Composition, structure and tensile biomechanical properties of equine articular cartilage during growth and maturation.

Articular cartilage undergoes structural and biochemical changes during maturation, but the knowledge on how these changes relate to articular cartilage function at different stages of maturation is lacking. Equine articular cartilage samples of four different maturation levels (newborn, 5-month-old, 11-month-old and adult) were collected (N = 25). Biomechanical tensile testing, Fourier transform infrared microspectroscopy (FTIR-MS) and polarized light microscopy were used to study the tensile, biochemical and structural properties of articular cartilage, respectively. The tensile modulus was highest and the breaking energy lowest in the newborn group. The collagen and the proteoglycan contents increased with age. The collagen orientation developed with age into an arcade-like orientation. The collagen content, proteoglycan content, and collagen orientation were important predictors of the tensile modulus (p < 0.05 in multivariable regression) and correlated significantly also with the breaking energy (p < 0.05 in multivariable regression). Partial least squares regression analysis of FTIR-MS data provided accurate predictions for the tensile modulus (r = 0.79) and the breaking energy (r = 0.65). To conclude, the composition and structure of equine articular cartilage undergoes changes with depth that alter functional properties during maturation, with the typical properties of mature tissue reached at the age of 5-11 months.

Application of second derivative spectroscopy for increasing molecular specificity of Fourier transform infrared spectroscopic imaging of articular cartilage.

OBJECTIVE: Fourier transform infrared (FT-IR) spectroscopic imaging is a promising method that enables the analysis of spatial distribution of biochemical components within histological sections. However, analysis of FT-IR spectroscopic data is complicated since absorption peaks often overlap with each other. Second derivative spectroscopy is a technique which enhances the separation of overlapping peaks. The objective of this study was to evaluate the specificity of the second derivative peaks for the main tissue components of articular cartilage (AC), i.e., collagen and proteoglycans (PGs). MATERIALS AND METHODS: Histological bovine AC sections were measured before and after enzymatic removal of PGs. Both formalin-fixed sections (n = 10) and cryosections (n = 6) were investigated. Relative changes in the second derivative peak heights caused by the removal of PGs were calculated for both sample groups. RESULTS: The results showed that numerous peaks, e.g., peaks located at 1202 cm(-1) and 1336 cm(-1), altered less than 5% in the experiment. These peaks were assumed to be specific for collagen. In contrast, two peaks located at 1064 cm(-1) and 1376 cm(-1) were seen to alter notably, approximately 50% or more. These peaks were regarded to be specific for PGs. The changes were greater in cryosections than formalin-fixed sections. CONCLUSIONS: The results of this study suggest that the second derivative spectroscopy offers a practical and more specific method than routinely used absorption spectrum analysis methods to obtain compositional information on AC with FT-IR spectroscopic imaging.

Maturation of collagen fibril network structure in tibial and femoral cartilage of rabbits.

OBJECTIVE: The structure and composition of articular cartilage change during development and growth, as well as in response to varying loading conditions. These changes modulate the functional properties of cartilage. We studied maturation-related changes in the collagen network organization of cartilage as a function of tissue depth. DESIGN: Articular cartilage from the tibial medial plateaus and femoral medial condyles of female New Zealand white rabbits was collected from six age-groups: 4 weeks (n=30), 6 weeks (n=30), 3 months (n=24), 6 months (n=24), 9 months (n=27) and 18 months (n=19). Collagen fibril orientation, parallelism (anisotropy) and optical retardation were analyzed with polarized light microscopy. Differences in the development of depth-wise collagen organization in consecutive age-groups and the two joint locations were compared statistically. RESULTS: The collagen fibril network of articular cartilage undergoes significant changes during maturation. The most prominent changes in collagen architecture, as assessed by orientation, parallelism and retardation were noticed between the ages of 4 and 6 weeks in tibial cartilage and between 6 weeks and 3 months in femoral cartilage, i.e., orientation became more perpendicular-to-surface, and parallelism and retardation increased with changes being most prominent in the deep zone. At the age of 6 weeks, tibial cartilage had a more perpendicular-to-surface orientation in the middle and deep zones than femoral cartilage (P<0.001) and higher parallelism throughout the tissue depth (P<0.001), while femoral cartilage exhibited more parallel-to-surface orientation (P<0.01) above the deep zone after maturation. Optical retardation of collagen was higher in tibial than in femoral cartilage at the ages of 4 and 6 weeks (P<0.001), while at older ages, retardation below the superficial zone in the femoral cartilage became higher than in the tibial cartilage. CONCLUSIONS: During maturation, there is a significant modulation of collagen organization in articular cartilage which occurs earlier in tibial than in femoral cartilage, and is most pronounced in the deep zone.

