Bovine Humeral Head Research Articles

Active Preservation of Osteochondral Graft under Cyclic Hydrostatic Pressure for Maintaining Cartilage Integrity and Subchondral Bone Quality

Objectives:Osteochondral allografts (OCA) are currently stored at 4˚C for an average of 24 days after procurement. However, chondrocyte viability, the major determinant of graft performance in-vivo, decreases and matrix integrity deteriorates with time under such conditions; for instance, chondrocyte viability falls below the acceptable level of 70% by 28 days. Hydrostatic pressure (HP) is a significant component in the mechanical loading environment and its application in vitro (0.5 - 15 MPa) elicited favorable effects on cartilage and bone. The purpose of this study was to investigate the effects of HP during osteochondral storage.Methods:Osteochondral (OC) explants (6x8 mm) were harvested from bovine humeral heads purchased from a local slaughterhouse (Fig. 1). OCs were randomly assigned to one of 4 groups: freshly harvested; stored at 4°C and atmospheric pressure (4˚C AP); stored at 37°C and AP (37˚C AP); and stored at 37°C with cyclic HP at 0-0.5 MPa, 0.5 Hz (37˚C HP). The explants were stored for 7, 14, 21, or 28 days in Dulbecco’s modified eagle medium/Ham’s nutrient mixture F12 (1:1), with antibiotics and with or without fetal bovine serum (FBS). Chondrocyte viability and density were assessed using LIVE/DEAD® staining in the entire tissue section and in the superficial, middle, and deep zones using the Image J program. In addition, we stained OC explants with safranin-O fast green; hematoxylin and eosin, sudan IV and antibodies against proliferating cell nuclear antigen (PCNA), keratan sulfate (KS). Furthermore, the microstructure and material composition of subchondral bone in the explants was quantified using micro-computed tomography (micro-CT, Scanco uCT 35) at a 12 micron nominal resolution.Results:Cartilage thickness was maintained at 37°C HP, significantly increased at 37°C AP and significantly decreased at 4°C AP (P < 0.05) with/without FBS at 28 days. Full-thickness chondrocyte viability was significantly higher at 21 and 28 days in the 37˚C HP group compared to 4°C or 37°C AP regardless of FBS (P<0.05). Specifically, chondrocyte viability in the superficial zone was maintained with 37˚C HP (Fig. 3) versus not in the other culture conditions. Cartilage matrices were similar between the conditions by 14 days (Fig. 4). However, surface fibrillation and degeneration were demonstrated in the 37 °C AP. KS seemed to be maintained in the superficial zone with HP by 28 days but decreased under other conditions (Fig. 5). More proliferating cells with 37°C HP were observed in the superficial zone compared to other conditions at 28 days. Utilization of FBS further increased the number of proliferating cells in each condition. The fatty granules were observed and distributed evenly throughout the subchondral bone and trabecular bone under all conditions at 28 days (Fig. 6). However, in the 4°C and 37°C AP groups, fewer fatty granules were observed in the subchondral bone and more fat in the trabecular bone (Fig. 6). Micro-CT evaluation revealed that the conditions produced similar tissue mineral density (Fig. 7). However, the trabecular number was significantly lower with 37°C AP compared to the other conditions (P<0.05).Conclusion:Osteochondral explants stored with 37°C HP maintained chondrocyte viability, histologically determined cartilage integrity, and subchondral bone quality.Figure 1.

Finite Element Modeling of Articular Cartilage to Characterize Biomechanical Properties: The Effect of Cartilage Surface Curvature

Degeneration and loss of articular cartilage in the synovial joint have been recognized as the main source of osteoarthritis which leads to pain, swelling and limit the joint mobility. Extensive experimental and computational studies have been performed to study the mechanical behavior and characterize the biomechanical properties of articular cartilage. However, a lack of attention was made on the curvature of the cartilage surface by assuming it was a flat surface. This assumption was inappropriate since the synovial joints possessed curved geometrical shape and may contribute to inaccuracies in characterizing the articular cartilage biomechanical properties. This study aims to examine the effects of the curvature of the cartilage surface in finite element modeling which incorporated with the experiment method to characterize biomechanical properties of articular cartilage. In this study, the biomechanical behavior of contact pressure and pore pressure were investigated at different radius of cartilage surface using the finite element method. The cartilage biomechanical properties of elastic modulus and permeability of the bovine humeral head were then characterized using a combination of indentation test and finite element method. It was found that the cartilage curvature produced a 6% difference in contact pressure and a 39% difference in pore pressure distribution compared to the flat surface cartilage in finite element analysis. Furthermore, significant observation in the characterized biomechanical properties was obtained where the differences of the cartilage curvature reached 33% for elastic modulus and 56% for permeability. Based on the results, the surface curvature of articular cartilage could play an important role in the computational modeling and characterization of its biomechanical properties.

