Biomechanical properties of knee articular cartilage

Structure and properties of knee articular cartilage are adapted to stresses exposed on it during physiological activities. In this study, we describe site- and depth-dependence of the biomechanical properties of bovine knee articular cartilage. We also investigate the effects of tissue structure and composition on the biomechanical parameters as well as characterize experimentally and numerically the compression-tension nonlinearity of the cartilage matrix. In vitro mechano-optical measurements of articular cartilage in unconfined compression geometry are conducted to obtain material parameters, such as thickness, Young's and aggregate modulus or Poisson's ratio of the tissue. The experimental results revealed significant site- and depth-dependent variations in recorded parameters. After enzymatic modification of matrix collagen or proteoglycans our results show that collagen primarily controls the dynamic tissue response while proteoglycans affect more the static properties. Experimental measurements in compression and tension suggest a nonlinear compression-tension behavior of articular cartilage in the direction perpendicular to articular surface. Fibril reinforced poroelastic finite element model was used to capture the experimentally found compression-tension nonlinearity of articular cartilage.     

Frictional behaviour of bovine articular cartilage

The experimentally measured indentation displacement and friction of normal and degraded (treated with chondroitinase AC) bovine articular cartilage plugs against a smooth steel plate were compared with the predictions based on the biphasic theory using the finite element method. It was found that the measured indentation displacement of both cartilage specimens could be predicted from the biphasic theory and the permeability for the degraded cartilage specimen was increased approximately three times. However, the measured friction coefficient was much lower for short period of loading, and the difference in the finite element prediction of friction coefficient between the normal and degraded cartilage specimens was not observed in the experiment. Therefore, it was concluded that both biphasic and other mechanisms were important in controlling the frictional and lubricating characteristics of articular cartilage in mixed and boundary lubrication regimes.     

Membrane transport properties of bovine articular cartilage

The thermodynamic parameters which define transport of nonelectrolytes through bovine articular cartilage membranes were evaluated. H³HO, glucose and sucrose were used as permeants. These solutes permeate more readily through the upper layers (near the articular surface) than through the denser deeper layers approaching bone. Cartilage is similar in many respects to a swollen cellulose gel. Viscous-flow contributes importantly to transprot within cartilage thus greatly enhancing the movement of nutrients.     

An in situ calibration of an ultrasound transducer: a potential application for an ultrasonic indentation test of articular cartilage

A change in mechanical properties of articular cartilage would be considered one of the most reliable signs of cartilage degeneration. While an indentation method has the potential to measure the cartilage properties in vivo, an accurate measurement of cartilage thickness in situ is technically difficult. An ultrasound transducer has often been used to measure the cartilage thickness. However, its accuracy is limited by the lack of an accurate measurement of the ultrasound speed of cartilage, for the ultrasound speed varies according to the pathological conditions of the tissue. Therefore, the objective of this study is to develop an in situ calibration method of predicting the true ultrasound speed of cartilage and thus allow the ultrasound transducer to measure the thickness of the tissue with great accuracy. By simultaneously implementing an indentation testing protocol using the ultrasound transducer as an indenter, this method can also provide an indentation stiffness measurement of cartilage.The feasibility of the proposed method was examined using normal and proteoglycan-depleted cartilage specimens. It was found that the true ultrasound speed measured by the in situ calibration method was sensitive to the proteoglycan depletion (1735+/-35 m/s for normal, and 1598+/-28 m/s for proteoglycan-depleted cartilage), and that the measured cartilage thickness was consistently accurate regardless of the tissue condition. The measured indentation stiffness of articular cartilage was also sensitive to the tissue condition. Thus, this study demonstrates that the proposed ultrasonic indentation technique can be used to accurately identify the abnormality of articular cartilage in situ.     