Quantitative analysis of spatial proteoglycan content in articular cartilage with Fourier transform infrared imaging spectroscopy: Critical evaluation of analysis methods and specificity of the parameters.

OBJECTIVE: To evaluate the specificity of the current Fourier transform infrared imaging spectroscopy (FT-IRIS) methods for the determination of depthwise proteoglycan (PG) content in articular cartilage (AC). In addition, curve fitting was applied to study whether the specificity of FT-IRIS parameters for PG determination could be improved. METHODS: Two sample groups from the steer AC were prepared for the study (n = 8 samples/group). In the first group, chondroitinase ABC enzyme was used to degrade the PGs from the superficial cartilage, while the samples in the second group served as the controls. Samples were examined with FT-IRIS and analyzed using previously reported direct absorption spectrum techniques and multivariate methods and, in comparison, by curve fitting. Safranin O-stained sections were measured with digital densitometry to obtain a reference for depthwise PG distribution. RESULTS: Carbohydrate region-based absorption spectrum methods showed a statistically weaker correlation with the PG reference distributions than the results of the curve fitting (subpeak located approximately at 1,060 cm(-1)). Furthermore, the shape of the depthwise profiles obtained using the curve fitting was more similar to the reference profiles than with the direct absorption spectrum analysis. CONCLUSIONS: Results suggest that the current FT-IRIS methods for PG analysis lack the specificity for quantitative measurement of PGs in AC. The curve fitting approach demonstrated that it is possible to improve the specificity of the PG analysis. However, the findings of the present study suggest that further development of the FT-IRIS analysis techniques is still needed.

Influence of exercise and joint topography on depth-related spatial distribution of proteoglycan and collagen content in immature equine articular cartilage.

REASONS FOR PERFORMING STUDY: There is ample evidence on topographical heterogeneity of the principal biochemical components of articular cartilage over the surface of the joint and the influence of loading thereon, but no information on depth-related zonal variation in horses. OBJECTIVES: To study depth-related zonal variation in proteoglycan (PG) and collagen content in equine articular cartilage. METHODS: Two techniques (safranin-O densitometry and Fourier transform infrared spectroscopy) were applied to sections of articular cartilage from the proximal phalangeal bone of the metacarpophalangeal joint of 18-month-old Thoroughbreds that had been raised at pasture from age 0-18 months without (PASTEX) and with (CONDEX) additional exercise. Two sites were investigated: site 1 at the joint margin that is unloaded at rest or at slow gaits, but subjected to high-intensity loading during athletic activity; and site 2, a continuously, but less intensively, loaded site in the centre of the joint. RESULTS: Proteoglycan values increased from the surface to the deep layers of the cartilage, collagen content showed a reverse pattern. PG content was significantly higher at site 2 in both PASTEX and CONDEX animals without an effect of exercise. In the PASTEX animals collagen content was significantly higher at site 1, but in the CONDEX group the situation was reversed, due to a significant exercise effect on site 1, leading to a reduced collagen content. CONCLUSIONS: Collagen and PG content gradients agree with findings in other species. The observations on PG levels suggest that the exercise level was not strenuous. The collagen results in the PASTEX group confirmed earlier findings, the lower levels at site 1 in the CONDEX group being possibly due to an advancement of the physiological maturation process of collagen remodelling. POTENTIAL RELEVANCE: This study confirms earlier observations that even moderate variations in exercise level in early age may have significant effects on the collagen network of articular cartilage.

Influence of intensity and changes of physical activity on bone mineral density of immature equine subchondral bone.

