ARTHRITIS & RHEUMATISMVol. 58, No. 12, December 2008, pp 3831–3842DOI 10.1002/art.24069© 2008, American College of Rheumatology

Increased Hydraulic Conductance of Human Articular Cartilageand Subchondral Bone Plate With Progression of Osteoarthritis

Jennifer Hwang, Won C. Bae, Wendy Shieu, Chad W. Lewis,William D. Bugbee, and Robert L. Sah

Objective. Osteoarthritis (OA) is characterized byprogressive degeneration of articular cartilage and re-modeling of the subchondral bone plate, comprisingcalcified cartilage and underlying subchondral bone.Calcified cartilage remodeling due to upward invasionby vascular canals or to calcified cartilage erosion maycontribute to biomechanical alteration of the osteochon-dral tissue and its subchondral bone plate component.The study hypothesis was that hydraulic conductance ofosteochondral tissue and subchondral bone plate in-creases with structural changes indicative of increasingstages of OA.

Methods. Osteochondral cores were harvestedfrom the knees of cadaveric tissue donors and fromdiscarded fragments from patients with OA undergoingknee surgery. The osteochondral cores from tissue do-nors were macroscopically normal, and the cores frompatients with OA had partial-thickness or full-thicknesserosion to bone. The cores were perfusion-tested todetermine the hydraulic conductance, or ease of fluidflow, in their native state and after enzymatic removal ofcartilage. Adjacent portions were analyzed by3-dimensional histology for calcified cartilage, subchon-dral bone, and subchondral bone plate thickness andvascular canal density.

Results. Hydraulic conductance of native osteo-

chondral tissue and subchondral bone plate was higher(2,700-fold and 3-fold, respectively) in fully erodedsamples than in normal samples. The calcified cartilagelayer was thicker (1.5-fold) in partially eroded samplesthan in normal samples but thinner and incomplete infully eroded samples. Subchondral bone plate vascular-ity was altered with increasing stages of OA.

Conclusion. During joint loading, increased hy-draulic conductance of the osteochondral tissue andsubchondral bone plate could have deleterious biome-chanical consequences for cartilage. Increased fluidexudation from overlying and opposing cartilage, in-creased fluid depressurization, and increased cartilagetissue strains could lead to chondrocyte death andcartilage damage.

Articular cartilage is a low-friction, load-bearingmaterial joined to the subchondral bone plate at theends of long bones that form a synovial joint. Duringjoint loading, pressurization of interstitial fluid withincartilage protects articular cartilage from high compres-sive strains (1,2). The ability of cartilage to support loadthrough interstitial fluid pressurization is dependent onits low hydraulic permeability. Hydraulic permeabilitydescribes the ease of fluid flow through a material, whichfor articular cartilage is governed by the extracellularmatrix. Degenerative changes in cartilage that occurwith osteoarthritis (OA) have been correlated withincreased hydraulic permeability of cartilage (3–5).However, hydraulic permeability has been determinedonly for cartilage slices separated from the subchondralbone, not for full-thickness osteochondral tissue. Al-though fluid transport and resistance to fluid flowthrough osteochondral tissue are normally governed bycartilage, the contribution of the subchondral bone plateboundary may become increasingly important as thecartilage is eroded during the progression of OA.

In OA, progressive degeneration of the articular

Supported by the NIH, the National Science Foundation, andthe Musculoskeletal Transplant Foundation. Ms Hwang was recipientof a National Science Foundation Graduate Research Fellowship. Dr.Sah’s work was supported by an award to the University of California,San Diego under the Howard Hughes Medical Institute ProfessorsProgram.

Jennifer Hwang, MS, Won C. Bae, PhD, Wendy Shieu, ChadW. Lewis, PhD, William D. Bugbee, MD, Robert L. Sah, MD, ScD:University of California–San Diego, La Jolla.

Address correspondence and reprint requests to Robert L.Sah, MD, ScD, Department of Bioengineering, MC 0412, University ofCalifornia, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0412.E-mail: rsah@ucsd.edu.

Submitted for publication February 15, 2008; accepted inrevised form August 24, 2008.

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cartilage matrix is associated with remodeling of thesubchondral bone plate. While cartilage decreases inthickness and mechanical integrity with the progressionof OA, subchondral bone increases in thickness andstiffness, undergoes accelerated turnover (6), and exhib-its altered trabecular architecture (7–9) and cysts (10).Although it is clear that progression of OA involvesstructural and mechanical changes in both cartilage andbone, it is unknown whether such changes are associatedwith altered fluid transport characteristics of the osteo-chondral tissue.