Turnover of proteoglycans in cultures of bovine articular cartilage

Proteoglycans in cultures of adult bovine articular cartilage labeled with [35S]sulfate after 5 days in culture and maintained in medium containing 20% fetal calf X serum had longer half-lives (average 11 days) compared with those of the same tissue maintained in medium alone (average 6 days). The half-lives of proteoglycans in cultures of calf cartilage labeled after 5 days in culture and maintained in medium with serum were considerably longer (average 21 days) compared to adult cartilage. If 0.5 mM cycloheximide was added to the medium of cultures of adult cartilage, or the tissue was maintained at 4 degrees C after labeling, the half-lives of the proteoglycans were greater, 24 and greater than 300 days, respectively. Analyses of the radiolabeled proteoglycans remaining in the matrix of the tissue immediately after labeling the tissue and at various times in culture revealed two main populations of proteoglycans; a large species eluting with Kav of 0.21-0.24 on Sepharose CL-2B, of high bouyant density and able to form aggregates with hyaluronate, and a small species eluting with a Kav of 0.63-0.70 on Sepharose CL-2B, of low buoyant density, containing only chondroitin sulfate chains, and unable to form aggregates with hyaluronate. The larger proteoglycan had shorter half-lives than the smaller proteoglycan; in cartilage maintained with serum, the half-lives were 9.8 and 14.5 days, respectively. Labeling cartilage with both [3H]leucine and [35S]sulfate showed the small proteoglycan to be a separate synthetic product. The size distribution of 35S-labeled proteoglycans lost into the medium was shown to be polydisperse on Sepharose CL-2B, the majority eluting with a Kav of 0.27 to 0.35, of high buoyant density, and unable to aggregate with hyaluronate. The size distribution of glycosaminoglycans from 35S-labeled proteoglycans appearing in the medium did not differ from that associated with labeled proteoglycans remaining in the matrix.     

Influence of aspect ratios on the creep behaviour of articular cartilage in indentation

Indentation tests are commonly used to determine the mechanical behaviour of articular cartilage with varying properties, thickness, and geometry. This investigation evaluated the effect of changing geometric parameters on the properties determined from creep indentation tests. Finite element analyses simulated the indentation behaviour of two models, an excised cylindrical specimen of cartilage with either normal and repair qualities and an osteochondral defect represented as a cylindrical region of repair cartilage integrated with a surrounding layer of normal tissue. For each model, the ratios of indenter radius to cartilage height (a/h=0.5,1.5) and cartilage radius to indenter radius (r/a=2,5) were varied. The vertical displacement of the cartilage under the indenter obtained through finite element analysis was fitted to a numerical algorithm to determine the aggregate modulus, permeability, and Poisson's ratio. Indentation behaviours of cartilage specimens for either model with a/h=1.5 were not affected by r/a for values of 2 and 5. Aggregate modulus was not greatly affected by the geometric changes studied. Permeability was affected by changes in the ratio of specimen to indenter radii for a/h=0.5. These findings suggest that experimental configurations of excised cylindrical specimens, also representing osteochondral defects with no or unknown degree of integration, where the cartilage layer has a/h=0.5 should not have r/a values on the order of 2 for confidence in the mechanical properties determined. Indentation of osteochondral defects where the repair cartilage is fully integrated to the surrounding cartilage can be performed with confidence for all cases tested.     

Synthesis of hyaluronate in cultured bovine articular cartilage.

The synthesis and distribution of hyaluronate and proteoglycan were studied in bovine articular cartilage in short-term explant culture with [3H]acetate and H2(35)SO4 as precursors. The incorporation of [3H]acetate into hyaluronate and sulphated glycosaminoglycans was linear with time, except that hyaluronate synthesis showed a marked lag at the beginning of the incubation. [3H]Hyaluronate represented 4-7% of the total [3H]glycosaminoglycans synthesized over a 6 h period. However, the distributions of [3H]hyaluronate and 3H-labelled sulphated glycosaminoglycans were different: about 50% of the newly synthesized [3H]hyaluronate appeared in the medium, compared with less than 5% of the 3H-labelled sulphated proteoglycans. A pulse-chase experiment revealed that the release of newly synthesized [3H]hyaluronate from cartilage was rapid. No difference was observed in the distribution of [3H]hyaluronate between medium and tissue by cartilage from either the superficial layer or the deep layer of articular cartilage. When articular cartilage was incubated with 0.4 mM-cycloheximide, proteoglycan synthesis was markedly inhibited, whereas the synthesis of hyaluronate was only partially inhibited and resulted in more of the newly synthesized hyaluronate being released into the medium. Analysis of the hydrodynamic size of [3H]hyaluronate isolated from cartilage on Sephacryl-1000 revealed one population that was eluted as a broad peak (Kav. less than 0.7), compared with two populations (Kav. greater than 0.5 and less than 0.5) appearing in the medium of cultures. These data suggest that hyaluronate is synthesized in excess of proteoglycan synthesis and that the hyaluronate that is not complexed with proteoglycans is rapidly lost from the tissue.     