REASONS FOR PERFORMING STUDY: Subchondral bone provides structural support to overlying articular cartilage and plays an important biomechanical role in osteochondral diseases. Mechanical features of bone correlate strongly with bone mineral density, which is directed by the loading conditions to which the tissue is subjected. OBJECTIVE: To investigate the influence of physical activity levels on subchondral bone mineral density (sBMD) in foals during early development. METHODS: Three groups of foals were subjected to different physical activity levels from birth until age 5 months. A proportion of these foals were subjected to euthanasia at 5 months while remaining foals were subjected to similar physical activity levels for 6 months until euthanasia at 11 months. Osteochondral specimens were collected for measurement of sBMD with peripheral quantitative computed tomography at 2 differently loaded anatomical sites of the proximal phalangeal bone at 1, 2, 3, 4 and 5 mm depth from the osteochondral junction. RESULTS: Growth significantly increased sBMD but by a different amount depending on anatomical location and physical activity level. Significantly higher sBMD was found at the habitually loaded central area in comparison to the intermittently peak loaded marginal site. Exercise increased sBMD throughout the whole depth of analysed tissue, but changes were generally more obvious at a depth of 2 mm. Interestingly, foals subjected to additional sprint training preserved the exercise-induced sBMD increase at the habitually loaded central area during the 6 months of the second phase of the study. CONCLUSIONS: Habitual low-intensity loading elicits a greater response in sBMD in quantitative terms than high-intensity low-frequency loading at the sites investigated in this study. Future sBMD may be influenced by means of well-tailored exercise regimens at young age. POTENTIAL RELEVANCE: Specific physical activity levels during early development may potentially reduce the prevalence of osteochondral injury later in life.

Biomechanical, biochemical and structural correlations in immature and mature rabbit articular cartilage.

OBJECTIVE: The structure and composition of articular cartilage change during development and growth. These changes lead to alterations in the mechanical properties of cartilage. In the present study, biomechanical, biochemical and structural relationships of articular cartilage during growth and maturation of rabbits are investigated. DESIGN: Articular cartilage specimens from the tibial medial plateaus and femoral medial condyles of female New Zealand white rabbits were collected from seven age-groups; 0 days (n=29), 11 days (n=30), 4 weeks (n=30), 6 weeks (n=30), 3 months (n=24), 6 months (n=24) and 18 months (n=19). The samples underwent mechanical testing under creep indentation. From the mechanical response, instantaneous and equilibrium moduli were determined. Biochemical analyses of tissue collagen, hydroxylysylpyridinoline (HP) and pentosidine (PEN) cross-links in full thickness cartilage samples were conducted. Proteoglycans were investigated depth-wise from the tissue sections by measuring the optical density of Safranin-O-stained samples. Furthermore, depth-wise collagen architecture of articular cartilage was analyzed with polarized light microscopy. Finite element analyses of the samples from different age-groups were conducted to reveal tensile and compressive properties of the fibril network and the matrix of articular cartilage, respectively. RESULTS: Tissue thickness decreased from approximately 3 to approximately 0.5mm until the age of 3 months, while the instantaneous modulus increased with age prior to peak at 4-6 weeks. A lower equilibrium modulus was observed before 3-month-age, after which the equilibrium modulus continued to increase. Collagen fibril orientation angle and parallelism index were inversely related to the instantaneous modulus, tensile fibril modulus and tissue thickness. Collagen content and cross-linking were positively related to the equilibrium compressive properties of the tissue. CONCLUSIONS: During maturation, significant modulation of tissue structure, composition and mechanical properties takes place. Importantly, the present study provides insight into the mechanical, chemical and structural interactions that lead to functional properties of mature articular cartilage.

Changes in spatial collagen content and collagen network architecture in porcine articular cartilage during growth and maturation.

OBJECTIVES: The present study was designed to reveal changes in the collagen network architecture and collagen content in cartilage during growth and maturation of pigs. METHODS: Femoral groove articular cartilage specimens were collected from 4-, 11- and 21-month-old domestic pigs (n=12 in each group). The animal care conditions were kept constant throughout the study. Polarized light microscopy was used to determine the collagen fibril network birefringence, fibril orientation and parallelism. Infrared spectroscopy was used to monitor changes in the spatial collagen content in cartilage tissue. RESULTS: During growth, gradual alterations were recorded in the collagen network properties. At 4 months of age, a major part of the collagen fibrils was oriented parallel to the cartilage surface throughout the tissue. However, the fibril orientation changed considerably as skeletal maturation progressed. At 21 months of age, the fibrils of the deep zone cartilage ran predominantly at right angles to the cartilage surface. The collagen content increased and its depthwise distribution changed during growth and maturation. A significant increase of the collagen network birefringence was observed in the deep tissue at the age of 21 months. CONCLUSIONS: The present study revealed dynamic changes of the collagen network during growth and maturation of the pigs. The structure of the collagen network of young pigs gradually approached a network with the classical Benninghoff architecture. The probable explanation for the alterations is growth of the bone epiphysis with simultaneous adaptation of the cartilage to increased joint loading. The maturation of articular cartilage advances gradually with age and offers, in principle, the possibility to influence the quality of the tissue, especially by habitual joint loading. These observations in porcine cartilage may be of significance with respect to the maturation of human articular cartilage.