Although the ease of fluid flow through cartilageis traditionally described by hydraulic permeability, it isuseful to describe the ease of fluid flow through osteo-chondral tissue and subchondral bone plate by therelated structural property of hydraulic conductance.Because hydraulic conductance describes flow through astructure rather than a material, it can be used tocharacterize ease of fluid flow through irregular struc-tures, such as osteochondral tissue with cartilage erosionor the undulating calcified cartilage layer of subchondralbone plate. Thus, hydraulic conductance can include thecontribution from subchondral bone plate in determin-ing fluid flow through an osteochondral structure, in-cluding how that role may change with erosion ofcartilage in OA.

Although hydraulic permeability has been mea-sured for cartilage (11), cortical bone (12), and cancel-lous bone (13), the fluid transport properties of the zoneof calcified cartilage at the osteochondral interfaceremain unknown. In studies of cartilage nutrition, calci-fied cartilage has been considered an impermeablebarrier to material transport from the subchondral bone,particularly after maturation of the joint (14). However,in vivo magnetic resonance imaging studies have dem-onstrated penetration by intravenous Gd-DTPA2�

across the osteochondral interface into the deep regionsof human articular cartilage (15). Furthermore, at theosteochondral interface of the intervertebral disc (stud-ied ex vivo), fluid flows between the marrow cavity andthe cartilaginous disc through vascular channels (16).This suggests that vascular channels in the subchondralbone plate may contribute to the fluid flow through, andhydraulic conductance of, the osteochondral interface.

The subchondral bone plate is the underlyingsupport structure for articular cartilage in synovial joints.The subchondral bone plate begins at the tidemark,which separates uncalcified cartilage from calcified car-tilage, and consists of both the calcified cartilage layerand the underlying subchondral bone. Calcified cartilageis composed of hypertrophic chondrocytes enveloped in

a calcified matrix and is vascular (17,18), whereas artic-ular cartilage is normally avascular. The calcified carti-lage layer attaches cartilage to subchondral bone (19,20)and provides a transitional zone of intermediate stiff-ness, reducing stress concentrations at the cartilage–bone interface (21). Below the calcified cartilage is thecement line or ossification front, which marks the begin-ning of the porous and vascular subchondral bone.

The calcified cartilage layer may play a criticalrole in OA pathogenesis by mediating interactions be-tween cartilage and bone. Calcified cartilage becomes“activated” in OA, increasing in thickness with theformation of new zones of calcified cartilage, duplica-tions of the tidemark, and vascular invasion into thetidemark (20,22,23). Vascular invasion in the initiationor development of OA has been speculated to occurthrough microcracks that extend between the bonemarrow space and the calcified cartilage after repeatedphysiologic loading (24,25). The vascular canals in thecalcified cartilage may affect the fluid pressurizationload-bearing behavior of the articular cartilage by affect-ing the hydraulic permeability of the underlying sub-chondral bone plate. Alteration of fluid flow across thecartilage–bone interface could affect the mechanical andchemical environment in ways that promote the progres-sion of OA.

The hypothesis of this study was that the hydrau-lic conductance of osteochondral tissue and subchondralbone plate increases with structural changes indicative ofincreasing stages of OA. The objectives of this studywere to determine, for the human medial femoral con-dyle with different grades of OA erosion, the following:the hydraulic conductance for osteochondral tissue be-fore and after removal of cartilage and the thickness andvascularity of the calcified cartilage and subchondralbone plate, as possible structural determinants of hy-draulic conductance of subchondral bone plate.

MATERIALS AND METHODS

Sample harvest. Osteochondral cores (9 mm diameter)were harvested from the medial femoral condyles of cadaverictissue bank donors and discarded knee fragments from pa-tients undergoing total knee replacement surgery; harvestingreceived institutional review board approval. Each core wasobtained from a different donor, and adjacent osteochondralfragments were obtained for histologic analysis. Tissue bankdonor cores (obtained from 12 individuals with a mean � SEMage of 24 � 3 years) were macroscopically normal, and OAcores were graded by visual inspection as having partial erosionof cartilage (15 patients, mean � SEM age 71 � 3 years) or fullerosion of cartilage with exposure of bone (16 patients,mean � SEM age 71 � 2 years).

3832 HWANG ET AL

Experimental design. Cores from normal, partiallyeroded, and fully eroded subchondral bone plate wereperfusion-tested first in the intact state at the time of harvestand again after removal of cartilage by papain (Sigma-Aldrich,St. Louis, MO). To prepare samples, an IsoMet Low Speedsaw (Buehler, Lake Bluff, IL) was used to trim the bone side ofcores to a subchondral bone plate thickness of 5 mm, leavingthe cartilage intact. Core diameter was measured with digitalcalipers, and all samples were perfusion-tested. Next, uncalci-fied cartilage was enzymatically removed from all cores bypapain digestion (125 �g/ml papain, 0.005M cysteine HCl,0.1M sodium phosphate, pH 6.2) at 60°C for 18–24 hours (18).Marrow was removed from papain-digested cores by incuba-tion in an ultrasonic bath in saline for 2–5 hours, followed byrinsing via perfusion of 50 ml saline through the core in eachdirection. After rinsing, samples were perfusion-tested again.Additional 9-mm–diameter cores (n � 3) of normal andpartially eroded subchondral bone plate were used to assessthe effect of papain digestion on hydraulic conductance ofbone. Uncalcified cartilage was removed from these samplesby mechanical debridement with a curette to isolate subchon-dral bone plate, followed by trimming and marrow removal.Samples were perfusion-tested for hydraulic conductance,papain-digested, and retested.

