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Biomed Tech 2007; 52:216222 2007 by Walter de Gruyter Berlin New York. DOI 10.1515/BMT.2007.039

Mechanical behavior of intact and low-grade degenerated cartilage


Mechanische Eigenschaften von intaktem und niedriggradig geschadigtem Knorpel

Gunter Spahn1,*, Enrico Kahl2, Hans Michael Klinger2, Thomas Muckley3, Manfred Gunther4 and Gunther O. Hofmann5
Center of Traumatology and Orthopedic Surgery, Eisenach, Germany 2 Orthopedic Clinic, Georg August University Gottingen, Gottingen, Germany 3 Department of Traumatology, Friedrich Schiller University of Jena, Jena, Germany 4 fzmb-Forschungszentrum fur Medizintechnik und Biotechnologie e.V., Bad Langensalza, Germany 5 Department of Traumatology, Friedrich Schiller University of Jena, Jena, Germany and Trauma Center ,,Bergmannstrost, Halle, Germany
1

Zusammenfassung
Ziel der Untersuchung war es, das Elastizitatsmodul (E Modul), die elastische und plastische Deformierung, die mechanische Harte und die Versagenslast in intaktem und niedriggradig geschadigtem hyalinem Schweinege lenkknorpel zu bestimmen. Unmittelbar nach Totung der 30 weiblichen Schlachtschweine wurden osteochondrale Plugs aus dem medialen Femurkondyl entnommen. Die Klassifikation der Knorpelschaden erfolgte entsprechend den Richtlinien der International Cartilage Repair Society (ICRS). Die mechanische Harte wurde mit einem Shore A-Tester bestimmt. Anschlieend wurden die Knorpelfla chen mit einem 5 mm-Intender zwischen 50 und 200 N belastet, um die Steifigkeit und die plastische Deformation zu bestimmen. Abschlieend wurden die Proben bis zur chondralen Fraktur belastet. Bei Knorpelschaden mit einem ICRS Grad I fand sich eine im Vergleich zum gesunden Knorpel signifikant verminderte Steifigkeit. Gesunder Knorpel zeigte bis zu einer Belastung von 100 N kaum eine plastische Deformation, wahrend ICRS Grad I Knorpel bereits bei einer Belastung von 50 N deutlich plastisch deformiert wurde. Das E-Modul fur gesun den Knorpel betrug 18,8 MPa und war signifikant hoher als bei ICRS Grad I (11,1 MPa) und ICRS Grad II (10,5 MPa). Gesunder Knorpel hatte eine signifikant hohere mechanische Harte (Shore A) und eine signifikant hohere Versagenslast als degenerierter Knorpel. Dabei korrelierte die Verminderung der Versagenslast signifikant mit der Verminderung des E-Moduls. Die Untersuchungen bestatigen, dass bereits die fruhe Knorpeldegene ration mit einer signifikanten Verminderung von Elastizitat und Bruchfestigkeit einhergeht. Dabei ist die Shore-Har te-Messung ein geeignetes Verfahren, diese biomechanischen Veranderungen in Knorpelpraparaten schnell zu erfassen. Schlusselworter: E-Modul; Knorpel; Plastizitat; Shore Harte-Steifigkeit; Versagenslast.

Abstract
Youngs modulus, elastic and plastic deformation, mechanical hardness and load at failure were determined for low-grade degenerated hyaline cartilage in a porcine model. Osteochondral plugs from the medial condyle of 30 female pigs were used. Cartilage defects were classified using the International Cartilage Repair Society (ICRS) protocol. Mechanical hardness was measured using a Shore A testing device. Total stiffness and plastic deformation was evaluated in the range 50200 N using a 5-mm indenter. The load at failure was then determined. ICRS grade I specimens showed significantly lower stiffness than grade 0 specimens. ICRS grade 0 specimen showed no significant plastic deformation within the load range 25100 N. In degenerated cartilage, plastic deformation started at a significantly lower load (50 N). The Youngs modulus at 25 N in ICRS grade 0 specimens (18.8 MPa) was significantly higher than in grade I (11.1 MPa) or grade II (10.5 MPa) specimens. Intact cartilage showed significantly higher tension at failure and mechanical Shore A hardness. Youngs modulus and tension at failure showed strong correlation. Cartilage degeneration is associated with a significant loss of elasticity and mechanical stress resistance. Shore hardness measurement is an adequate method for rapid biomechanical evaluation of cartilage specimens. Keywords: cartilage; failure; plasticity; Shore hardness; stiffness; Youngs modulus.
*Corresponding author: Dr. med. Gunter Spahn, Center of Traumatology and Orthopedic Surgery Eisenach, Sophienstr. 16, 99817 Eisenach, Germany E-mail: spahn@pk-eisenach.de

