.

RESULTSSafranin-O staining revealed a progressive removal ofproteoglycans near the articular surface after collage-nase digestion with the depth of proteoglycan deple-tion increasing with time (Fig. 3A–D). Similarlyconfocal reflectance microscopy revealed degradationof the collagen network with a sharp border betweendegraded and undegraded regions (Fig. 3E–H). Thedepth of matrix removed increased from 150mm at15min, 300mm at 30min, and 450mm at 90min. Thedepth of proteoglycan and collagen removal was simi-lar at all times (Fig. 3I).

In contrast to collagenase, trypsin removed onlyproteoglycans (Fig. 4A–D) but not collagen (Fig. 4E–H). The depth of proteoglycan removal progressedfrom 100mm at 15min, 150mm at 30min, and 200mmat 90min (Fig. 4I). Confocal reflectance showed littleto no change after trypsin treatment.

As previously reported shear modulus |G�| forhealthy undigested samples exhibited a distinct spa-tial pattern12,13,14,18,19 (Fig. 5A). The shear moduluswas low in a narrow band (�400mm) near the articu-lar surface, with a well-defined minimum at �100mm.Beyond 500mm in depth the shear modulus showedless variation. Collagenase treatment dramaticallylowered the shear modulus at the surface. By 30min|G�| at 50mm was reduced by 20% relative to control,

and by 90min had decreased by more than 50-fold.The mechanical effect of degradation was confined to350mm at 15min, 420mm at 30min, and 500mm at90min. Notably the characteristic minimum |G�| wasnot present after 90min of collagenase exposure(Fig. 5A). Collagenase treatment made the tissuemore compliant and more viscous at the articularsurface leading to an increase in both the local phaseangle and local energy dissipation (Fig. 5B,C).

Similarly to collagenase, trypsin treatment samplesdisplayed similar reduction in |G�| within the first400mm near the articular surface (Fig. 5D). Thespatial pattern of change in |G�| was differentbetween collagenase and trypsin. Trypsin-treated sam-ples had the lowest |G�| at z �100mm from surfaceeven after 90 min of exposure, similar to undegradedtissue (Fig. 5D). This is likely related to the fact thattrypsin leaves the collagen network largely intact, asindicated by CRM data. The local phase angle afterproteoglycan removal alone increased d up to 2-foldlocally in higher and longer exposure times. A similarmechanical trend was seen with DE, by 15mindisplaying higher energy dissipation with longertrypsin exposure times.

DISCUSSIONThis study evaluated the effect of progressive proteo-glycan and collagen degradation on the depth-dependent viscoelastic shear properties of articularcartilage. In these experiments, enzymatic digestionby trypsin and collagenase at the surface resulted inlocal structural changes along with a significant reduc-tion in the magnitude of the local shear modulus and acorresponding increase in the energy dissipation andlocalization. Safranin-O staining revealed a decreasein proteoglycan concentration near the articular sur-face after either trypsin or collagenase treatment, withproteoglycan depletion increasing with time. Enzymatic

Figure 3. (A–D) Saf-O histology and (E–H) confocal reflectance micrographs of articular cartilage before digestion and after 15, 30,and 90min of digestion with collagenase, (I) normalized intensity curves as a function of depth for SAF-O and CRM values (n¼ 6 ateach time point).

MICROSCALE MECHANICS OF CARTILAGE DEGRADATION 3


treatments decreased the local shear modulus up to50-fold, but increased tissue viscoelastic phase lag byup to 2-fold locally, and energy dissipation by up to100-fold locally (Fig. 5). Because our measurementresolution was 20mm, we were able to detect suchmechanical changes in the tissue near the articularsurface where enzymatic degradation occurred.

Histologic examination (Figs. 3 and 4) showedthat the initial loss of proteoglycans and collagenwere primarily near the articular surface and pro-gressed deeper into the tissue with time. Thisprogressive front of matrix removal by trypsin20 and

collagenase21 has been previously documented. Con-focal reflectance microscopy produced clear imagesof the collagen network near the articular surfaceand similarly, showed a progressive removal aftercollagenase exposure. In contrast, attenuation ofconfocal reflectance was associated with trypsintreatment; however, the collagen network appearedintact (Fig. 4E–H). As noted previously22 collagenremoval appeared to also cause proteoglycan remov-al, as evidence by the fact that collagenase treat-ment produced fronts that were co-located to within�25mm.

Figure 4. (A–D) Saf-O histology and (E–H) confocal reflectance micrographs of articular cartilage before digestion and after 15, 30,and 90min of digestion with trypsin, (I) normalized intensity curves as a function of depth for SAF-O and CRM values (n¼ 4 at eachtime point).

Figure 5. (A, B) Shear modulus G) vs. depth z from the surface (C, D) Phase angle (d) vs. depth z (E, F) Energy dissipated (DE) vs.depth z (control n¼5, collagenase n¼7, and trypsin n¼6).