Perfusion testing. Darcy’s law was used to estimate thehydraulic conductance constant, c, using a least squares fit oflinear fluid velocity, U, versus pressure drop across the sample,‚P. Darcy’s law describes how easily fluid flows through aporous solid and is expressed as follows:

U �QA �

kp�Ph � c�P

where U is the linear flow rate, with dimensions of m/second,Q is the volumetric flow rate, with dimensions of m3/second,and A is the sample cross-sectional area in the direction offlow, with dimensions of m2. Ease of fluid flow through astructure can be described by c, the hydraulic conductance withdimensions of m/(Pa � second), or by kp, the hydraulic perme-ability with dimensions of m2/(Pa � second), and h, the samplethickness with dimensions of m. For subchondral bone plate, cwas assessed because the undulating calcified cartilage layerhas an irregular thickness, making it difficult to identify a valuefor h to allow calculation of the kp value. Thus, ease of fluidflow through samples was characterized as a 2-hour hydraulicconductance constant (c2h) for short-term perfusion and asc0.5h for subchondral bone plate samples (representing equili-brium values).

Each sample was inserted into Tygon tubing (Cole-Parmer, Vernon Hills, IL), sealed circumferentially, and testedfor hydraulic conductance (Figure 1A). Phosphate bufferedsaline (PBS) was perfused through each sample at constantflow rates controlled by a syringe pump (Harvard PHD 2000;Harvard Apparatus, Holliston, MA), and pressure drop acrossthe sample was measured using a low-range pressure trans-ducer (�P � 0–55 kPa) (Validyne DP45; Validyne Engineer-ing, Northridge, CA). Different flow rates were used forosteochondral and subchondral bone plate cores, to keeppressures within the range of the transducer. Osteochondralsamples were perfused for 2 hours at flow rates, U, of 0.0013mm/second and 0.0026 mm/second (or equivalent Q, 0.083mm3/second and 0.17 mm3/second, normalized to A, 64 mm2).

Subchondral bone plate samples were perfused for 0.5 hour atflow rates starting from 0.026 mm/second and increased inincrements of 0.65 mm/second up to 3.9 mm/second. Thepressure drop across the sample at each flow rate was mea-sured, averaging readings over 5 seconds at 2 Hz. Samples weretested both with flow outward from the joint (from cartilage tobone) and inward into the joint (from bone to cartilage).Conductance estimates from different flow directions werereproducible (�16% to �20%) and were averaged for eachsample. Conductance estimates were also reproducible withinthe same sample in repeated measurements. Thus, 3 trials persample per flow direction were used to obtain a best-fit

Figure 1. A, Schematic diagram of perfusion test setup and represen-tative Darcy plot to estimate hydraulic conductance constant, c. B,Effect of osteoarthritis erosion and papain digestion on hydraulicconductance of osteochondral tissue and subchondral bone plate.Conductance values were obtained after 2-hour perfusion of normal,partially eroded, and fully eroded osteochondral tissue before andafter removal of cartilage by papain digestion. Bars show the mean andSEM. ● � P � 0.017; Œ � P � 0.0033.

HYDRAULIC CONDUCTANCE OF ARTICULAR CARTILAGE AND BONE 3833

conductance estimate for each sample, using a least squaresanalysis (typical r2 � 0.98).

Histologic analysis. Osteochondral fragments adjacentto each core site were obtained; one was left intact, and theother was papain-digested along with the core. Paired sampleswere fixed in 4% paraformaldehyde in PBS, pH 7.4, at 4°C for3 days and then decalcified in 15% EDTA, pH 8.5, at 37°C for3 weeks. For 3-dimensional (3-D) histologic analysis, samples(n � 3) were cut to �3-mm3 blocks and fluorescently stainedwith 0.1% eosin Y (pH 5.0) for 12–16 hours, embedded inSpurr’s resin, and imaged at 2.24 �m3 voxel resolution with aNikon E600 fluorescence microscope (Nikon, Tokyo, Japan)(26). Three-dimensional image data sets were visualized usingRESView 3.0 software (Resolution Sciences, Corte Madera,CA), and 2-D cross-sections were exported for qualitativeviewing and stereologic analysis of structural features, usingAdobe Photoshop (Adobe Systems, San Jose, CA) and ImageJ(National Institutes of Health, Bethesda, MD).