Introduction
During joint activity, hyaline cartilage is exposed to complex mechanical stress in the form of compressive load, shear stress, friction and wear. The ability of cartilage to resist stress depends on the morphological structure and the properties of the extracellular matrix w2, 23x. For the mechanical behavior of cartilage, the most important factor is the water-containing proteoglycan-collagen network. Collagen fibrils are

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G. Spahn et al.: Mechanical behavior of intact and low-grade degenerated cartilage 217

responsible for mechanical stress resistance. During loading, collagen fibrils align and stretch along the direction of stress. Proteoglycans have a high water-binding capacity. Compressive force causes an outflow of water. Therefore, the compressive force is neutralized to hydrodynamic intermolecular friction. This is responsible for absorption of mechanical shocks and distributes mechanical loads more evenly across the subchondral bone. Cartilage degeneration can be classified into different stages. Early degeneration is associated with edema, softening and superficial fibrillation. In later stages, partial- or full-thickness cartilage defects are observed. Physiologically, cartilage has high stiffness and elasticity, but low plastic deformation. The proportionality between loading stress and deformation is described as the modulus of elasticity (Youngs modulus). A high Youngs modulus reflects the elasticity of intact cartilage. Youngs modulus in healthy cartilage has been found to vary from 5 to 30 MPa w5, 7, 12, 13, 16, 17, 19, 21x. The values depend on the location, test species, and test design. Conservation of cartilage specimens (fresh specimens, freezing) can also influence the measurements w25x. It is generally accepted that increased mechanical load causes plastic deformation of cartilage. Under physiological conditions, plastic deformation is reversible (viscoelasticity). Cartilage degeneration is associated with changes in mechanical behavior. Previous investigations demonstrated a significant decrease in stiffness and Youngs modulus in degenerated cartilage w14, 22x. Healthy cartilage exhibits high stress resistance against breakdown w23x. However, little is known about the plastic deformation, mechanical hardness (resistance to permanent deformation) and tension at failure in low-grade degenerated cartilage. This study was undertaken to evaluate the biomechanical behavior of intact and low-grade degenerated cartilage in a porcine model. It was hypothesized that changes in mechanical hardness and loss of viscoelastic properties are correlated with a decrease in mechanical stress resistance against breakdown at failure.

Figure 1 Porcine femoral condyles with a grade 1 lesion within the medial surface.

Measurement of mechanical hardness (Shore A) The mechanical hardness (Shore A) was measured three times by an independent medical assistant using an instrument from Zwick (Ulm, Germany). The highest value was used for final analysis. Thin osteochondral plugs from the weight-bearing area (approx. 10 mm=10 mm) including some of the subchondral bone layer (approx. 10 mm) were prepared with a chisel. Mechanical tests Osteochondral specimens were embedded in a plastercontaining steel cylinder. Mechanical tests were performed with a model Z010 material tester (Zwick). Specimens were loaded by a steel indenter with a diameter of 5 mm (As19.6 mm2). All tests were performed at a constant speed of 20 mm/s. After preloading (0.1 N), specimens were stressed from 25 and 50 N in 25-N steps up to 200 N. The load-dependent compression was recorded on the testing machine (Dltotal). After relief, the specimens were subjected to a new preload. The difference in length was determined for evaluation of plastic deformation (Dlplastic). Finally, the load at failure (chondral fracture) was determined. The detailed testing protocol is listed in Table 1. Measurement of the chondral layer thickness The thickness of the chondral layer (d) close to the area tested was measured by an independent medical assistant using a precision measurement instrument (Kraftfix, Schmalkaldia GmbH, Schmalkalden, Germany). The instrument had a tolerance of 0.1 mm. Calculations and statistics The results for mechanical measurements were recorded by the software (testXpert, Zwick) of the testing machine. Total compression was calculated as the thickness under compression divided by the thickness of the chondral layer (sDltotal/d wmm/mmx). Plastic compression was calculated as plastic deformation divided by the thickness (plsDlplastic/d wmm/mmx). Elastic compression was expressed as the difference between total compression and plastic compression: elasticstotal-plastic wmm/mmx. Tension (pressure) was calculated as the quotient of force (F) to the area (A) of the steel indenter (ssF/A

