Journal of Structural Biology 181 (2013) 264–273

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Journal of Structural Biology

journal homepage: www.elsevier .com/ locate/y jsbi

Age-related nanostructural and nanomechanical changes of individualhuman cartilage aggrecan monomers and their glycosaminoglycan side chains

Hsu-Yi Lee a,1, Lin Han b,c,1, Peter J. Roughley d, Alan J. Grodzinsky a,e,f,⇑, Christine Ortiz b,⇑a Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United Statesb Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United Statesc School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, United Statesd Genetics Unit, Shriners Hospital for Children, McGill University, Montréal, Canada, QC H3G 1A6e Department of Mechanical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United Statesf Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, United States

a r t i c l e i n f o

Article history:Received 4 October 2012Received in revised form 10 December 2012Accepted 11 December 2012Available online 25 December 2012

Keywords:CartilageAggrecanGlycosaminoglycanUltrastructureAging

1047-8477/$ - see front matter � 2012 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.jsb.2012.12.008

⇑ Corresponding authors. Fax: +1 617 258 6936 (C.(A.J. Grodzinsky).

E-mail addresses: alg@mit.edu (A.J. Grodzinsky), co1 Denotes equal contribution.

a b s t r a c t

The nanostructure and nanomechanical properties of aggrecan monomers extracted and purified fromhuman articular cartilage from donors of different ages (newborn, 29 and 38 year old) were directly visu-alized and quantified via atomic force microscopy (AFM)-based imaging and force spectroscopy. AFMimaging enabled direct comparison of full length monomers at different ages. The higher proportion ofaggrecan fragments observed in adult versus newborn populations is consistent with the cumulative pro-teolysis of aggrecan known to occur in vivo. The decreased dimensions of adult full length aggrecan(including core protein and glycosaminoglycan (GAG) chain trace length, end-to-end distance and exten-sion ratio) reflect altered aggrecan biosynthesis. The demonstrably shorter GAG chains observed in adultfull length aggrecan monomers, compared to newborn monomers, also reflects markedly altered biosyn-thesis with age. Direct visualization of aggrecan subjected to chondroitinase and/or keratanase treatmentrevealed conformational properties of aggrecan monomers associated with chondroitin sulfate (CS) andkeratan sulfate (KS) GAG chains. Furthermore, compressive stiffness of chemically end-attached layers ofadult and newborn aggrecan was measured in various ionic strength aqueous solutions. Adult aggrecanwas significantly weaker in compression than newborn aggrecan even at the same total GAG density andbath ionic strength, suggesting the importance of both electrostatic and non-electrostatic interactions innanomechanical stiffness. These results provide molecular-level evidence of the effects of age on the con-formational and nanomechanical properties of aggrecan, with direct implications for the effects of aggre-can nanostructure on the age-dependence of cartilage tissue biomechanical and osmotic properties.

� 2012 Elsevier Inc. All rights reserved.

1. Introduction

Aggrecan, the most abundant proteoglycan in the extracellularmatrix (ECM) of articular cartilage, is composed of a �250 kDa coreprotein (Tortorella et al., 2002) substituted with �100 chondroitinsulfate (CS) and �30 keratan sulfate (KS) glycosaminoglycan (GAG)chains, as well as N-linked and O-linked oligosaccharides (Dudhia,2005). In vivo, aggrecan monomers form high molecular weightaggregates (>200 MDa) by noncovalently binding to hyaluronan(HA) stabilized by link protein (LP), which are enmeshed within areinforcing collagen fibrillar network (Muir, 1979). This hierarchi-cally structured ECM determines the unique biomechanical prop-

ll rights reserved.

Ortiz), fax: +1 617 258 5239

rtiz@mit.edu (C. Ortiz).

erties of cartilage, including load bearing and lubrication insynovial joints (Maroudas, 1979). Age and disease-induced deteri-oration of human cartilage (Hudelmaier et al., 2001) is character-ized by significant structural heterogeneity of aggrecan, includingdifferences in core protein and GAG side chain length, KS and CSsubstitution, and CS sulfate-ester substitution, which are criticaldeterminants of cartilage charge density and distribution (Baylissand Ali, 1978; Dudhia, 2005; Plaas et al., 2001; Plaas et al., 1997;Roughley and White, 1980). Progressive C-terminal truncation ofthe core protein by proteolytic enzymes takes place with increas-ing maturation (Sandy and Verscharen, 2001), and variations inaggrecan structure, in turn, can further affect its susceptibility toproteolytic digestions and development of osteoarthritis (Roughleyet al., 2006).