Stereologic analysis. Calcified cartilage and subchon-dral bone thickness. The thickness of the calcified cartilage andsubchondral bone was measured by averaging individual thick-ness measurements from histologic sections of a control tissuevolume. Ten random 2-D cross-sections uniformly spaced by0.1 mm were exported for a volume of tissue 1 mm2 � 2 mmdeep, encompassing the osteochondral interface for eachsample. On each vertical 1 mm � 2 mm cross-section, a grid of4 lines (each 0.2 mm apart) was overlaid on the image, with thelines normal to the tissue surface. The thickness of the calcifiedcartilage and subchondral bone was measured along each ofthe 4 lines, for a total of 40 individual measurements across all10 cross-sections, and averaged to produce 1 value for calcifiedcartilage thickness and 1 value for subchondral bone thicknessfor each sample. The calcified cartilage thickness was definedas the distance between the tidemark and the cement line, andthe subchondral bone thickness was defined as the distancebetween the cement line and the bottom edge of the corticalbone plate, or the boundary between the solid bone matrix andthe large void space associated with trabecular bone porosity.Subchondral bone plate thickness was the sum of the calcifiedcartilage and subchondral bone thicknesses. In partially erodedOA samples in which multiple tidemarks were present, thecalcified cartilage thickness was measured between the lowesttidemark (closest to the bone) and the cement line.

Vascular canal density. The number of vessels percross-sectional area was counted within the same tissue volumeused for the thickness measurements. Fifty vertical 2-D sec-tions (1 mm � 2 mm) uniformly spaced 0.02 mm apart wereexported from the tissue volume. Vessels were defined as voidspace sheathed by bone that started from the subchondralbone space and ended within the calcified cartilage or beyondthe calcified cartilage into the cartilage deep zone (in partiallyeroded samples) or up to the bony subchondral bone platesurface (in fully eroded samples). Larger vessels appearing inmultiple neighboring sections were counted only once.

Statistical analysis. Data are presented as the mean �SEM. Conductance data were log10 transformed to improvehomoscedasticity (27). Log10-transformed data were con-firmed to be normally distributed (P � 0.01) by the Anderson-Darling test (28). To assess the effect of erosion and papaindigestion on conductance, a repeated-measures analysis ofvariance (ANOVA) was used with a fixed factor of the degree

of OA erosion (normal, partial, full) and with repeated mea-sures for before and after digestion. Post hoc comparisonswere performed using unpaired t-tests between each group andits 3 relevant comparison groups (1 to compare before versusafter papain digestion, 2 to compare normal versus partialerosion versus full erosion). For these t-tests, significance wasadjusted (P � 0.05/3). To assess structural changes in thicknessand vascularity, a repeated-measures ANOVA was used with afixed factor of the degree of OA erosion and with repeatedmeasures for paired samples with and without papain diges-tion. Tukey’s post hoc comparisons were used. P values lessthan 0.05 were considered significant.

RESULTS

Hydraulic conductance. Hydraulic conductancewas dependent on both OA erosion (P � 0.01, byANOVA) and papain digestion (P � 0.001, byANOVA), with no interactive effect. With increasingseverity of OA erosion, the hydraulic conductance ofosteochondral samples and subchondral bone plate in-creased significantly (P � 0.017) (Figure 1B). The effectof OA erosion on conductance was evident both in thenative state and after cartilage removal in subchondralbone plate samples. In osteochondral samples in theirnative state, hydraulic conductance increased from 0.065mm/(MPa � second) for normal cartilage and 0.16 mm/(MPa � second) for partially eroded cartilage to 176mm/(MPa � second) for cartilage fully eroded down toexposed bone. Conductance in fully eroded samples wasincreased 2,700-fold compared with normal samples(P � 0.0033) and 1,000-fold compared with partiallyeroded samples (P � 0.017). Similar trends were evidentafter papain digestion of all samples to remove cartilageand isolate the subchondral bone plate. Subchondralbone plate conductance followed an increasing trend,from 702 mm/(MPa � second) for normal subchondralbone plate to 1,673 mm/(MPa � second) for partiallyeroded subchondral bone plate and 2,316 mm/(MPa �second) for fully eroded subchondral bone plate. Therewas a significant 3-fold increase in hydraulic conduc-tance in fully eroded subchondral bone plate comparednormal subchondral bone plate (P � 0.017).