Comparison of proteoglycans from bovine articular cartilage.

Four bovine articular cartilages have been compared with regard to the chemical composition of the whole cartilages, the amount of proteoglycan selectively extracted with 3 M MGCl2 or with 3 M guanidine-HCl, and the compositions and physical properties of the isolated proteoglycans. The whole cartilages differ but slightly in composition. Occipital condylar cartilage, a thin cartilage from the smallest joint, contains 4% more collagen and proportionately less proteoglycan than proximal humeral, the thickest cartilage from the largest joint. Each cartilage contains a pool of proteoglycan that resists extraction with 3 M MgCl2 but is extracted with 3 M guanidine-HCl. The proteoglycan extracted from each cartilage with 3 M guanidine-HCl contains a high molecular weight proteoglycan-collagen complex demonstrated by analytical ultracentrifugation and by the turbidity of its visible and ultra-violet spectra. The four cartilages appear to differ most remarkably in the fraction of total proteoglycan extracted from each as proteoglycan-collagen complex.     

Determination of Poisson's ratio of articular cartilage by indentation using different-sized indenters

Articular cartilage is often characterized as an isotropic elastic material with no interstitial fluid flow during instantaneous and equilibrium conditions, and indentation testing commonly used to deduce material properties of Young's modulus and Poisson's ratio. Since only one elastic parameter can be deduced from a single indentation test, some other test method is often used to allow separate measurement of both parameters. In this study, a new method is introduced by which the two material parameters can be obtained using indentation tests alone, without requiring a secondary different type of test. This feature makes the method more suitable for testing small samples in situ. The method takes advantages of the finite layer effect. By indenting the sample twice with different-sized indenters, a nonlinear equation with the Poisson's ratio as the only unknown can be formed and Poisson's ratio obtained by solving the nonlinear equation. The method was validated by comparing the predicted Poisson's ratio for urethane rubber with the manufacturer's supplied value, and comparing the predicted Young's modulus for urethane rubber and an elastic foam material with modulii measured by unconfined compression. Anisotropic and nonhomogeneous finite-element (FE) models of the indentation were developed to aid in data interpretation. Applying the method to bovine patellar cartilage, the tissue Young's modulus was found to be 1.79 +/- 0.59 MPa in instantaneous response and 0.45 +/- 0.26 MPa in equilibrium, and the Poisson's ratio 0.503 +/- 0.028 and 0.463 +/- 0.073 in instantaneous and equilibrium, respectively. The equilibrium Poisson's ratio obtained in our work was substantially higher than those derived from biphasic indentation theory and those optically measured in an unconfined compression test. The finite element model results and examination of viscoelastic-biphasic models suggest this could be due to viscoelastic, inhomogeneity, and anisotropy effects.     

A comparison of healthy human and swine articular cartilage dynamic indentation mechanics