Gel filtration chromatography and other biochemical assayshave characterized age-related changes in the mean values ofaggrecan and GAG molecular weight, composition and

H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273 265

hydrodynamic size of large ensembles of cartilage-extractedaggrecan (Bayliss and Ali, 1978; Dudhia, 2005; Plaas et al., 2001;Plaas et al., 1997; Roughley and White, 1980). Such measures,however, cannot define the structure of individual aggrecan mono-mers, the degree of GAG variability within individual aggrecan, andthe consequences of such ultrastructure on aggrecan molecularmechanics, all of which can regulate the macroscopic biomechan-ical and osmotic properties of articular cartilage as a function ofage. This understanding could fill the knowledge gap betweenthe conventional macroscopic tissue measurements and the under-lying fundamental molecular-level properties of the matrix (Hanet al., 2011; Hunziker et al., 2002; Stolz et al., 2007), issues of greatimportance to disease progression and tissue regeneration. Highresolution imaging techniques, such as electron microscopy(Buckwalter and Rosenberg, 1982; Buckwalter and Rosenberg,1983; Buckwalter et al., 1994; Mörgelin et al., 1988; Rosenberget al., 1975; Thyberg, 1977; Wiedemann et al., 1984) and atomicforce microscopy (AFM) (Fritz et al., 1997; Jarchow et al., 2000;Todd et al., 2003), have shown the potential for examining aggre-can ultrastructure at the molecular level. We have recently demon-strated that high resolution AFM imaging can directly visualize andquantify the animal age and species-related variations in aggrecanultrastructural features, such as the spatial distribution and lengthheterogeneity of GAG side chains (Kopesky et al., 2010; Lee et al.,2010; Ng et al., 2003). In addition, we have shown that AFM-basedforce spectroscopy can relate these structural features to aggrecannanomechanical properties and their physical origins, such as ionicstrength and [Ca2+] dependence, and thereby provide direct molec-ular evidence on the age-related changes in cartilage tissue func-tions (Dean et al., 2006; Han et al., 2007a; Han et al., 2007b; Hanet al., 2008).

Toward this end, the goal of this study was to quantify age-re-lated changes in the structure and nanomechanical properties ofhuman aggrecan, and the role of CS- versus KS-GAGs in aggrecanconformation. Firstly, AFM-based high resolution imaging was uti-lized to quantify the structural and conformational parameters ofindividual aggrecan monomers extracted from newborn and adulthuman articular cartilage. The age-related differences in chondro-cyte-mediated proteolytic and biosynthetic activities were distin-guished for the first time via direct visualizing of individualmonomers. Secondly, via selective removal of KS- and/or CS-GAGchains, we were able to distinguish the contributions of theseGAG components to the molecular structure and stiffness of aggre-can. Thirdly, the compressive nanomechanical properties of aggre-can at different ages were evaluated via AFM-based forcespectroscopy to correlate molecular mechanical behavior to theobserved aggrecan nanostructures.

2. Materials and methods

2.1. Isolation of human articular cartilage aggrecan

Macroscopically normal human articular cartilage sampleswere obtained at autopsy from the femoral condyles of one new-born, one 29 year-old and one 38 year-old adult with whom therewas no evidence of arthritic disease or joint damage. Aggrecan waspurified by dissociative CsCl density gradient centrifugation from4 M guanidine HCl extracts of the cartilage, as described previously(Roughley and White, 1980). The initial dissociative (D1) fractionsof the newborn and 38 year-old human samples were subjected toa second dissociative CsCl density gradient centrifugation, whereaspart of the 29 year-old human initial D1 fractions was treated withkeratanase II or chondroitinase ABC prior to the second dissociativedensity gradient centrifugation (see Section 2.2). The D1D1 frac-

tions were then dialyzed exhaustively against water, lyophilized,dissolved in water, and stored at �20 �C in 1 mg/ml aliquots.

2.2. Keratanase and chondroitinase treatments of human aggrecan

Part of the initial D1 fractions of the 29 year-old human aggre-can samples were treated with keratanase II or chondroitinase ABCin order to remove the KS- or CS-GAGs. The D1 fractions were firstdissolved at 2 mg/ml in buffer. Aggrecan solution was then incu-bated at 37 �C for overnight with 5 mUnits keratanase II (Sei-kagaku, Tokyo, Japan) per mg aggrecan and 50 mM sodiumacetate at pH 6.0 for KS-GAG removal, and 50 mUnits chondroitin-ase ABC (Seikagaku) per mg aggrecan, 100 mM Tris–HCl, 100 mMsodium acetate at pH 7.3 for CS-GAG removal. The digested sam-ples were subsequently subjected to the second dissociative CsCldensity gradient centrifugation, dialyzed, and stored at �20 �C in1 mg/ml aliquots.

2.3. AFM-based imaging and quantification of aggrecan structure

Aggrecan samples for AFM imaging were prepared followingthe procedures described previously (Ng et al., 2003). Briefly,50 ll aggrecan aliquots (�100 lg/ml) were deposited on 3-amino-propyltriethoxysilane (APTES, Sigma Aldrich, St. Louis, MO) freshlytreated muscovite mica surfaces (SPI Supplies, West Chester, PA,#1804 V-5) for 20–30 min at room temperature, rinsed gently withMilliQ water (18 MX cm resistivity, Purelab Plus UV/UF, US Filter,Lowell, MA) and air dried. Tapping mode AFM imaging was carriedout in ambient conditions using rectangular Si AFM probe tips(AC240TS-2, Olympus, nominal spring constant k = 2 N/m, nominaltip radius R < 10 nm) and the Nanoscope IIIA Multimode AFM (Vee-co, Santa Barbara, CA). All imaging parameters were optimized toacquire high quality images.