Effect of hydration on the frequency-dependent viscoelastic properties of articular cartilage

The aim of this study was to determine the effect of tissue hydration on the frequency-dependant viscoelastic properties of articular cartilage. Such changes were determined at frequencies associated with normal (1-10 Hz) and impulsive/traumatic (90 Hz) heel-strike times. Cartilage on bone samples, obtained from bovine humeral heads, was tested when hypo-hydrated and hyper-hydrated using dynamic mechanical analysis. Dynamic mechanical analysis was performed at a range of frequencies between 1 and 90 Hz. Hypo-hydration increased the stiffness of cartilage as compared to hyper-hydrated cartilage; this increase was greater at higher frequencies. The storage modulus and stiffness increased in hypo-hydrated cartilage as compared to hyper-hydrated cartilage. However, the loss modulus and stiffness increased when cartilage was hypo-hydrated as compared to hyper-hydrated, but these increases were not frequency dependent. An impulsive heel-strike time may result in a greater increase of stiffness in hypo-hydrated cartilage, compared with hyper-hydrated cartilage. However, the ratio of storage to loss stiffness was greater for hyper-hydrated cartilage, thereby, reducing the tissue's ability to dissipate energy and increasing the likelihood of cartilage rupture.

Evaluation of Negative Fixed-Charge Density in Tissue-Engineered Cartilage by Quantitative MRI and Relationship With Biomechanical Properties

Applying tissue-engineered cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of the cartilage. Magnetic resonance imaging (MRI) of articular cartilage has been widely accepted and applied clinically in recent years. In this study, we evaluated the negative fixed-charge density (nFCD) of tissue-engineered cartilage using gadolinium-enhanced MRI and determined the relationship between nFCD and biomechanical properties. To reconstruct cartilage tissue, articular chondrocytes from bovine humeral heads were embedded in agarose gel and cultured in vitro for up to 4 weeks. The nFCD of the cartilage was determined using the MRI gadolinium exclusion method. The equilibrium modulus was determined using a compressive stress relaxation test, and the dynamic modulus was determined by a dynamic compression test. The equilibrium compressive modulus and dynamic modulus of the tissue-engineered cartilage increased with an increase in culture time. The nFCD value--as determined with the [Gd-DTPA(2-)] measurement using the MRI technique--increased with culture time. In the regression analysis, nFCD showed significant correlations with equilibrium compressive modulus and dynamic modulus. From these results, gadolinium-enhanced MRI measurements can serve as a useful predictor of the biomechanical properties of tissue-engineered cartilage.

Two-dimensional strain fields on the cross-section of the bovine humeral head under contact loading

The objective of this study was to provide a detailed experimental assessment of the two-dimensional cartilage strain distribution on the cross-section of immature and mature bovine humeral heads subjected to contact loading at a relatively rapid physiological loading rate. Six immature and six mature humeral head specimens were loaded against glass and strains were measured at the end of a 5 s loading ramp on the textured articular cross-section using digital image correlation analysis. The primary findings indicate that elevated tensile and compressive strains occur near the articular surface, around the center of the contact region. Few qualitative or quantitative differences were observed between mature and immature joints. Under an average contact stress of ∼1.7 MPa, the peak compressive strains averaged −0.131±0.048, which was significantly less than the relative change in cartilage thickness, −0.104±0.032 ( p<0.05). The peak tensile strains were significantly smaller in magnitude, at 0.0325±0.013. These experimental findings differ from a previous finite element analysis of articular contact, which predicted peak strains at the cartilage–bone interface even when accounting for the porous-hydrated nature of the tissue, its depth-dependent inhomogeneity, and the disparity between its tensile and compressive properties. These experimental results yield new insights into the local mechanical environment of the tissue and cells, and suggest that further refinements are needed in the modeling of contacting articular layers.