Articular cartilage is a multicomponent, poroviscoelastic tissue with nonlinear mechanical properties vital to its function. A consequent goal of repair or replacement of injured cartilage is to achieve mechanical properties in the repair tissue similar to healthy native cartilage. Since fresh healthy human articular cartilage (HC) is not readily available, we tested whether swine cartilage (SC) could serve as a suitable substitute for mechanical comparisons. To a first approximation, cartilage tissue and surgical substitutes can be evaluated mechanically as viscoelastic materials. Stiffness measurements (dynamic modulus, loss angle) are vital to function and are also a non-destructive means of evaluation. Since viscoelastic material stiffness is strongly strain rate dependent, stiffness was tested under different loading conditions related to function. Stiffness of healthy HC and SC specimens was determined and compared using two non-destructive, mm-scale indentation test modes: fast impact and slow sinusoidal deformation. Deformation resistance (dynamic modulus) and energy handling (loss angle) were determined. For equivalent anatomic locations, there was no difference in dynamic modulus. However, the HC loss angle was ~35% lower in fast impact and ~12% higher in slow sinusoidal mode. Differences seem attributable to age (young SC, older HC) but also to species anatomy and biology. Test mode-related differences in human-swine loss angle support use of multiple function-related test modes. Keeping loss angle differences in mind, swine specimens could serve as a standard of comparison for mechanical evaluation of e.g. engineered cartilage or synthetic repair materials.     

Friction coefficients for mechanically damaged bovine articular cartilage

We used a pin-on-disc tribometer to measure the friction coefficient of both pristine and mechanically damaged cartilage samples in the presence of different lubricant solutions. The experimental set up maximizes the lubrication mechanism due to interstitial fluid pressurization. In phosphate buffer solution (PBS), the measured friction coefficient increases with the level of damage. The main result is that when poly(ethylene oxide) (PEO) or hyaluronic acid (HA) are dissolved in PBS, or when synovial fluid (SF) is used as lubricant, the friction coefficients measured for damaged cartilage samples are only slightly larger than those obtained for pristine cartilage samples, indicating that the surface damage is in part alleviated by the presence of the various lubricants. Among the lubricants considered, 100 mg/mL of 100,000 Da MW PEO in PBS appears to be as effective as SF. We attempted to discriminate the lubrication mechanism enhanced by the various compounds. The lubricants viscosity was measured at shear rates comparable to those employed in the friction experiments, and a quartz crystal microbalance with dissipation monitoring was used to study the adsorption of PEO, HA, and SF components on collagen type II adlayers pre-formed on hydroxyapatite. Under the shear rates considered the viscosity of SF is slightly larger than that of PBS, but lower than that of lubricant formulations containing HA or PEO. Neither PEO nor HA showed strong adsorption on collagen adlayers, while evidence of adsorption was found for SF. Combined, these results suggest that synovial fluid is likely to enhance boundary lubrication. It is possible that all three formulations enhance lubrication via the interstitial fluid pressurization mechanism, maximized by the experimental set up adopted in our friction tests.     

Ultrasound speed and attenuation in progressive trypsin digested articular cartilage

Subtle changes of articular cartilage (AC) can lead to tissue degeneration and even osteoarthritis (OA). The early degeneration of AC is closely related to a change in proteoglycans (PG) content. The observation of PG is therefore an appropriate way of studying OA and evaluating the degree of AC degeneration. In this study, 20 cartilage-bone samples were prepared from normal porcine femoral condyle cartilage and 10 samples were digested over 2 h using 0.25% trypsin solution. The dynamic process of PG-digestion was explored using a conventional A-mode ultrasound (US) experimental system with a 10 MHz center frequency. Quantitative acoustic parameters were calculated from ultrasonic radio-frequency echo signals and included US speed (USS), US amplitude attenuation coefficient (UAA) and broadband US attenuation coefficient (BUA). The experimental results showed that the conventional A-mode ultrasound is valuable for tracking the degree of PG-digestion. Histology also confirmed the validity of the ultrasound observations. For every AC sample, the degree of PG-digestion within a given time was different and was affected by individual differences. After two hours of degeneration, USS showed a mean decrease of 0.4% (P<0.05). UAA was significantly lower after a two-hour PG depletion period (from (2.45±0.23) to (2.28±0.41) dB mm⁻¹). BUA showed no significant differences during this process. In conclusion, conventional ultrasound can provide useful information about trypsin-induced progressive PG depletion in AC and can reflect variations of PG content via the quantitative acoustic parameters USS and UAA. The results of this study may be used to identify an indirect indicator of cartilage matrix integrity and OA disease progression.     