The AFM height images were digitized into pixels and theaggrecan core protein structure features, including the trace lengthof core proteins LCP, and the end-to-end distance, Ree, were tracedautomatically with a custom Matlab (Mathworks) program (Kope-sky et al., 2010). The trace length of the core protein, LCP, and theend-to-end distance, Ree, were calculated according to the spatialcoordinates of the traces. The trace lengths of the GAG chains weretraced manually with the SigmaScan Pro image analysis software(SPSS Science, Chicago, IL). The extension ratio, Ree/LCP, was calcu-lated as a measure to demonstrate the degree of extension of eachmolecule. Nonparametric Mann–Whitney U-test was performed totest the difference of their mean values between each pair of pop-ulations without the assumption of normal distribution, with sig-nificance set at p < 0.05.

2.4. AFM-based nanomechanical test of aggrecan

Compressive nanomechanical properties of full length aggrecanwere measured following the procedures described previously(Dean et al., 2005). In short, micropatterned surfaces with denselypacked, chemically end-attached aggrecan and neutral, hydrophilicself-assembled monolayer (OH-SAM, 11-mercaptoundecanol,HS(CH2)11OH, Sigma Aldrich) were prepared via microcontactprinting (Wilbur et al., 1994). A Multimode IV AFM with the Pico-Force piezo (Veeco) was utilized to perform compressive nanome-chanical tests with an OH-SAM functionalized, gold-coatedspherical borosilicate colloidal probe tip (Novascan, Ames, IA,k � 0.12 N/m, R � 2.5 lm).

Two measurements were employed to assess the mechanicalstiffness of aggrecan at various ionic strengths. First, the com-pressed aggrecan layer height H was measured as a function of ap-plied normal force via contact mode AFM scanning across theaggrecan-OH-SAM patterned surface at a given range of applied

266 H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273

normal forces (0–30 nN) in NaCl aqueous solutions (ionic strength,IS = 0.001–1.0 M, pH � 5.6, tip lateral displacement rate = 60 lm/s). Secondly, aggrecan compression was performed by having theprobe tip approach perpendicularly to the end-grafted aggrecanlayer (z-piezo displacement velocity = 2 lm/s). The pH values ofthe aqueous solutions used in the nanomechanical tests were mea-sured to remain�5.6 during the experiment (buffers were not usedso that IS could be controlled down to 0.001 via NaCl alone). Rawdata were converted to normal force as a function of tip-to-sub-strate distance, D, based on the height measurement (Dean et al.,2006). Normal stresses were calculated from the normal forceusing the surface element integration method (Bhattacharjee andElimelech, 1997; Dean et al., 2006). The initial sulfated GAG (sGAG,including both CS-GAG and KS-GAG) density prior to compressionas measured via the dimethylmethylene blue dye assay (Farndaleet al., 1986), was �20 mg/ml (one aggrecan molecule per25 nm � 25 nm surface area) at 0.1 M NaCl for both newborn andadult cartilage aggrecan samples. sGAG density during compres-sion was calculated as a function of compressed height by normal-izing the total sGAG content under the probe tip to the reduced,compressed volume.

LGAG

LCP

aa

bb

cc

dd

ee

ff

Ree

newborn human

Fig. 1. Tapping mode AFM height images of (a, b, c) newborn and (d, e, f) 38-year old aduAn example of core protein trace length, LCP, end-to-end distance, Ree, and GAG chain tr

3. Results

3.1. Structural dimensions and heterogeneity of human aggrecan andconstituent GAG chains

Structure and conformation of individual aggrecan monomerswere directly visualized and the constituent GAG chains wereclearly resolved in the AFM height images (Fig. 1). The globular do-mains at the core protein N- or C-terminals are distinct in theseimages (Fig. 1a and d, indicated by arrows). Aggrecan monomersconsisting of globular domains at both ends were defined as fulllength aggrecan. The characterization of the full-length aggrecanwill be described in Section 3.2.

In all the obtained images, newborn aggrecan were in generalmore uniform in size, whereas adult aggrecan exhibited greatervariations in terms of LCP. A greater portion of short fragments(�100 nm) bearing globular domains were also observed in the29 and 38 year-old adult aggrecan populations (Fig. 1d and e).The short fragments are presumably the accumulated fragmentsfrom the proteolytic processing (Lark et al., 1997; Yasumotoet al., 2003).

300 nm

150 nm

0.7 nm

0 nm

0.7 nm

0 nm

0.7 nm

0 nm

100 nm

adult human (38 yr)

lt human aggrecan monomers. Globular domains are indicated by arrows (a and d).ace length, LGAG, are shown in (c).

Table 1Summary of measured aggrecan structural parameters from AFM images (mean ± 95% confidence interval of means).

All aggrecan Full length aggrecan

LCP (nm) n LCP (nm) Ree (nm) Ree/LCP (%) n

Newborn 477 ± 16 139 554 ± 10 420 ± 23 76 ± 4% 6238 year-old 246 ± 13 450 510 ± 24 324 ± 35 64 ± 6% 2629 year-old untreated 216 ± 20 193 486 ± 12 314 ± 29 65 ± 6% 5329 year-old KS-removed 207 ± 21 152 443 ± 18 304 ± 28 68 ± 5% 6229 year-old CS-removed 145 ± 9 377 331 ± 17 188 ± 19 57 ± 6% 42

H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273 267

The mean and 95% confidence interval of the aggrecan struc-tural parameters LCP, Ree and Ree/LCP for each population are re-ported in Table 1. These data suggest that the newbornpopulation exhibits significantly larger values in all these parame-ters than either the 38 or 29 year old adult aggrecan populations(p < 0.01). In this study, we focused on the comparison betweenthe newborn and 38 year old populations (Fig. 2), as the compari-son with the 29 year old population yielded the same conclusions.