With removal of uncalcified cartilage by papaindigestion, hydraulic conductance increased significantlyfor all grades of samples (Figure 1B). In particular,hydraulic conductance of normal and partially erodedosteochondral samples increased 10,800-fold (P �0.0033) and 10,500-fold (P � 0.0033), respectively, whilehydraulic conductance of fully eroded samples increased13-fold (P � 0.0033). For normal and partially erodedsubchondral bone plate samples isolated by mechanical

3834 HWANG ET AL

debridement, subchondral bone plate hydraulic conduc-tance increased 2-fold (P � 0.05) after papain digestion.

Histology. Three-dimensional histology showeddifferences in calcified cartilage and subchondral boneplate structure associated with the degree of degenera-tion. In all groups, calcified cartilage was histologicallydistinct from surrounding tissue. In normal and partiallyeroded samples, the calcified cartilage layer borderedboth the overlying uncalcified cartilage with a gently

undulating tidemark and the underlying subchondralbone with a highly interdigitated cement line (Figures2A, B, D, and E). In fully eroded samples, the calcifiedcartilage was an incomplete layer, appearing in irregularpockets at the smooth bony surface (Figure 2C).

OA samples with partial-thickness erosion ofcartilage exhibited fibrillated cartilage, multiple tide-marks, and a thickened calcified cartilage layer (Figures2B and E). Papain digestion removed uncalcified carti-

Figure 2. Normal (A and D), partially eroded (B and E), or fully eroded (C and F) osteochondral sampleswithout (A, B, and C) and with (D, E, and F) articular cartilage removal by papain digestion. OA � osteoarthritis.

HYDRAULIC CONDUCTANCE OF ARTICULAR CARTILAGE AND BONE 3835

lage but appeared to preserve the entire calcified carti-lage below the lowest tidemark (Figures 2D and E). OAsamples with full-thickness erosion of cartilage andexposure of bone had a smooth bony surface and a thick,dense subchondral bone plate (Figures 2C and F), withan incomplete calcified cartilage layer present in irreg-ular pockets at the surface (Figure 2C). Papain digestionappeared to open up more void space and channelsthrough the subchondral bone plate (Figure 2F).

Vascularity was present in normal subchondralbone plate as well as in OA samples with partial- orfull-thickness erosion of the subchondral bone plate butdiffered in appearance. In normal subchondral boneplate, vascular canals were most commonly seen as longfinger-like protrusions sheathed by bone originatingfrom the marrow space and ending within the calcifiedcartilage (Figure 3A). In partially eroded subchondralbone plate, vascular canals appeared greater in size andnumber, either ending in the calcified cartilage or pro-truding above the calcified cartilage into deep-zonecartilage nearing the duplicate tidemark (Figure 3B). In

fully eroded subchondral bone plate, vascularity ap-peared as large, interconnected void spaces or channelsstarting from within the thickened bone plate and endingat the smooth, bony surface (Figures 2F and 3C).

Stereologic measurements. Calcified cartilageand subchondral bone thickness varied with the degreeof OA erosion but were not altered by papain digestion.Papain digestion did not have an effect on calcifiedcartilage thickness (P � 0.46) or subchondral bonethickness (P � 0.41), which was consistent with qualita-tive histologic findings (Figure 4A). The grade of OAerosion did have a significant effect on both calcifiedcartilage thickness (P � 0.01) and subchondral bonethickness (P � 0.01). The calcified cartilage thickness inpartially eroded samples was 157 �m, �1.5-fold greaterthan that in normal samples (P � 0.01), while thecalcified cartilage thickness in fully eroded samples was54 �m, �2-fold less than that in normal samples (P �0.01) and �3-fold less than that in the partially erodedsamples (P � 0.001). The subchondral bone thickness offully eroded samples was 911 �m, �2-fold greater than

Figure 3. Typical vascular canals penetrating into calcified cartilage in normal subchondral bone plate (A), intodeep zone cartilage in partially eroded subchondral bone plate (B), and to the surface of fully eroded subchondralbone plate (C). OA � osteoarthritis.

3836 HWANG ET AL

that of both normal (P � 0.01) and partially erodedsamples (P � 0.01).

The density of vessels penetrating into the calci-fied cartilage layer or subchondral bone plate surfacevaried with the degree of OA erosion but was notaffected by papain digestion. Papain digestion did nothave a significant effect on the density of vessels pene-trating into or beyond the calcified cartilage (P � 0.27)(Figure 4B). The grade of OA erosion did have asignificant effect on the density of vessels (P � 0.01),

displaying a trend similar to that seen for calcifiedcartilage thickness. Partially eroded subchondral boneplate tended to have the greatest density of vesselspenetrating the calcified cartilage (19 vessels/mm2).