Up-regulation of matrix in bovine articular cartilage explants by electric fields

Electric stimulation has long been used as a tool to promote connective tissue healing, but the mechanism(s) by which this is accomplished are not yet known. We have previously determined, using mass cultures of fetal bovine articular chondrocytes, a specific set of capacitively coupled electrical stimulation parameters (e.g., duration of stimulation, response time, amplitude, frequency, and duty cycle) that significantly elevated production of collagen and proteoglycan, and up-regulated type II collagen and aggrecan mRNA expression in vitro. In the present study, we applied our best signal parameters (30-min continuous stimulation (100% duty cycle) followed by a pulsed (1h on, 5h off, 4x/day) 50% (1 min on, 1 min off) duty cycle) to cultures of adult bovine articular cartilage explants and obtained similar results. Since the latter system more closely mimics the in vivo chondrocyte environment, these data argue for the utility of electric stimulation for the maintenance of adult cartilage matrix in situ.     

Measuring fixed charge density of goat articular cartilage using indentation methods and biochemical analysis

An important indicator of osteoarthritis (OA) progression is the loss of proteoglycan (PG) aggregates from the cartilage tissue. Using the indentation creep test, two analytical methods, as previously developed by Lu et al. [Lu, X. L., Miller, C., Chen, F. H., Guo, X. E., Mow, V. C., 2007. The generalized triphasic correspondence principle for simultaneous determination of the mechanical properties and proteoglycan content of articular cartilage by indentation. Journal of Biomechanics 40, 2434-2441 (EPub).], for predicting the fixed charge density (FCD) of goat knee articular cartilage in the normal (control) and degenerated states were compared: (1) a "dual-stage" method to calculate FCD from the mechanical properties of the tissue when tested in isotonic and hypertonic solutions; and (2) a "single-stage" method to predict FCD (as in (1)) assuming an intrinsic Poisson's ratio of 0.05 in the hypertonic state. A biochemical analysis using 1,9-dimethylmethylene blue (DMMB) assay was conducted to directly measure PG content, and hence FCD. The association between the FCD and the aggregate modulus of the tissue was also explored. The mean (+/-S.D.) FCD values measured using the dual-stage method were the closest (control: 0.129+/-0.039, degenerated: 0.046+/-029) to the DMMB results (control: 0.125+/-0.034, degenerated: 0.057+/-0.024) as compared to those of the single-stage method (control: 0.147+/-0.035, degenerated: 0.063+/-0.026). The single-stage method was more reliable (r(2)=0.81) when compared to the dual-stage method (r(2)=0.79). A prediction of FCD from the aggregate modulus generated the least reliable FCD prediction (r(2)=0.68). Because both the dual- and single-stage methods provided reliable FCD estimates for normal and degenerated tissue, the less time-consuming single-stage method was concluded to be the ideal technique for predicting FCD and hence PG content of the tissue.     

Injurious mechanical compression of bovine articular cartilage induces chondrocyte apoptosis

A bovine cartilage explant system was used to evaluate the effects of injurious compression on chondrocyte apoptosis and matrix biochemical and biomechanical properties within intact cartilage. Disks of newborn bovine articular cartilage were compressed in vitro to various peak stress levels and chondrocyte apoptotic cell death, tissue biomechanical properties, tissue swelling, glycosaminoglycan loss, and nitrite levels were quantified. Chondrocyte apoptosis occurred at peak stresses as low as 4.5 MPa and increased with peak stress in a dose-dependent manner. This increase in apoptosis was maximal by 24 h after the termination of the loading protocol. At high peak stresses (>20 MPa), greater than 50% of cells apoptosed. When measured in uniaxial confined compression, the equilibrium and dynamic stiffness of explants decreased with the severity of injurious load, although this trend was not significant until 24-MPa peak stress. In contrast, the equilibrium and dynamic stiffness measured in radially unconfined compression decreased significantly after injurious stresses of 12 and 7 MPa, respectively. Together, these results suggested that injurious compression caused a degradation of the collagen fibril network in the 7- to 12-MPa range. Consistent with this hypothesis, injurious compression caused a dose-dependent increase in tissue swelling, significant by 13-MPa peak stress. Glycosaminoglycans were also released from the cartilage in a dose-dependent manner, significant by 6- to 13-MPa peak stress. Nitrite levels were significantly increased above controls at 20-MPa peak stress. Together, these data suggest that injurious compression can stimulate cell death as well as a range of biomechanical and biochemical alterations to the matrix and, possibly, chondrocyte nitric oxide expression. Interestingly, chondrocyte programmed cell death appears to take place at stresses lower than those required to stimulate cartilage matrix degradation and biomechanical changes. While chondrocyte apoptosis may therefore be one of the earliest responses to tissue injury, it is currently unclear whether this initial cellular response subsequently drives cartilage matrix degradation and changes in the biomechanical properties of the tissue.     