The CS-GAG chain length LGAG was found to be quite heteroge-neous within one individual newborn and adult aggrecan mono-mer (Fig. 3a), and the mean LGAG of this individual newbornmonomer was significantly greater than that of the adult monomer(Table 2, Fig. 3a, p < 0.0001). These individual monomer LGAG distri-butions were then compared to LGAG for the entire populations ofnewborn and 38 year old aggrecan (Fig. 3b), as well as the identi-fied full length monomers from these newborn and 38 year oldaggrecan populations (Fig. 3c). The distributions and mean valuesof the individual, full length and all observed aggrecan populationswere quite similar (Fig. 3), where GAG chains from the newbornaggrecan were significantly longer (p < 0.0001, Table 2).

3.2. Nanostructure of keratanase and chondroitinase treated aggrecan

The structural analysis of the 29 year old aggrecan populationfocused on the roles of KS- and CS-GAG chains on aggrecanstructure and conformation. The keratanase II-treated aggrecan

0

10

20

30

0 100 200 300 400 500 600 7000

10

20

Core Protein Trace Length LCP (nm)

Freq

uenc

y (%

)

0

200

400

600

800

newborn adultCor

e Pr

otei

n Tr

ace

Len

gth L C

P (nm

)

*newborn477 ± 96 nm(n = 139)

adult (38 yr)246 ± 144 nm

(n = 450)

a all observed aggrecan

0

200

400

600

800

0 100 200 300 400 500 600 7000

102030

010203040

End-to-end Distance Ree (nm)

Freq

uenc

y (%

)

newborn adult

End-

to-e

nd d

ista

nce R

ee (n

m)

*newborn420 ± 92 nm(n = 62)

adult (38 yr)324 ± 89 nm

(n = 26)

c full length aggrecan

Fig. 2. Histograms and box-and-whisker plots of structural parameter distributions of nlength, LCP, of all observed aggrecan, (b) trace length, LCP, of full length aggrecan, (c) endlength aggrecan. ⁄p < 0.01 between the newborn and adult human aggrecan populations

appeared globally similar to the untreated aggrecan (Fig. 4a–d),consistent with the presence of the many CS chains which arelarger than KS. The chondroitinase ABC treated aggrecan had arelatively long, GAG-free region of core protein, corresponding tothe position of the CS1 and CS2 domains, which occupy �70% ofthe total aggrecan contour length (Fig. 4e–f). However, shorterGAG chains were still visible, located near the globular G1–G2domains, corresponding to one of the putative locations of KS(Fig. 5).

Both KS- and CS-GAG removal resulted in significantly shorterLCP for aggrecan with the full core proteins (Table 2 and Fig. 6a).Interestingly, the KS-removed aggrecan population showed valuesof Ree and the extension ratio, Ree/LCP, similar to the those of the un-treated population, both of which were significantly greater thanthe CS-removed population (Table 2 and Fig. 6b and c). The com-parison of LCP between untreated and KS-removed aggrecanincluding all observed aggrecan populations resulted in no statisti-cal difference (data not shown), likely because the variations in LCP

introduced by the fragments overshadow the shortening effects ofKS-removal on LCP.

3.3. Compressive nanomechanics of full-length aggrecan under fullyhydrated conditions

Aggrecan height, H, was measured as a function of applied nor-mal force in electrolyte baths of 0.001–1.0 M NaCl. Consistent with

0

200

400

600

800

0 100 200 300 400 500 600 7000

20

400

20

40

Core Protein Trace Length LCP (nm)

Freq

uenc

y (%

)

newborn adultCor

e Pr

otei

n Tr

ace

Leng

th L

CP (

nm)

*newborn555 ± 41 nm(n = 62)

adult (38 yr)510 ± 62 nm(n = 26)

b full length aggrecan

0

20

40

60

80

100

0 20 40 60 80 1000

20

40

0

20

40

60

Extention Ratio Ree/LCP (%)

Freq

uenc

y (%

)

newborn adult

Exte

ntio

n R

atio

Ree/L

CP (

%) *newborn

76 ± 16%(n = 62)

adult (38 yr)64 ± 16%(n = 26)

d full length aggrecan

ewborn and 38-year-old adult human aggrecan monomer core proteins: (a) trace-to-end distance, Ree, of full length aggrecan, and (d) extension ratio, Ree/LCP, of fullvia Mann–Whitney U test.

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 900

20

400

20

40

GAG Chain Trace Length LGAG (nm)

Freq

uenc

y (%

)

newborn adultGAG

Cha

in T

race

Len

gth L G

AG (n

m)

*newborn55 ± 16 nm(n = 102)

adult (38 yr)33 ± 11 nm

(n = 117)

b all observed aggrecan

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 900

20

400

20

40

GAG Chain Trace Length LGAG (nm)

Freq

uenc

y (%

)

newborn adultGAG

Cha

in T

race

Len

gth L G

AG (n

m)

*newborn57 ± 15 nm(n = 34)

adult (38 yr)35 ± 11 nm

(n = 25)

c full length aggrecan

0

20

40

60

80

100

0 10 20 30 40 50 60 70 80 900

20

400

20

40

GAG Chain Trace Length LGAG (nm)

Freq

uenc

y (%

)

newborn adultGAG

Cha

in T

race

Len

gth L G

AG (n

m)

*newborn52 ± 10 nm(n = 31)

adult (38 yr)35 ± 9 nm(n = 38)

a individual aggrecan

(Fig. 1c)

(Fig. 1f)

Fig. 3. Histograms and box-and-whisker plots of newborn and 38-year-old adulthuman aggrecan monomer GAG side chain trace length, LGAG, distributions: (a)individual aggrecan, (b) all observed aggrecan, (c) full length aggrecan. ⁄p < 0.0001between the newborn and adult human aggrecan populations via Mann–Whitney Utest.