Biomechanical characterization and in vitro mechanical injury of elderly human femoral head cartilage: comparison to adult bovine humeral head cartilage

In vitro mechanical injury of articular cartilage is useful to identify events associated with development of post-traumatic osteoarthritis (OA). To date, many in vitro injury models have used animal cartilage despite the greater clinical relevance of human cartilage. We aimed to characterize a new in vitro injury model using elderly human femoral head cartilage and compare its behavior to that of an existing model with adult bovine humeral head cartilage. Mechanical properties of human and bovine cartilage disks were characterized by elastic modulus and hydraulic permeability in radially confined axial compression, and by Young's modulus, Poisson's ratio, and direction-dependent radial strain in unconfined compression. Biochemical composition was assessed in terms of tissue water, solid, and glycosaminoglycan (GAG) contents. Responses to mechanical injury were assessed by observation of macroscopic superficial tissue cracks and histological measurements of cell viability following single injurious ramp loads at 7 or 70%/s strain rate to 3 or 14 MPa peak stress. Confined compression moduli and Young's moduli were greater in elderly human femoral cartilage vs adult bovine humeral cartilage whereas hydraulic permeability was less. Radial deformations of axially compressed explant disks were more anisotropic (direction-dependent) for the human cartilage. In both cartilage sources, tissue cracking and associated cell death during injurious loading was common for 14 MPa peak stress at both strain rates. Despite differences in mechanical properties, acute damage induced by injurious loading was similar in both elderly human femoral cartilage and adult bovine humeral cartilage, supporting the clinical relevance of animal-based cartilage injury models. However, inherent structural differences such as cell density may influence subsequent cell-mediated responses to injurious loading and affect the development of OA.

Assessment of Fixed Charge Density in Regenerated Cartilage by Gd-DTPA-enhanced MRI

Applying regenerated cartilage in a clinical setting requires noninvasive evaluation to detect the maturity of cartilage tissue. Magnetic resonance (MR) imaging of articular cartilage is well accepted and has been applied clinically in recent years. We attempt to establish a noninvasive method to evaluate the maturity of regenerated cartilage tissue using gadolinium-enhanced MR imaging. To reconstruct cartilaginous tissue, we embedded articular chondrocytes harvested from bovine humeral head in agarose gel and cultured the cells in vitro up to 4 weeks. The fixed charge density (FCD) of the cartilage was determined using MRI gadolinium exclusion method. The sulfated glycosaminoglycan (sGAG) content was determined by dimethylmethylene blue dye-binding assay. The sGAG content and FCD of the regenerated cartilage increased with duration of culture. In the T1Gd maps, the [Gd-DTPA(2-)] in the specimen decreased, and the boundary between the sample disk and the bath solution of phosphate buffered saline (PBS) became clearer as time in culture increased. In the linear regression analysis, FCD and sGAG content correlated significantly. Gadolinium-enhanced MR imaging measurements can be useful predictors of the degree of cartilaginous tissue formation.

Effect of tissue maturity on cell viability in load-injured articular cartilage explants

During joint maturation, articular cartilage undergoes compositional, structural, and biomechanical changes, which could affect how the chondrocytes within the cartilage matrix respond to load-induced injury. The objective of this study was to determine the effects of tissue maturity on chondrocyte viability when explanted cartilage was subjected to load-induced injury. Cartilage explants from immature (4-8-week-old) and mature (1.5-2-year-old) bovine humeral heads were cyclically loaded at 0.5 hertz in confined compression with a stress of 1 or 5 megapascals for 0.5, 1, 3, 6 and 16 h. Cell death was assessed at 0, 24 and 48 h after load removal using cell viability dyes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The organization of pericellular matrix (PCM), biochemical composition and biomechanical properties of the cartilage were also determined. For the immature and mature cartilage, cell death began at the articular surface and increased in depth with loading time up to 6h. No increase of cell death was found after load removal for up to 48 h. In both groups, cell death increased at a faster rate with the increase of stress level. The depth of cell death in the immature cartilage was greater than the mature cartilage, despite the immature cartilage having a higher bulk aggregate modulus. A less organized PCM in immature cartilage was found as indicated by the weak staining of type VI collagen. Cells in the mature cartilage are less vulnerable to load-induced injury than those in immature cartilage.