Materials and methods


Specimens A total of 30 fresh porcine knee joints were obtained from a local abattoir after normal slaughtering. Because the animals had undergone regular slaughter, no approval from a veterinarian authority was needed according to German law w1x. The female pigs (Sus scrofa domestica) were approximately 12 months old. The joints, which had intact capsule, were stored at -208C and were thawed overnight before use. The medial femoral condyles were exposed directly before the tests. Cartilage of the weight-bearing area within the medial condyle (Figure 1) was graded (visual inspection and palpation using an arthroscopic hook) by two independent and experienced arthroscopic surgeons (E.K. and H.M.K) according to the ICRS w3x criteria.

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218 G. Spahn et al.: Mechanical behavior of intact and low-grade degenerated cartilage

Table 1 Mechanical test protocol. Force (N) Preload Load Relief Preload Stepwise increase (75, 100, 125, 150, 175, 200 N) Load Relief Preload Load at failure 0.1 25 0 0.1 Time (min) 1 1 1 1 Parameter recorded Total compression (Dltotal) Plastic deformation (Dlplastic)

200 0 0.1

1 1 1

Total compression (Dltotal) Plastic deformation (Dlplastic) Test stopped after achieving 30% of Fmax

All tests were performed at a constant speed (20 mm/min) using a steel indenter (diameter 5 mm).

wN/m2x). According to Hookes law, Youngs modulus was calculated as Ess/ wMPax. SPSS version 13.3 (SPSS, Chicago, IL, USA) was used for statistical evaluation. All values are presented as mean (SD). The Kolmogorov-Smirnov test was used to assess the normality of distributions. ANOVA was used for comparison of means.

Mechanical hardness ICRS grade I specimens showed significantly lower mechanical hardness than intact grade 0 specimens (Table 2). No differences in mechanical hardness were observed between ICRS grade I and II specimens (ps0.362). Stiffness

Results
Frequency of cartilage defects In a total of 16 condyles (53.3%), the cartilage was macroscopically intact. ICRS grade I defects were detected in 10 (33.3%) condyles. The cartilage exhibited superficial fissures and local softening. ICRS grade II defects were observed in four condyles (13.3%), with fraying and lesions extending to -50% of the depth. The thickness of the chondral layers for intact and defective cartilage was not significantly different (Table 2).

Specimens with a cartilage defect showed significantly lower stiffness than those with intact cartilage (ps0.000). No differences in stiffness were observed between ICRS grade I and II specimens (Table 3). The load-displacement curves are shown in Figure 2. Elastic and plastic deformation, and Youngs modulus In intact cartilage (ICRS grade 0), no significant plastic deformation was observed at a compressive force of 25100 N (ss1.3-5.1 MPa). However, a higher force of

Table 2 Mechanical properties of intact and defective cartilage specimens. ICRS grade 0 Thickness d (mm) Hardness (Shore A) Load at failure (N) Tension at failure (MPa) Youngs modulus at 25 N (MPa) SD values in parentheses. 2.6 (0.3) 88.4 (10.3) 607.9 (107.9) 31.0 (5.5) 18.8 (3.9) I 2.5 (0.4) 55.6 (10.5) 429.4 (107.2) 21.9 (5.4) 11.1 (3.1) II 2.7 (0.7) 49.7 (6.5) 343.0 (43.3) 17.5 (2.2) 10.5 (1.7) 0.547 0.000 0.000 0.000 0.000 p

Table 3 Compression (total) in relation to compressive force and ICRS grade. F (N) s (MPa) ICRS grade 0 25 50 75 100 125 150 175 200 1.3 2.6 3.9 5.1 6.4 7.7 9.0 10.3 0.07 0.09 0.10 0.13 0.14 0.19 0.21 0.24 (0.01) (0.02) (0.02) (0.03) (0.03) (0.04) (0.04) (0.05) I 0.12 0.15 0.17 0.23 0.25 0.32 0.36 0.41 (0.03) (0.03) (0.04) (0.05) (0.05) (0.07) (0.08) (0.09) II 0.13 0.16 0.19 0.23 0.26 0.35 0.38 0.44 (0.02) (0.03) (0.04) (0.05) (0.06) (0.07) (0.07) (0.08) 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 p

SD values in parentheses.

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G. Spahn et al.: Mechanical behavior of intact and low-grade degenerated cartilage 219

Figure 2 Load-displacement curves in intact and defective cartilage.