Table 2Summary of measured aggrecan glycosaminoglycan (GAG) side chain trace lengthsfrom AFM images of untreated aggrecan (mean ± 95% confidence interval of means).

All aggrecan Full length aggrecan Individual aggrecan

LGAG (nm) n LGAG (nm) n LGAG (nm) n

Newborn 55 ± 3 102 57 ± 5 34 52 ± 4 3138 year-old 33 ± 2 117 35 ± 5 25 35 ± 3 3829 year-old 29 ± 1 127 32 ± 3 29 –

268 H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273

previous observations on aggrecan from bovine and equine carti-lage (Dean et al., 2006; Lee et al., 2010), H decreased monotonicallywith increasing applied normal force and ionic strength (Fig. 7a–d).The aggrecan heights measured at low forces reflect the relativedifferences in the lengths and the degrees of extension of differentaggrecan populations observed via AFM imaging (Fig. 7a and b). Atall the tested ionic strengths, the newborn human aggrecan exhib-ited significantly greater height than the 38-year-old adult humanaggrecan (Fig. 7a–d), consistent with the AFM imaging measure-ments on the distribution of LCP, Ree and Ree/LCP (Fig. 2b–d).

In the force spectroscopy measurements, normal forces werethen measured as a function of the distance, D, between the AFMprobe tip and the substrate. The normal force was found to in-crease nonlinearly with decreasing the tip-substrate separationdistance, D (Fig. 7e and f), also consistent with previous observa-tions (Dean et al., 2006; Lee et al., 2010). The newborn aggrecanalso displayed longer range repulsive forces than the adult aggre-can at any given ionic strength, suggesting more superior compres-sive nanomechanical properties of newborn aggrecan molecules.The compressive behavior assessed via the height and force mea-surements coincide with each other, as shown for the measure-ments at 0.01 M ionic strength (Fig. 7g). The differences betweenH and D at small normal forces (<5 nN) may be due to the tare force(�100 pN) necessary to enable stable feedback, and the additionalweaker shear resistance of aggrecan during the contact modeheight measurement (Han et al., 2007a).

The compressive behavior of newborn and 38 year old aggrecanpopulations were directly compared at near-physiologic ionicstrength, 0.10 M. The force-distance curves were converted tostress versus sGAG concentration (see Section 2) (Fig. 8). The nor-mal compressive resistance increased with increasing sGAG con-centration for both populations; however, the newborn aggrecanexhibited a much stronger resistance to compressive stress thanthe adult aggrecan, even at the same sGAG concentration.

4. Discussion

In this study, direct visualization of individual human aggrecanmolecules enabled separation of the proteolytic and biosyntheticvariations in aggrecan ultrastructural and nanomechanical proper-ties between age groups. Accumulated proteolytic activity, charac-terized by the increased heterogeneity and fragmentation ofaggrecan was seen in both adult aggrecan populations. The varia-tion in cell biosynthetic processes with age was reflected especiallyin the difference in GAG chain lengths, and to additional degrees bythe full length aggrecan core protein length, end-to-end distanceand extension ratio. In particular, the length of GAG side chainswithin single aggrecan molecules significantly decreased withage. In addition, the roles of various GAG side chain componentswere quantified by selective removal of KS-GAG and CS-GAG con-stituents. As a result of these structural changes, the uncompressedaggrecan volume (height) and compression resistance were alsoreduced in adult aggrecan populations.

4.1. Age-related aggrecan structural changes due to proteolyticvariations

The progressive proteolytic modification of aggrecan duringaging is evident in the presence of smaller aggrecan fragments inboth newborn and adult aggrecan populations (Fig. 1). The adultaggrecan population contained markedly more fragmented aggre-can monomers (LCP < 300 nm, Fig. 2a), showing the accumulationof enzymatically cleaved aggrecan with increasing age, e.g., mono-mers with partial CS-GAG domains or free G1-domains. In vivo,aggrecan core proteins are cleaved by a number of enzymes, pri-marily aggrecanases (a disintegrin and metalloproteinase withthrombospondin motifs, ADAMTS-4 and ADAMTS-5) and matrixmetalloproteinases (MMPs) (Flannery et al., 1992; Sandy et al.,1991; Tortorella et al., 2002). Aggrecanases cleave the aggrecancore protein in the interglobular domain (IGD) to produce freeG1 domains (G1-NITEGE) (Sandy et al., 1991), and more efficiently(Tortorella et al., 2002) at distinctive sites within the CS region toleave fragments with partially retained GAG chains and core pro-tein lengths �25–75% of the full length aggrecan. MMPs cleavethe core protein in the IGD (Flannery et al., 1992) to produce

LCP

G3

G1

bb dd ff

untreated keratanase II-treated chondroitinase ABC-treated0.7 nm

0 nm

200 nm

aa

0.7 nm

0 nm

50 nm

cc ee

Ree

Fig. 4. Tapping mode AFM height images of (a, b) untreated, (c, d) keratanase II-treated and (e, f) chondroitinase ABC-treated 29 year old adult human aggrecan monomers.An example of core protein trace length, LCP, and end-to-end distance, Ree, are shown in (b).