Microscale frictional response of bovine articular cartilage from atomic force microscopy

The objective of this study was to compare micro- and macroscale friction coefficients of bovine articular cartilage. Microscale measurements were performed using standard atomic force microscopy (AFM) techniques, using a 5 μm spherical probe tip. Twenty-four cylindrical osteochondral plugs were harvested in pairs from adjacent positions in six fresh bovine humeral heads (4–6 months old), and divided into two groups for AFM and macroscopic friction measurements. AFM measurements of friction were observed to be time-independent, whereas macroscale measurements demonstrated the well-documented time-dependent increase from a minimum to an equilibrium value. The microscale AFM friction coefficient ( μ AFM, 0.152±0.079) and macroscale equilibrium friction coefficient ( μ eq, 0.138±0.036) exhibited no statistical differences ( p=0.50), while the macroscale minimum friction coefficient ( μ min , 0.004±0.001) was significantly smaller than μ eq and μ AFM ( p<0.0001). Variations in articular surface roughness ( R q=462±216 nm) did not correlate significantly with μ AFM, μ eq or μ min. The effective compressive modulus determined from AFM indentation tests using a Hertz contact analysis was E *=45.8±18.8 kPa. The main finding of this study is that μ AFM is more representative of the macroscale equilibrium friction coefficient, which represents the frictional response in the absence of cartilage interstitial fluid pressurization. These results suggest that AFM measurements may be highly suited for exploring the role of boundary lubricants in diarthrodial joint lubrication independently of the confounding effect of fluid pressurization to provide greater insight into articular cartilage lubrication.

Estimation of the Young's modulus of articular cartilage using an arthroscopic indentation instrument and ultrasonic measurement of tissue thickness

We evaluated whether the use of cartilage thickness measurement would improve the ability of the arthroscopic indentation technique to estimate the intrinsic stiffness of articular cartilage. First, cartilage thickness and ultrasound reflection from the surface of bovine humeral head were registered in situ using a high-frequency ultrasound probe. Subsequently, cartilage was indented in situ at the sites of the ultrasound measurements using arthroscopic instruments with plane-ended and spherical-ended indenters. Finally, full-thickness cartilage disks ( n=30) were extracted from the indented sites (thickness=799–1654 μm) and the equilibrium Young's modulus was determined with a material testing device in unconfined compression geometry. We applied analytical and numerical indentation models for the theoretical correction of experimental indentation measurements. An aspect-ratio (the ratio of indenter radius to cartilage thickness) correction improved the correlation of the indenter force with the equilibrium Young's modulus from r 2=0.488 to r 2=0.642–0.648 ( n=30) for the plane-ended indenter (diameter=1.000 mm, height=0.300 mm) and from r 2=0.654 to r 2=0.684–0.692 ( n=30) for the spherical-ended indenter (diameter=0.500 mm, height=0.100 mm), depending on the indentation model used for the correction. The linear correlation between the ultrasound reflection and the Young's modulus was r 2=0.400 ( n=30). These results suggest that with large indenters, knowledge of the cartilage thickness improves the reliability of the indentation measurements, especially in pathological situations where cartilage thickness may be significantly lower than normal. Ultrasound measurements also provide diagnostically important information about cartilage thickness as well as knowledge of the integrity of the superficial zone of cartilage.

A Conewise Linear Elasticity mixture model for the analysis of tension-compression nonlinearity in articular cartilage.

A biphasic mixture model is developed that can account for the observed tension-compression nonlinearity of cartilage by employing the continuum-based Conewise Linear Elasticity (CLE) model of Curnier et al. (J. Elasticity, 37, 1-38, 1995) to describe the solid phase of the mixture. In this first investigation, the orthotropic octantwise linear elasticity model was reduced to the more specialized case of cubic symmetry, to reduce the number of elastic constants from twelve to four. Confined and unconfined compression stress-relaxation, and torsional shear testing were performed on each of nine bovine humeral head articular cartilage cylindrical plugs from 6 month old calves. Using the CLE model with cubic symmetry, the aggregate modulus in compression and axial permeability were obtained from confined compression (H-A = 0.64 +/- 0.22 MPa, k2 = 3.62 +/- 0.97 x 10(-16) m4/N.s, r2 = 0.95 +/- 0.03), the tensile modulus, compressive Poisson ratio, and radial permeability were obtained from unconfined compression (E+Y = 12.75 +/- 1.56 MPa, v- = 0.03 +/- 0.01, kr = 6.06 +/- 2.10 x 10(-16) m4/N.s, r2 = 0.99 +/- 0.00), and the shear modulus was obtained from torsional shear (mu = 0.17 +/- 0.06 MPa). The model was also employed to predict the interstitial fluid pressure successfully at the center of the cartilage plug in unconfined compression (r2 = 0.98 +/- 0.01). The results of this study demonstrate that the integration of the CLE model with the biphasic mixture theory can provide a model of cartilage that can successfully curve-fit three distinct testing configurations while producing material parameters consistent with previous reports in the literature.