DISCUSSION

These results indicate that hydraulic conductanceof both osteochondral tissue and the isolated subchon-dral bone plate is increased with increasing stages of OAcartilage erosion, in association with structural changesin calcified cartilage and subchondral bone thicknessand vascularity. Increased hydraulic conductance allowsfor greater ease of interstitial fluid flow through theosteochondral tissue and subchondral bone plate. Com-pared with normal osteochondral tissue and subchondralbone plate, partially eroded osteochondral tissue andsubchondral bone plate exhibited no significant differ-ences in hydraulic conductance (Figure 1). However,structural changes were evident in partially eroded sub-chondral bone plate, with increased calcified cartilagethickness (1.5-fold), a trend for increased density ofvascular canals penetrating the calcified cartilage, andlarger vascular canals (Figures 2A and B, 3A and B, and4). Compared with normal tissue, fully eroded osteo-chondral tissue and subchondral bone plate exhibited amarked increase (2,700-fold and 3-fold, respectively) inhydraulic conductance (Figure 1).

Structural changes were also evident in fullyeroded subchondral bone plate, with the appearance ofdiscontinuities in calcified cartilage, increased subchon-dral bone thickness (2-fold), decreased density of vascu-lar canals penetrating the subchondral bone plate sur-face, and larger void spaces within the subchondral boneplate (Figures 2B and C, 3B and C, and 4). In theprogression from normal osteochondral tissue into thepartially and fully eroded stages of OA, changes incalcified cartilage thickness and vascularity occur to-gether with increases in hydraulic conductance andsubchondral bone thickness. These results suggest thatthe calcified cartilage structural and vascular remodelingthat occurs with OA contributes to increased ease offluid flow across the osteochondral structure, a factorthat may play a role in advancing OA degeneration(Figure 5).

The subchondral bone plate was isolated fromosteochondral tissue using a method that may haveaffected the hydraulic conductance results. In humansubchondral bone plate preparations, papain digestionselectively removes uncalcified cartilage down to thetidemark while maintaining calcified cartilage thickness

Figure 4. Stereologic measurements of subchondral bone plate struc-ture. A, Thickness of calcified cartilage (CC; solid portion of bars) andunderlying subchondral bone (ScB; open portion of bars). The totalheight of each column is the subchondral bone plate thickness. B,Number of vessels penetrating the calcified cartilage–bone interfaceper cross-sectional area in normal, partially eroded, and fully erodedosteochondral samples before and after cartilage removal by papaindigestion. Bars show the mean and SEM. ● � P � 0.01 for within-group or between-group comparisons; Œ � P � 0.001 for between-group comparison. OA � osteoarthritis.

HYDRAULIC CONDUCTANCE OF ARTICULAR CARTILAGE AND BONE 3837

and contour but not creating perforations through thesubchondral bone plate (18). Such subchondral boneplate preparations are devoid not only of cartilage butalso of marrow and soft tissue linings of vascular canals,which could participate in modulating fluid transportacross the subchondral bone plate. Papain digestion mayalso remove proteins such as collagen from bone, whichcould affect the subchondral bone plate hydraulic con-ductance. Papain digestion of normal and partiallyeroded subchondral bone plate (which had been isolatedmechanically) increased conductance 2-fold, whereaspapain digestion of fully eroded samples increased con-ductance �13-fold (Figure 1B). Thus, the estimate ofhydraulic conductance of subchondral bone plate, asperformed in the current study with papain digestion, islikely to be higher than subchondral bone plate conduc-tance in vivo. Nevertheless, papain digestion was usefulas a repeatable, nondestructive method for removinguncalcified cartilage while preserving the structure ofcalcified cartilage.

The 2-hour hydraulic conductance constant (c2h)

evaluated in the present study is an upper bound esti-mate of the overall conductance of full-thickness osteo-chondral tissue. Hydraulic conductance of osteochon-dral tissue is determined by the low fluid permeability ofthe cartilage matrix, which dominates the fluid pressur-ization behavior and resistance to flow. As fluid entersthe cartilage and flows from the superficial zone towardthe deep zone, it exerts drag forces to pull the solidmatrix with it. The drag forces result in matrix compac-tion, which decreases porosity and permeability withinthe tissue in a depth-varying, nonuniform manner (29–31). A new steady state is reached when the matrixconsolidation and fluid flow through the tissue arebalanced, maintaining a constant pressure across thetissue. In the present study, perfusion flow time wasstandardized to 2 hours, as a physiologically relevantperiod of loading, and to allow calculation of the c2h. At2 hours, full-thickness osteochondral tissue would not befully compacted, and the c2h is higher than that at steadystate (cSS). For normal osteochondral tissue, the c2h isgreater than the cSS by �5-fold (data not shown). For

Figure 5. Schematic presentation of normal (A), partially eroded (B), and fully eroded (C) osteochondral tissueand potential deleterious effects of increased subchondral bone plate permeability leading to increased fluid loss.Wavy arrows indicate fluid flow within cartilage and also fluid loss from overlying (B) and opposing (C) articularcartilage during loading. Plus signs indicate the magnitude of osteochondral and subchondral bone platehydraulic conductance. See Figure 4 for definitions.