Fig. 5. Tapping mode AFM height images of chondroitinase ABC-treated aggrecanmonomers. The presence of short GAG side chains, presumably KS-GAGs, arevisualized near one end of the core proteins (arrows), likely within the KS-domainalong the core protein.

H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273 269

another type of free G1 domain (G1-VDIPEN). These proteolyticactivities are directly reflected in the observed high concentrationof aggrecan fragments with reduced LCP within the adult popula-tion (Fig. 2a). While G1-free fragments are expected to diffuseout of cartilage after cleavage, G1-associated fragments retain thecapability of binding to hyaluronan. Thus, these fragments areknown to accumulate in cartilage tissue with age (Lark et al.,1997; Yasumoto et al., 2003). Compared to intact aggrecan witha half-life of �3.5 years in the inter-territorial matrix, the free-G1domains have a much longer resident time in the matrix of 19–25 years (Maroudas et al., 1998; Verzijl et al., 2001). The accumu-lation of free G1-domains has been hypothesized to be detrimentalto cartilage, as they can worsen its biomechanical properties bycompeting with full length aggrecan on the hyaluronan bindingsites (Mercuri et al., 1999).

The accumulation of proteolytically-derived aggrecan frag-ments is diminished for the newborn aggrecan, reflected by thegreater values of average LCP (Fig. 2a), and smaller difference withits full aggrecan subpopulation (Fig. 2b). This aggrecan structurecomparison thus provided direct observation on the cumulativeage-related proteolytic effects on cartilage aggrecan heterogeneityand polydispersity at the individual molecular level. In vivo, thedifferences of cartilage aggrecan polydispersity between these

two age groups are expected to be more pronounced than theobservation here (Figs. 1 and 2), as the density-based D1D1 samplepreparation procedure has removed a large portion of GAG-depleteaggrecan fragments (Roughley and White, 1980), and thus, reducedthe polydispersity to a larger extent in the adult aggrecan popula-tion (Bayliss and Ali, 1978; Dudhia et al., 1996; Plaas et al., 2001;Roughley and White, 1980).

4.2. Age-related aggrecan core protein structural changes due tobiosynthesis variations

With the advantages of direct visualization, we were able tospecifically pick the full length aggrecan subpopulation, whichhad globular domains identified at both the N- and C-termini(Fig. 1c and f). The full length aggrecan monomers have not under-gone enzymatic modifications and, hence, only reflect the results ofchondrocyte biosynthetic processes. Moreover, the metabolic halflife of full length aggrecan is �3.4 years (Maroudas et al., 1998),shorter than the age difference between the newborn and adult(both 29 and 38 year old) human subjects. Hence, the full lengthaggrecan found in the adult populations are most likely synthe-sized later in the subject’s life time. In the absence of proteolyticmodifications, the significantly greater values of full length aggre-can core protein trace length, LCP, provided direct evidence of theage-related variations in the chondrocyte biosynthetic activities.It is likely that this age-related decrease in core protein length isnot a direct result of variation in core protein synthesis, but ratheran indirect consequence of variations in the sulfation pattern,number and length of GAG side chains (Dudhia, 2005; Roughleyand White, 1980).

4.3. Age-related glycosaminoglycan chain structural changes due tobiosynthesis variations

Although previous chromatography (Carney et al., 1985;Deutsch et al., 1995; Inerot et al., 1978; Plaas et al., 1997) and elec-tron microscopy (Buckwalter et al., 1994) studies have suggestedsignificant shortening of GAG chain lengths with increasing age,

007006005004003002001000

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ated

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Exte

nsio

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atio

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G (%

)

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CS-removed188 ± 62 nm

(n = 42)

CS-removed57 ± 18%(n = 42)

Fig. 6. Histograms and box-and-whisker plots of the structural parameter distri-butions of 29-year-old full length adult human aggrecan monomers: (a) coreprotein trace length, LCP, (b) end-to-end distance, Ree, and (c) extension ratio, Ree/LCP. Only full length aggrecan monomers with indentifiable G1 and G3 globulardomains are included. ⁄p < 0.05 for each population compared with the other twopopulations via Mann–Whitney U test.

270 H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273

this study for the first time provided detailed ultrastructural char-acterization and comparison of GAGs attached to human aggrecanbased on the information from individual molecules (Fig. 3). With-in each individual full length aggrecan (Fig. 1c and f), GAG chains ofthe adult aggrecan are considerably shorter than those of the new-born aggrecan (Fig. 3a).

For the newborn population, most of the GAG chains are CS-GAGs, whereas for the adult population, the observed GAG chains

are likely a mixture of CS- and KS-GAGs. The differences in bothchain lengths (Brown et al., 1998; Huckerby et al., 1999; Santeret al., 1982) and number of chains (Elliott and Gardner, 1979) be-tween KS and CS decrease with age. The narrower distribution ofLGAG seen in the individual adult aggrecan monomers (Fig. 3a)may also reflect the decreased GAG length heterogeneity due tothe decreased length differences between CS and KS with age(Brown et al., 1998; Huckerby et al., 1999; Santer et al., 1982), orbetween CS chains in the CS1 and CS2 regions (Rodriguez et al.,2006).