Optimization of the arthroscopic indentation instrument for the measurement of thin cartilage stiffness

Structural alterations associated with early, mostly reversible, degeneration of articular cartilage induce tissue softening, generally preceding fibrillation and, thus, visible changes of the cartilage surface. We have already developed an indentation instrument for measuring arthroscopic stiffness of cartilage with typical thickness >2 mm. The aim of this study was to extend the applicability of the instrument for the measurement of thin (<2 mm) cartilage stiffness. Variations in cartilage thickness, which will not be known during arthroscopy, can nonetheless affect the indentation measurement, and therefore optimization of the indenter dimensions is necessary. First, we used theoretical and finite element models to compare plane-ended and spherical-ended indenters and, then, altered the dimensions to determine the optimal indenter for thin cartilage measurements. Finally, we experimentally validated the optimized indenter using bovine humeral head cartilage. Reference unconfined compression measurements were carried out with a material testing device. The spherical-ended indenter was more insensitive to the alterations in cartilage thickness (20% versus 39% in the thickness range 1.5-5 mm) than the plane-ended indenter. For thin cartilage, the optimal dimensions for the spherical-ended indenter were 0.5 mm for diameter and 0.1 mm for height. The experimental stiffness measurements with this indenter correlated well with the reference measurements (r = 0.811, n = 31, p<0.0001) in the cartilage thickness range 0.7-1.8 mm. We conclude that the optimized indenter is reliable and well suited for the measurement of thin cartilage stiffness.

Mature Full-thickness Articular Cartilage Explants Attached to Bone are Physiologically Stable over Long-term Culture in Serum-free Media

Mature tissue explants containing the entire depth of articular cartilage, calcified and uncalcified, attached to a thin layer of subchondral bone were isolated from bovine humeral heads of 1-2-year-old steers. These explants were placed in defined serum-free culture medium for a period of 3 weeks to investigate their biological and mechanical stability and thus to determine their potential utility in studies of cartilage physiology. Tissue mass remained constant over the culture period and no evident tissue swelling or distortion was observed. Chondrocytes were viable in all zones at the time of tissue isolation and throughout the culture period, with the exception of a thin layer of cells at the articular surface and the cut radial edge of the disks. Proteoglycan metabolism attained a steady state after 5 days of culture when the rate of loss of proteoglycan to culture media was compensated by new synthesis to maintain a stable proteoglycan content. Collagen metabolism was also stable with a constant content of type II collagen and a constant content of denatured collagen II throughout culture; the content of the C-propeptide of type II procollagen as a measure of procollagen synthesis, dropped slightly during the first week to attain a steady state after that time. Dynamic and equilibrium mechanical properties of these explant disks were also stable confirming maintenance of these tissue properties during long-term culture. In addition, the disk geometry of the system, with the cut surface in the bone parallel to the intact articular surface, is well-suited to study tissue regulation by mechanical load. Taken together, the stability of these indicators of tissue physiology indicates the maintenance in serum-free conditions of normal metabolism for organ cultures containing full-depth mature articular cartilage attached to bone.

Optical and mechanical determination of poisson's ratio of adult bovine humeral articular cartilage