)100 N (s)5.1 MPa) led to an increase in plastic deformation (Figure 2A and Table 5). Compressive loads )175 N (ss9.0 MPa) were associated with a nearly total loss of elasticity (Table 4). In defective cartilage, a low compressive load (Fs25 N, ss1.3 MPa) did not cause significant plastic deformation. Significant plastic deformation occurred at

a significantly lower compressive load (Fs50 N, ss2.6 MPa) compared to intact cartilage (Figure 2B,C, Table 5). ICRS grade II specimens tended to show higher plastic deformation than grade I specimens. Compressive loads )100 N (ss5.1 MPa) were associated with a nearly complete loss of elasticity in ICRS grade I and II specimens.

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220 G. Spahn et al.: Mechanical behavior of intact and low-grade degenerated cartilage

Table 4 Elastic compression (elastic) in relation to compressive force and ICRS grade. F (N) s (MPa) ICRS grade 0 25 50 75 100 125 150 175 200 1.3 2.6 3.9 5.1 6.4 7.7 9.0 10.3 0.06 0.08 0.09 0.12 0.10 0.12 0.07 0.05 (0.01) (0.02) (0.02) (0.03) (0.02) (0.03) (0.05) (0.04) 1 0.11 0.13 0.06 0.05 0.01 0.03 0.04 0.03 (0.00) (0.03) (0.02) (0.04) (0.06) (0.05) (0.06) (0.08) 2 0.12 0.12 0.05 0.00 0.02 0.02 0.04 0.05 (0.03) (0.01) (0.01) (0.03) (0.07) (0.08) (0.06) (0.07) 0.000 0.000 0.000 0.000 0.000 0.000 0.298 0.783 p

SD values in parentheses. Table 5 Plastic compression (plastic) in relation to compressive force and ICRS grade. F (N) s (MPa) ICRS grade 0 25 50 75 100 125 150 175 200 1.3 2.6 3.9 5.1 6.4 7.7 9.0 10.3 0.01 0.01 0.01 0.01 0.04 0.07 0.10 0.19 (0.00) (0.00) (0.00) (0.01) (0.01) (0.01) (0.03) (0.02) I 0.01 0.02 0.11 0.18 0.23 0.30 0.31 0.38 (0.00) (0.02) (0.05) (0.08) (0.10) (0.07) (0.10) (0.09) II 0.01 0.04 0.13 0.22 0.28 0.33 0.34 0.39 (0.01) (0.02) (0.02) (0.06) (0.12) (0.12) (0.11) (0.09) 0.158 0.004 0.000 0.000 0.000 0.000 0.000 0.000 p

SD values in parentheses.

Hookes law is only applicable in ICRS grade 0 specimens up to a compressive force of 100 N (ss5.1 MPa). For specimens with chondral defects, application was only possible up to a load of 25 N (ss1.3 MPa). Youngs modulus was therefore calculated at 25 N (ss1.3 MPa). Youngs modulus in cartilage defects was significantly (ps0.000) lower, with no differences between ICRS grade I and II defects (Table 2). Force (tension) at failure and mechanical (Shore A) hardness Specimens were subjected to compressive stress to determine the load and tension at failure. Intact cartilage specimens showed significantly (ps0.000) higher resistance against breakdown than specimens with a defect. ICRS grade II specimens showed a trend (ps0.152) to lower tension at failure than grade I specimens (Table 2). The thickness of the cartilage layer did not correlate with ICRS grade or mechanical properties. A significant

correlation was observed between ICRS grade and mechanical properties (Table 6). The mechanical hardness showed strong correlation to Youngs modulus (Figure 3) and the tension (force) at failure (Figure 4).

Discussion
It was surprising that approximately half of the healthy pigs were suffering from cartilage degeneration. This should be considered in further experiments. The advantage of this porcine model is the good comparability of the specimens without variance in age and sex. The osteochondral specimens showed low variance of the thickness of the cartilage layer. The use of osteochondral layers reflects the biological properties of the unit involving cartilage and subchondral bone. Mechanical hardness is a term used in materials science. It is a property of a solid material that reflects its