Compressive fatigue and endurance of juvenile bovine articular cartilage explants

Articular cartilage is an enduring tissue. For most individuals, articular cartilage facilitates a lifetime of pain-free ambulation, supporting millions of loading cycles from activities of daily living. Although early studies into osteoarthritis focused on the role of mechanical fatigue in articular cartilage degeneration, much is still unknown regarding its strength and endurance characteristics. The compressive strength of juvenile, bovine articular cartilage explants was determined to be loading rate-dependent, reaching a maximum strength of 29.5 ± 4.8 MPa at a strain rate of 0.10 %/sec. The fatigue and endurance properties of articular cartilage were characterized utilizing a material testing system, as well as a custom, validated instrument termed the two degrees-of-freedom endurance meter (endurometer). These instruments characterized fatigue in articular cartilage explants at loading levels ranging from 10 to 80 % strength (%S), up to 100,000 cycles. Cartilage explants displayed characteristics of fatigue – fatigue life increased as the loading magnitude decreased. All explants failed within 14,000 cycles at loading levels between 50 and 80 %S. At 10 and 20 %S, all explants endured 100,000 loading cycles. There was no significant difference in equilibrium compressive modulus between run-out explants and unloaded controls, although the pooled modulus increased in response to testing. Histological staining and biochemical assays revealed no material changes in collagen, sulfated glycosaminoglycan, or hydration content between unloaded controls and explants cyclically loaded to run-out. These results suggest articular cartilage may have a putative endurance limit of 20 %S (5.86 MPa), with implications for articular cartilage biomechanics and mechanobiology.     

Comparison of the equilibrium response of articular cartilage in unconfined compression,confined compression and indentation

At mechanical equilibrium, articular cartilage is usually characterized as an isotropic elastic material with no interstitial fluid flow. In this study, the equilibrium properties (Young's modulus, aggregate modulus and Poisson's ratio) of bovine humeral, patellar and femoral cartilage specimens (n=26) were investigated using unconfined compression, confined compression, and indentation tests. Optical measurements of the Poisson's ratio of cartilage were also carried out. Mean values of the Young's modulus (assessed from the unconfined compression test) were 0.80+/-0.33, 0.57+/-0.17 and 0.31+/-0.18MPa and of the Poisson's ratio (assessed from the optical test) 0.15+/-0.06, 0.16+/-0.05 and 0.21+/-0.05 for humeral, patellar, and femoral cartilages, respectively. The indentation tests showed 30-79% (p<0.01) higher Young's modulus values than the unconfined compression tests. In indentation, values of the Young's modulus were independent of the indenter diameter only in the humeral cartilage. The mean values of the Poisson's ratio, obtained indirectly using the mathematical relation between the Young's modulus and the aggregate modulus in isotropic material, were 0.16+/-0.06, 0.21+/-0.05, and 0.26+/-0.08 for humeral, patellar, and femoral cartilages, respectively. We conclude that the values of the elastic parameters of the cartilage are dependent on the measurement technique in use. Based on the similar values of Poisson's ratios, as determined directly or indirectly, the equilibrium response of articular cartilage under unconfined and confined compression is satisfactorily described by the isotropic elastic model. However, values of the isotropic Young's modulus obtained from the in situ indentation tests are higher than those obtained from the in vitro unconfined or confined compression tests and may depend on the indenter size in use.