The equilibrium stiffness of articular cartilage is controlled by flow-independent elastie properties (Young's modulus, E s, and Poisson's ratio, v s) of the hydrated tissue matrix. In the current study, an optical (microscopic) method has been developed for the visualization of the boundaries of cylindrical bovine humeral head articular cartilage disks ( n = 9), immersed in physiological solution, and compressed in unconfined geometry. This method allowed a direct, model-independent estimation of Poisson's ratio of the tissue at equilibrium, as well as characterization of the shape changes of the sample during the nonequilibrium dynamic phase. In addition to optical analyses, the equilibrium behavior of cartilage disks in unconfined and confined ramp-stress relaxation tests provided a direct estimation of the aggregate modulus, H a, and Young's modulus and, indirectly. Poisson's ratio for the articular cartilage. The mean value for Poisson's ratio obtained from the optical analysis was 0.185 ± 0.065 (mean ± S.D., n = 9). Values of elastic parameters obtained from the mechanical tests were 0.754 ± 0.198 MPa, 0.677 ± 0.223 MPa, and 0.174 ± 0.106 for H a, E s, and v s, respectively (mean ± S.D., n = 7). The similar v s-values obtained with optical and mechanical techniques imply that, at equilibrium, for these two tests, the isotropic model is acceptable for mechanical analysis. However, the microscopic techniques revealed that the lateral expansion, especially during the initial phase of relaxation, was inhomogeneous through the tissue depth. The superficial cartilage zone expanded less than the radial zone. The zonal differences in expansion were attributed to the known zonal differences in the fibrillar collagen architecture and proteoglycan concentration.

Surface and Subsurface Morphology of Bovine Humeral Articular Cartilage as Assessed by Atomic Force and Transmission Electron Microscopy

Maintenance of superficial structural integrity is essential for the load-bearing function of articular cartilage. In this study, we used atomic force microscopy (AFM) to image the 3-D surface and subsurface morphology of fresh bovine humeral head articular cartilage maintained in physiological solution. Complementary ultrastructural data were obtained by transmission electron microscopy (TEM) of cryoprocessed samples. The surface irregularities observed in previous scanning electron microscopic studies were not apparent with AFM. The most superficial layer, typically 200–500 nm thick, consisted of acellular and nonfibrous tissue. Occasionally, it exhibited local discontinuities through which the underlying network of collagen fibrils, oriented parallel to the surface and displaying the characteristic periodic banding, could been seen. Local variations in force-curve measurements indicate the existence of differences in micromechanical properties along the articular surface. AFM thus furnishes a new method for characterizing the surface structure and properties of freshly excised articular cartilage in physiologically relevant conditions. It confirms the existence of an amorphous, nonfibrous articular surface which may be vital for its normal lubrication and wearing propertiesin vivo.

A method of quantitative autoradiography for the spatial localization of proteoglycan synthesis rates in cartilage.

Incorporation of [35S]-sulfate into cartilage tissue indicates the synthesis of aggrecan, the large aggregating proteoglycan (PG) that endows cartilage with resistance to compression. Scintillation counting of tissue digests provides a quantitative measure of incorporated sulfate but does not provide information on the spatial location of synthesis within the tissue. Such spatially specific information is necessary to determine which cell populations respond to diffusible factors and to correlate local mechanical events (e.g., deformation, interstitial fluid stress) to cellular biosynthetic responses. The aim of this study was to develop and characterize a liquid emulsion autoradiography technique, including an automated grain counting procedure, to derive spatial profiles of aggrecan synthesis rates in cartilage. We chose mature 10-14-month-old bovine humeral head articular cartilage as a model system and applied a liquid emulsion autoradiography technique to [35S]-sulfate-labeled, resin-embedded, and semithin-sectioned tissue explants. High-magnification light microscopy color images were captured on a computer. Automated image analysis for grain number determination included a color thresholding procedure to discriminate grains from the lightly stained structural image and computation of the average area of a single grain from each image. Determination of grain number, whether originating from single grains or grain clusters, was performed by dividing the total grain area in the image by the average area of a single grain in the same image. This procedure largely eliminated the effects of variations of microscope light intensity, camera performance, image focus, section stain intensity, and thresholding on the resulting grain numbers. By altering the specific activity of the medium radiolabel and the emulsion exposure times, we demonstrated a linear dose dependence, without saturation, of grain number on radioactive content in the underlying section. By cutting specimens in half and performing liquid scintillation counting on one half and autoradiography on the other half, we found that each disintegration occurring in the section during exposure resulted in 0.67 +/- 0.21 grains (mean +/- SD; n = 58). Therefore, counted grain numbers can be directly converted to incorporated sulfate, largely reflecting the synthesis of the PG aggrecan. As an example of calculated intratissue profiles of aggrecan synthesis rates, we found for the mature bovine tissue in serum-free medium that aggrecan synthesis increases monotonically from the articular surface to the radial zone by as much as tenfold.