Table 6 Correlation between cartilage thickness, ICRS grade and mechanical properties. Thickness Thickness ICRS grade Mechanical hardness Youngs modulus Tension at failure Rs-0.033 ps0.862 Rs0.181 ps0.338 Rs0.257 ps0.170 Rs0.102 ps0.590 ICRS grade Rs-0.033 ps0.862 Mechanical hardness Rs0.181 ps0.338 Rs-0.820 ps0.000 Youngs modulus Rs0.257 ps0.170 Rs-0.730 ps0.000 Rs0.919 ps0.000 Tension at failure Rs0.102 ps0.590 Rs-0.720 ps0.000 Rs0.900 ps0.000 Rs0.846 ps0.000

Rs-0.820 ps0.000 Rs-0.730 p-0.000 Rs-0.720 ps0.000

Rs0.919 ps0.000 Rs0.900 ps0.000

Rs0.846 ps0.000

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G. Spahn et al.: Mechanical behavior of intact and low-grade degenerated cartilage 221

Figure 3 Correlation between mechanical stiffness (Shore A) and Youngs modulus.

resistance to permanent deformation. Hardness can be measured on various scales (Shore, Brinell, Rockwell and others). In the rubber and polymer industry, Shore measurement is well established. A hardened steel indenter with a diameter of 1.3 mm is used to apply a defined load. The penetration depth is recorded and expressed on a scale ranging from 0 (very soft) to 100 (very hard). Until now, measurement of Shore hardness was seldom performed in medical investigations. It is possible to determine the hardness of skin in the diagnosis of diabetic neuropathy w4, 20, 24x or for diagnosis of claw disease in veterinary medicine w11x. Foitzik et al. performed hardness measurements for the diagnosis of pancreatitis w9x. In neurosciences, hardness measurements are possible for determination of tactile sensitivity measurements w8x. This is the first report of hardness measurements in cartilage research. In our experiments we found a significant loss of mechanical hardness in specimens with cartilage defects. The good correlation between Shore hardness

and Youngs modulus (Shore hardness of 50 units correlates with Youngs modulus of ;10 MPa) suggests that this test is suitable for further mechanical investigations. The advantage of this measurement is its good practicality in use. The test is easy to perform without expensive equipment. Cartilage is very sensitive to water humidification. Cartilage specimens lose approximately 10% of their weight within 5 min at room temperature (personal observation). This can be a possible source of error in the evaluation of cartilage. It is even possible to use mechanical hardness measurements for clinical evaluations, e.g., during arthroscopies. In the past, some investigators performed stiffness measurements of cartilage during arthroscopies. Lyyra et al. designed an indentation instrument based on deformation of a cartilage surface as defined on a Wheatstone bridge w18x. In an arthroscopic evaluation, Duda et al. w6x observed a significant decrease in mechanical stiffness in degenerated cartilage using a new arthroscopic device. The stiffness was measured by reflection of a water jet at a constant pressure. A similar device could be designed based on hardness measurements in the future. Cartilage degeneration is associated with a significant loss of stiffness w15x. Specimens with a cartilage defect showed significantly higher compression than intact specimens. Intact cartilage has significantly higher elasticity. Under a compressive load (s-5 MPa), no significant plastic deformation was observed. This is associated with tension of the matrix collagen fibers. The pressure is buffered by the proteoglycan-collagen network, which has a high water content. A higher load causes an increase in plastic deformation. This results in a typical hysteresis curve (Figure 2A). Plastic deformation is associated with water outflow from the extracellular matrix into the joint space. Under physiological conditions this process is reversible by water backflow and an increase in the synthesis of matrix composites w2x. In cartilage defects, significantly earlier plastic deformation was observed. Plastic deformation occurred at a significantly lower load (s-2.6 MPa). The medial condyle of human knees is normally subject to a pressure range of 10002000 N w2x. The mean contact area amounts to approximately 10 mm2 w10x. Normally, the medial condyle cartilage is stressed at a mean pressure of 1020 MPa. Breakdown of healthy cartilage is not likely within this compressive load range. In the case of decreased regenerative potential, stepwise changes in cartilage are possible. Degenerated cartilage showed significantly lower mechanical stress resistance of 21.9 MPa for grade I and 17.5 MPa for grade II lesions. In this range, cartilage breakdown is possible at normal weight loads during physiological activities.

Conclusion
Cartilage degeneration is associated with a significant loss of elasticity and mechanical stress resistance. These important changes occur during early degeneration.

Figure 4 Correlation between Youngs modulus and mechanical resistance to failure.

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222 G. Spahn et al.: Mechanical behavior of intact and low-grade degenerated cartilage

Mechanical hardness measurement is adequate for evaluation of these mechanical properties.


w14x

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