4 GRIFFIN ET AL.


To assess the mechanical consequences of thisdegradation, we used confocal strain mapping tocharacterize the viscoelastic shear properties of thetissue. To extend our previous work,13,14,18,19 thisstudy investigated how removal of collagen and proteo-glycans affects the mechanical properties in thissurface region. Collagenase and trypsin treatmentsaltered the shear modulus |G�| differently. Collage-nase lowered |G�| dramatically at the surface(Fig. 5A). After 30min of digestion, |G�| at 20mmdropped by 30% at the surface, but by 90min, |G�|surface had dropped so much that there was no visibleminimum. In contrast, trypsin treatment lowered |G�| near surface, but the |G�| minimum was alwaysat 100mm, even after this region had been depletedfully of proteoglycans (Fig. 4D). These data suggestthat both collagen and proteoglycans contribute toshear properties, but both play a distinct role onbearing shear loads at the articular surface. Particu-larly, proteoglycan removal lowers the overall magni-tude of |G�| and increases the corresponding d, butpreserves the underlying microstructure that producesthe characteristic minimum near the surface. Con-versely, collagenase digestion, which is a harsherenzymatic treatment of the tissue, affects |G�| and d

the same way as trypsin but also destroys themicrostructure that produces the characteristic mini-mum of |G�| near the surface.

We note this strongly non-linear drop-off in |G�|arising from collagenase digestion (Fig. 4A) is consistentwith predictions of network-based models of cross-linkedpolymers.24–27 In these models, a fibrous network’sshear modulus is extremely sensitive to connectivitynear the rigidity percolation phase transition. Above thepercolation threshold, the cross-linked network is suffi-ciently connected that stresses can be transmittedthrough the system. Below the percolation threshold,the network is too fragmented, hence the system isunable to bears stresses and the shear modulus dropsdramatically. The data can therefore be interpreted asan enzymatically driven transition that decreases con-nectivity in the collagen fiber network, and moves thesystem as a whole toward the percolation phase transi-tion. The data also suggest the proteoglycan networkeffectively behaves as a simple viscoelastic medium thatmakes a contribution to |G�| in parallel to the collagennetwork. Indeed, the observations presented here areconsistent with other data that has explored thesemodels in greater detail.28

The local viscoelastic capacity of cartilage undershear has received little attention despite the fact thatshear forces in articular cartilage correlate with tissuedamage and disease.23 In cartilage, the osmotic pres-sure exerted by the proteoglycans keeps the collagennetwork prestressed with tensile forces.24 Further-more, previous studies have shown that cartilagestiffens and becomes less viscous locally when de-formed in shear.18 Both of these studies thereforesuggest that stressed or extended collagen networks

are more elastic and less viscous, implying that remov-ing such stress would make the network more compli-ant and more viscous. In the current study, specificremoval of proteoglycans by trypsin treatment removedprestress chemically, making the tissue more compli-ant and more viscous at the articular surface leadingto a very large local energy dissipation (Fig. 5F).Previous work reports that articular cartilage dissi-pates shear energy primarily near its surface.13 Theresults of this study suggest that cartilage degenera-tion compromises this energy-absorbing surface region,therefore increasing tissue susceptibility to shear-in-duced damage into the remainder of the tissue.

Proteoglycan removal increased viscous behavior,which runs counter to the idea that proteoglycan–proteoglycan friction or collagen–proteoglycan frictionis responsible for this viscous behavior. The combina-tion of proteoglycan and collagen removal increasedviscous behavior more than proteoglycan removalalone implying the removal of tension on the collagennetwork contributes to increased viscous behavior.Collagenase increased the rate of energy dissipationby cartilage up to 100-fold locally progressing higherat longer exposure times (Fig. 5C).

The mechanical effect of enzymatic digestion to thecartilage matrix was seen at depths beyond areaswhere histological staining was lost (�400mm). Localproteoglycan and collagen removal by collagenase de-creased |G�| up to �500mm with increasing mechani-cal effects in d and DE. Histology indicated initial lossof proteoglycans only up to �200mm for trypsin-treatedsamples (Fig. 4D); however, mechanical reductionswere seen up to �500mm in |G�|, with increasingvalues in d, and DE that extended beyond the boundaryof degraded tissue. This suggest that local mechanicalmeasurements are a sensitive measure of changes incartilage shear properties because they can resolvechanges in depth-wise variations as well as differencesin the overall magnitude. This provides an attractivecomplement to histological imaging, which offers moreexplicit static structural information, and can be usedin a synergistic fashion to develop a more robustunderstanding of healthy and disease tissue integrity.

Overall, our results provide novel information oncartilage mechanics and insights on the mechanismsfor changes in matrix composition and functionalproperties associated with matrix degradation. As inearly stages of OA, these treatments cause localsuperficial matrix damage, with concomitant reduc-tions of surface mechanical properties on the micro-scale. Detection of such changes could revealsignatures of early onset OA that are not currentlyavailable with traditional imaging techniques, present-ing new opportunities and new perspectives on carti-lage therapies and repair.

ACKNOWLEDGMENTSThe authors gratefully acknowledge Olufunmilayo Adebayo,Brandon Borde, and Eddie Bonnie for their helpful discussions

MICROSCALE MECHANICS OF CARTILAGE DEGRADATION 5


and suggestions. DJG was funded by Cornell University andthe NSF GRFP grant number DGE-0707428. This research wasfunded in part by Cornell University; NIH NIAMS grantnumbers: R21-AR054867, R21-AR062677, 3R01AR053571-0351.

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