4.4. Age-related variations in aggrecan conformation and compressivenanomechanical properties

The end-to-end distance, Ree, and extension ratio, Ree/LCP, of thenewborn aggrecan are markedly greater than the adult population(Fig. 2c and d). Newborn human aggrecan molecules thus adapt amore extended conformation compared to the adult aggrecan, ow-ing to the stronger steric and electrostatic repulsion as a result oflonger GAG chains and higher net charge densities. The conforma-tional entropic effects are less important, leading to a more ex-tended, less coil-like molecular conformation (Flory, 1953).Similarly, due to the stronger GAG–GAG repulsion, the newbornpopulation also demonstrates greater initial height and highercompressive stiffness compared to the adult aggrecan upon com-pression nanomechanical test at physiological-like conditions(Fig. 7). The higher electrostatic forces for the newborn populationarise from the greater number of charges per GAG chain, greaternumber of GAG chains, and possibly smaller GAG–GAG moleculespacing along the core protein. The higher nonelectrostatic forcesare related to the larger dimensions of aggrecan (i.e., LCP and LGAG).Macroscopically, the compressive modulus of cartilage correlateswell with the GAG fixed charge density (Williamson et al., 2001).At the nanoscale, we have shown that the molecular structureand dimension of aggrecan and GAGs also affect the compressivebehavior of aggrecan (Dean et al., 2006; Lee et al., 2010). At phys-iologic-like ionic strength (0.10 M), higher compressive stress wasobserved for the newborn aggrecan than the adult aggrecan evenat the same sulfated GAG (sGAG) density (Fig. 8). This differenceis due to the larger nonelectrostatic (steric and entropic) compo-nents (Bathe et al., 2005; Comper and Laurent, 1978), as well asvariation in the heterogeneity of charge and electrostatic potentialdistributions within the end-grafted aggrecan layer (Buschmannand Grodzinsky, 1995; Dean et al., 2003). This difference furtherdemonstrates the importance of aggrecan molecular architectureand associated heterogeneous, spatially nonuniform charge distri-bution in determining aggrecan molecular mechanics and cartilagetissue stiffness.

4.5. Structure and distribution of different GAG side chain components

Using the 29-year-old human aggrecan population as the modelsystem, we directly identified the spatial distribution of differentKS- and CS-GAG side chain components along the aggrecan coreprotein, and explored and identified their contributions to thestructure and properties of aggrecan by selective removal of eachconstituent (Fig. 4). The KS-GAGs only make up a small fractionof the side chains, as removal of KS-GAGs did not lead to markedchanges in the brush-like aggrecan structure (Fig. 4d). On the otherhand, the majority of the GAGs are CS-GAG chains, where removalof CS-GAGs essentially transforms aggrecan into a linear core pro-tein, with only traces of shorter KS-GAGs near the N-terminal(Figs. 4f and 5). This observation thus provides the direct experi-mental evidence that supports the previous hypothesis on thestructure and spatial distribution of aggrecan side chain constitu-ents (Hardingham and Fosang, 1992; Ng et al., 2003).

c dnewborn human aggrecan adult human aggrecan

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)

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0.01M NaCl

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Fig. 7. (a–d) Three dimensional height images (at �3 nN applied normal force) and compressed aggrecan layer height, H, versus applied normal force, F, curves of end-grafted(a, c) newborn and (b, d) 38-year-old adult human aggrecan layers, measured via contact mode AFM imaging on an aggrecan and hydroxyl-terminated self-assembledmonolayer (OH-SAM) micro-patterned surface (top right schematic). Each data point represents the average of eight different scan locations under the same normal force, theSDs are smaller than the size of the data symbols. (e–f) Normal force, F, versus distance, D, curves of end-grafted (e) newborn and (f) 38-year-old adult human aggrecan layers,measured via AFM-based colloidal force spectroscopy. The probe tip approached the substrate perpendicular to the plane of the substrate (bottom right schematic). Eachcurve represents the average of 30 different locations on the aggrecan pattern, the SDs are smaller than the width of the data curves. (g) Comparison of the H versus F (4,replotted from c and d) and F versus D (dashed lines, replotted from e and f) measurements at 0.01 M ionic strength. All the experiments were conducted in NaCl aqueoussolutions at 0.001–1.0 M ionic strengths using a gold-coated spherical colloidal probe tip (R � 2.5 lm), functionalized with OH-SAM.

0 20 40 60 80 1000

0.05

0.1

0.15

0.2newbornadult

sGAG concentration (mg/ml)

Stre

ss (M

Pa)

Fig. 8. Stress versus sGAG concentration curves converted from force-distancecurves in Fig. 7e and f (0.1 M NaCl). Each curve is an average of 30 approaches atdifferent locations on the aggrecan pattern. The 95% confidence intervals of eachaveraged curve are smaller than the width of the data curves.