Effects of glucosamine sulfate on intracellular UDP-hexosamine and UDP-glucuronic acid levels in bovine primary chondrocytes.

OBJECTIVE: To analyze the effects of exogenously added glucose (Glc), glucosamine (GlcN) and glucosamine sulfate (GS) on the intracellular UDP-hexoses (UDP-Hex), UDP-N-acetylhexosamines (UDP-HexN) and UDP-glucuronic acid (UDP-GlcA) levels in bovine primary chondrocytes. METHODS: Chondrocytes were incubated with different concentrations of Glc, GlcN and GS either in high- or low-glucose DMEM for up to 120min to analyze the intracellular levels of UDP-Hex, UDP-GlcA and UDP-HexN by a reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry analysis. Glycosaminoglycan (GAG) synthesis rate and aggrecan mRNA expression levels were quantified using (35)S-sulfate incorporation assay and quantitative real-time RT-PCR, respectively. The cells were cultivated for 2 days or 8 days before UDP-sugar analysis. RESULTS: Levels of UDP-HexN and UDP-GlcA were unchanged at 10microM concentration of GS in low-glucose DMEM, while addition of 1mM GlcN or GS in low-glucose DMEM for 10min increased UDP-HexN level. The highest intracellular level of UDP-HexN was reached at 30min after addition of 1mM GS to the cells. The intracellular contents of UDP-HexN and UDP-GlcA related to UDP-Hex were higher after prolonged cultivation of chondrocytes for 8 days compared with 2-day-old cultures. Aggrecan mRNA expression and GAG synthesis remained at control level after the cells were treated with 10, 100microM or 1mM of GS for 24h. CONCLUSION: Physiologically relevant level of GS could not increase the intracellular UDP-HexN and UDP-GlcA levels in bovine primary chondrocyte, while longer-time culture itself appeared to increase the intracellular UDP-HexN and UDP-GlcA levels.

Immature porcine knee cartilage lesions show good healing with or without autologous chondrocyte transplantation.

OBJECTIVE: The purpose of this study was to find out how deep chondral lesions heal in growing animals spontaneously and after autologous chondrocyte transplantation. METHODS: A 6mm deep chondral lesion was created in the knee joints of 57 immature pigs and repaired with autologous chondrocyte transplantation covered with periosteum or muscle fascia, with periosteum only, or left untreated. After 3 and 12 months, the repair tissue was evaluated with International Cartilage Repair Society (ICRS) macroscopic grading, modified O'Driscoll histological scoring, and staining for collagen type II and hyaluronan, and with toluidine blue and safranin-O staining for glycosaminoglycans. The repair tissue structure was also examined with quantitative polarized light microscopy and indentation analysis of the cartilage stiffness. RESULTS: The ICRS grading indicated nearly normal repair tissue in 65% (10/17) after the autologous chondrocyte transplantation and 86% (7/8) after no repair at 3 months. At 1 year, the repair tissue was nearly normal in all cases in the spontaneous repair group and in 38% (3/8) in the chondrocyte transplantation group. In most cases, the cartilage repair tissue stained intensely for glycosaminoglycans and collagen type II indicating repair tissue with true constituents of articular cartilage. There was a statistical difference in the total histological scores at 3 months (P=0.028) with the best repair in the spontaneous repair group. A marked subchondral bone reaction, staining with toluidine blue and collagen type II, was seen in 65% of all animals. CONCLUSIONS: The spontaneous repair ability of full thickness cartilage defects of immature pigs is significant and periosteum or autologous chondrocytes do not bring any additional benefits to the repair.

Functional consequences of cartilage degeneration in the equine metacarpophalangeal joint: quantitative assessment of cartilage stiffness.