3838 HWANG ET AL

porous, permeable materials with a stiff solid matrix,such as subchondral bone plate, the effect of flow-dependent matrix consolidation is negligible; therefore,steady state is reached on a shorter time scale. Forsubchondral bone plate, the c0.5h and cSS were similar(data not shown).

The observations of differences in permeabilityand structure between normal and OA articular carti-lage and subchondral bone plate extend the findings ofprevious studies. The present study extends permeabilitymeasurements, traditionally made on isolated cartilageand bone tissues, by directly analyzing the full thicknessof articular cartilage still attached to subchondral boneplate, providing an overall conductance for the osteo-chondral unit. Previous measurements of hydraulic per-meability on cartilage sections elucidated variations inzonal properties (32) and may have been influenced byalterations in matrix organization and water content dueto detachment from the underlying calcified cartilage(11,33).

The c2h and c0.5h of normal osteochondral tissueand subchondral bone plate determined in the presentstudy can be converted into corresponding apparenthydraulic permeability (kp) values, using average thick-nesses for cartilage and subchondral bone plate, allow-ing comparison with previous studies (Table 1). Theapparent kp value of normal cartilage attached to sub-chondral bone plate in the present study was greaterthan values for normal cartilage reported in the litera-ture (1,11,34). This can be attributed in part to the2-hour perfusion time, which did not allow the full-

thickness cartilage to reach steady-state hydrostatic pres-surization and associated matrix compaction, resulting ina higher apparent permeability and precluding directcomparison with kp values in the literature, which reflectsteady state. The kp value of normal subchondral boneplate observed in the current study, which reachedsteady state on a shorter time scale (�0.5 hour), waswithin the range of values reported for normal corticaland cancellous bone (13,35,36) (Table 1).

The trend toward a small increase in hydraulicconductance of OA osteochondral tissue is consistentwith the slight increases in hydraulic permeability of OAarticular cartilage reported previously (3–5). Increasedhydraulic conductance of osteochondral tissue occurringwith OA may be attributable primarily to decreasedcartilage thickness, increased hydraulic permeability ofthe remaining OA cartilage, or a combination of both.However, in the current study, hydraulic conductance ofpartially eroded osteochondral tissue was not signifi-cantly different from that of normal tissue, despitecartilage thickness being 30% lower. Because fluid pres-surization is normally maintained by the low hydraulicpermeability of deep zone cartilage (31,32,37), the over-all hydraulic conductance of osteochondral tissue mayremain close to normal for cases of partial erosion inwhich the deep zone remains intact. For osteochondraltissue with full-thickness erosion of cartilage, the rela-tively high hydraulic conductance may be determined byany residual cartilage in the subchondral bone plate.

Increased hydraulic conductance of osteochon-dral tissue and the subchondral bone plate could have

Table 1. Hydraulic permeability (kp) (mm2/[MPa � second]) of articular cartilage and subchondral boneplate

Tissue/type kp Average thickness, mm Ref.

CartilageNormal 0.0001–0.002 0.3–0.8 1, 11, 32

Cartilage plus ScBPNormal 0.15 2.5 Present studyOA, partially eroded* 0.27 1.8 Present study

Cortical boneNormal (canine) 40–80 0.5–1.0 33, 34

ScBPNormal 90 0.47 Present studyOA, partially eroded 140 0.46 Present studyOA, fully eroded 190 1.1 Present study

ScBP, papain-digestedNormal 350 0.49 Present studyOA, partially eroded 830 0.50 Present studyOA, fully eroded 2,010 0.87 Present study

Cancellous boneNormal 20,000–8,000,000 1.0 13, 33

* OA � osteoarthritis; ScBP � subchondral bone plate.

HYDRAULIC CONDUCTANCE OF ARTICULAR CARTILAGE AND BONE 3839

deleterious biomechanical consequences for cartilageduring joint loading (Figure 5). Normally, articularcartilage is able to support joint loads through interstitialfluid pressurization due to its low hydraulic conductance(Figure 5A). Fluid pressurization in cartilage plays amajor role in providing load support for a prolongedduration after contact, preventing the solid matrix fromdeforming to an equilibrium strain state (38). Theduration of load support by fluid pressurization can becharacterized using a time constant to reach equilibriumthat depends on factors affecting the rate of fluid lossfrom cartilage, including the radial path length, com-pressive modulus, and hydraulic permeability (39). Fornormal human cartilage with a contact radius, a, of 10mm, a compressive modulus, HA, of 0.5 MPa, and a kp

value of 2 � 10�15 m4/(N � second), the characteristictime constant, a2/(HA � kp), is �28 hours, thus protectingcartilage for long durations of loading. In OA, increasedfluid loss from cartilage during joint loading may occurdue to fluid exudation into the underlying subchondralbone plate, causing shorter times to equilibrium andpotentially leading to large deformations and acceler-ated cartilage degeneration (Figure 5B).