H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273 271

4.6. Contribution of GAG side chains to aggrecan conformation

Removal of both KS-GAGs and CS-GAGs significantly reduced thecore protein trace length, LCP (Fig. 6a). Because neither keratanase II

nor chondroitinase ABC modifies the amino acid sequence andphysical contour length of the core protein (Oike et al., 1980), thedecrease in the trace length, LCP, can be attributed to a less extendedshort range conformation of the core protein, and the local coilingand aggregation of amino acid units due to hydrophobic interac-tions or electrostatic attraction. These short range conformationchanges can take place at the length scale beyond the spatialresolution of AFM imaging (�2 nm), and thus, result in reducedmeasured values of LCP. This effect is more pronounced for the CS-removed aggrecan, given the presence of strikingly more CS-GAGsthan KS-GAGs within each aggrecan.

Interestingly, removal of KS-GAG chains had negligible effectson the distribution of the end-to-end distance, Ree, or the extensionratio, Ree/LCP (Fig. 6b and c). This observation suggests that theKS-GAG constituents play minor roles in determining aggrecanmolecular conformation and deformation, given the small numberof KS-GAGs, the shorter chain lengths and much smaller number ofnegative charges compared to CS-GAGs. By comparison, removal ofCS-GAG chains led to significant decreases in Ree and Ree/LCP

(Fig. 6b and c). Since CS-GAGs contribute to most of the negativecharges of aggrecan, CS-GAG-absent aggrecan behaves more simi-larly to linear protein molecules, with only traces of shorter GAG

272 H.-Y. Lee et al. / Journal of Structural Biology 181 (2013) 264–273

chains visible near one end of the core protein, presumably the KS-GAG chains close to the N-terminal (Fig. 5). These few short KS-GAG chains thus have minimal impact on the intra-molecular ste-ric, electrostatic repulsion, and nanomechanical properties ofaggrecan, consistent with the observation that the KS-removedaggrecan behave similar to the untreated aggrecan (Fig. 6).

4.7. Implications regarding age-related cartilage tissue degradationand repair

Age-related weakening of aggrecan is known to be directly re-lated to function changes in articular cartilage. Decreases in themodulus and increases in the hydraulic permeability of cartilageswith age may be attributed to the weaker molecular stiffness of fulllength aggrecan (Figs. 7 and 8), as well as the higher concentrationof enzymatically cleaved G1-associated fragments that are boundto hyaluronan. In addition, in vivo, aggrecan molecules are en-trapped within the collagen fibrillar network in a pre-strainedstate, with molecular strains �40–60% for macroscopically uncom-pressed cartilage (Wight et al., 1991). Under these finite molecularstrains, differences in compressive resistance between the new-born and adult aggrecan at the same GAG concentration are evenmore prominent (Fig. 8). These results give direct experimentalevidence of progressive changes in human cartilage properties thatmay result from changes at the molecular-level, further elucidatingthe relation between aggrecan monomer structure and biome-chanical properties of cartilage.

Interestingly, the biosynthesis-related structural and mechani-cal differences observed here between the extracted newborn ver-sus adult aggrecan are similar to the differences observed withaggrecan synthesized by adult equine bone marrow stromal cells(BMSCs) versus chondrocytes from the same animals (Lee et al.,2010). The BMSC-synthesized aggrecan monomers show nano-structural and nanomechanical properties superior to the aggrecansynthesized by adult equine chondrocytes, largely due to differ-ences in GAG chain length. Consequently, BMSC-aggrecan alsoexhibits higher compressive stresses than adult cartilage aggrecanat the same sGAG concentration (Kopesky et al., 2010; Lee et al.,2010).

5. Conclusions

In this study, the age-related structure and property changes ofaggrecan monomers from human articular cartilage were quanti-fied at the length scales of individual molecules and physiologi-cal-like molecular assemblies. While previous biochemicalstudies have reported age-related changes in large ensembles ofhuman cartilage aggrecan, this study provides direct evidence ofthe modifications by proteolytic and biosynthetic processes onthe structure, conformation and mechanical behavior of aggrecanrelated to individual molecules. These molecular-level changes,including accumulation of fragmented aggrecan, reduction inaggrecan and CS-GAG size (LCP, LGAG), changes in conformation(Ree, Ree/LCP), and decrease in aggrecan compressive stiffness arecharacteristics of cartilage aging in vivo, and directly affect the tis-sue function of cartilage and the risk of osteoarthritis. AFM-baseddirect visualization of individual aggrecan has enabled us to sepa-rate the full-length and fragmented aggrecan populations, andtherefore de-convolute the effects of changes in proteolytic cleav-age and chondrocyte biosynthesis mechanisms. In addition, theroles of CS-GAG and KS-GAG side chains on aggrecan conformationwere distinguished via this direct visualization method. This studyprovides the knowledge basis to link certain age- and osteoarthri-tis-related changes in cartilage tissue to fundamental molecularprocesses that take place at the nanometer length scale. It is hoped

that the information presented here will aid further progress in thefield of osteoarthritis research, including detecting early-stage dis-ease, documenting the disease progression, as well as assessingand optimizing treatment interventions.

Acknowledgments

This work was supported by the National Science Foundation(grant CMMI-0758651), the National Institutes of Health (grantAR33236 and AR60331), the National Security Science and Engi-neering Faculty Fellowship (grant N00244-09-1-0064), and theShriners of North America. The authors thank the Institute forSoldier Nanotechnologies at MIT, funded through the U.S. ArmyResearch Office, for the use of instruments.

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