For full-thickness cartilage erosion with exposureof the subchondral bone plate, the articular cartilage ofthe opposing joint surface may be damaged throughfluid exudation from its superficial zone into andthrough the eburnated and permeable joint surface(Figure 5C). The depth of the opposing cartilage thatundergoes fluid depressurization and concomitant tissueconsolidation would depend on the time characteristicsof loading. The characteristic time to equilibrium for theopposing cartilage, using confined compression analysisof normal human cartilage with 2-mm thickness, is �1hour (40). At the relatively high physiologic frequency ofgait (0.5 Hz), the characteristic depth of fluid depressur-ization extends �40 �m from the articular surface. Witha prolonged loading duration typical of standing, e.g., forperiods of 15 minutes (0.001 Hz), the fluid depressur-ization extends �1 mm from the surface (34). Even fora short loading duration of 2 minutes and a conservativecontact stress of 0.5 MPa, the opposing cartilage surfacewould experience a deformation of �0.2 mm (40), whichcould lead to localized cell death, as when the superficialzone is compressed against a porous platen in vitro (41).Thus, the biomechanical consequences of increased sub-chondral bone plate permeability in OA may contributeto overlying cartilage degeneration as well as the spread-ing of OA onto the opposing joint surface.

In the normal joint, the calcified cartilage inter-

face may function as a zone of intermediate hydraulicpermeability between articular cartilage and subchon-dral bone, similar to its role as a zone of intermediatestiffness for the transfer of mechanical loads. Althoughthe calcified cartilage layer in mature joints has previ-ously been considered impermeable (14,42), this percep-tion may have been attributable to low rates of solutediffusion and convection into the adjacent cartilage deepzone rather than low hydraulic permeability of thecalcified cartilage layer itself. In the present study,eburnated subchondral bone plate with eroded calcifiedcartilage had higher hydraulic conductance than normalsubchondral bone plate with intact calcified cartilage, inspite of the greater subchondral bone thickness anddensity from eburnation, which would be expected todecrease conductance. Thus, an intact calcified cartilagelayer appears to be more permeable to fluid than isarticular cartilage but less permeable than is corticalbone, making it a zone of intermediate permeabilitybetween deep zone cartilage and subchondral bone inthe normal osteochondral junction. With the onset ofOA, the relative permeabilities of cartilage, calcifiedcartilage, and subchondral bone in the osteochondraljunction may be altered, which may disrupt the ho-meostasis of the osteochondral unit and affect progres-sion of the disease.

OA subchondral bone plate may undergo addi-tional structural changes due to the intrusion of fluidinto eroded or permeable areas, including the develop-ment of cartilaginous pockets and bone marrow lesions.Hydraulic conductance of fully eroded subchondralbone plate increased 10-fold, along with the appearanceof void pockets and channels after papain digestion,consistent with residual cartilage within the subchondralbone plate. Cartilaginous pockets in OA joints withexposed bone have been documented both within and atthe surface of the subchondral bone plate and mayparticipate in the repair process (43–45). The void areasmay also represent bone cysts or bone marrow lesionsfound in sclerotic bone, which are associated with pain inOA (46,47). Bone cysts may arise from intrusion ofpressurized fluid into bone at the joint surface, assuggested by openings between cysts and the joint cavityand their rounded morphology (10).

The increase in hydraulic conductance of osteo-chondral tissue and subchondral bone plate in OAallows more direct fluid movement between cartilageand bone compared with that in the normal joint. Fluidmovement can, in turn, act as a mechanical signal to cellsand enhance the transport of diffusible factors, pro-

3840 HWANG ET AL

cesses that may mediate the crosstalk between cartilageand bone. In OA, altered boundary conditions at theosteochondral interface may disrupt the mechanical andchemical interactions between the microenvironments ofcartilage and bone, which may affect tissue homeostasisand cell physiology. Understanding how altered bound-ary conditions affect the mechanical and chemical inter-actions between cartilage and bone will lead to a morecomplete picture of the multi-tissue pathogenesis of OA.

AUTHOR CONTRIBUTIONS

Dr. Sah had full access to all of the data in the study and takesresponsibility for the integrity of the data and the accuracy of the dataanalysis.Study design. Hwang, Lewis, Sah.Acquisition of data. Hwang, Bae, Shieu.Analysis and interpretation of data. Hwang, Bugbee, Sah.Manuscript preparation. Hwang, Bugbee, Sah.Statistical analysis. Hwang, Sah.

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