REASONS FOR PERFORMING STUDY: No quantitative data currently exist on the relationship of the occurrence of cartilage degeneration and changes in site-specific biomechanical properties in the metacarpophalangeal (MCP) joint in the horse. OBJECTIVES: To gain insight into the biomechanical consequences of cartilage deterioration at 2 differently loaded sites on the proximal articular surface of the proximal phalanx (P1). HYPOTHESIS: Static and dynamic stiffness of articular cartilage decreases significantly in degenerated cartilage. METHODS: Cartilage degeneration index (CDI) values were measured at the lateral dorsal margin (Site 1), lateral central fovea (Site 2) and entire joint surface of P1 (CDIP1) in 30 horses. Group 1 contained joints without (CDIP1 values <25 %, n = 22) and Group 2 joints with (CDIP1 values >25 %, n = 8) signs of cartilage degeneration. Cartilage thickness at Sites 1 and 2 was measured using ultrasonic and needle-probe techniques. Osteochondral plugs were drilled out from Sites 1 and 2 and subsequently tested biomechanically in indentation geometry. Young's modulus at equilibrium and dynamic modulus were determined. RESULTS: Cartilage thickness values were not significantly different between the 2 groups and sites. Young's modulus at Site 1 was significantly higher in Group 1 than in Group 2; at Site 2, the difference was not significant. Dynamic modulus values were significantly higher in Group 1 than in Group 2 at both sites. CONCLUSIONS: Degenerative cartilage changes are clearly related to loss of stiffness of the tissue. Absolute changes in cartilage integrity in terms of CDI are greatest at the joint margin, but concomitant changes are also present at the centre, with a comparable decrease of the biomechanical moduli at the 2 sites. Therefore, significant cartilage degradation at the joint margin not only reflects local deterioration of biomechanical properties, but is also indicative of the functional quality in the centre. POTENTIAL RELEVANCE: These findings may be important for improving prognostication and developing preventative measures.

Abnormal response to physical activity in femurs after heterozygous inactivation of one allele of the Col2a1 gene for type II collagen in mice.

The objective of this study was to evaluate the influence of heterozygous inactivation of one allele of the type II collagen gene (Col2a1) on biomechanical properties and mineral density of bone under physical loading conditions. C57BL/6-TGN mice with heterozygous knockout (HZK) inactivation of Col2a1 gene and their nontransgenic littermate controls were housed in individual cages with running wheels for 9 and 15 months. The running activity of each mouse was monitored continuously throughout the experiment. Bone mineral density (BMD) of mice femora was measured using dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT). Biomechanical properties were determined using three-point bending tests. Vertebral bone samples were prepared for quantitative polarized light microscopy and digital densitometry of proteoglycans. The concentration of total collagen and collagen cross-links were analyzed using high-performance liquid chromatograpy (HPLC). The average daily running distance was shorter for the HZK mice between the age of 4 and 15 months as compared with normal runners (P < 0.05). The ultimate breaking force was 14.8% and 23.6% (9 vs. 15 months) lower in HZK-runners than in wild-type runners. BMD of the femur was 6.1% lower in HZK-runners at the age of 9 months (P < 0.05). Physical activity increased cortical BMD in wild-type runners but not in the HZK runners at the age of 9 months. The collagen network of the HZK mice was less organized. There were only minor changes in BMD and mechanical and structural properties between sedentary HZK mice and their wild-type controls. Increased physical activity induced significantly lower bone density, mechanical properties, and organization of collagen fibers in male HZK mice. However, there were no major differences in biomechanical parameters between sedentary HZK and wild-type male mice. This suggests an important guiding role of collagen type II in bone remodelling and maturation.

Functional adaptation of articular cartilage from birth to maturity under the influence of loading: a biomechanical analysis.

REASONS FOR PERFORMING STUDY: The concept of functional adapatation of articular cartilage during maturation has emerged from earlier biochemical research. However, articular cartilage has principally a biomechanical function governed by joint loading. OBJECTIVES: To verify whether the concept of functional adaptation can be confirmed by direct measurement of biomechanical properties of cartilage. HYPOTHESIS: Fetuses have homogeneous (i.e. site-independent) cartilage with regard to biomechanical properties. During growth and development to maturity, the biomechanical characteristics adapt according to functional (loading) demands, leading to distinct, site-dependent biomechanical heterogeneity of articular cartilage. METHODS: Osteochondral plugs were drilled out of the surface at 2 differently loaded sites (Site 1: intermittent impact-loading during locomotion, Site 2: low-level constant loading during weightbearing) of the proximal articular cartilage surface of the proximal phalanx in the forelimb from stillborn foals (n = 8), horses of age 5 (n = 9) and 18 months (n = 9) and mature horses (n = 13). Cartilage thickness was measured using ultrasonic, optical and needle-probe techniques. The osteochondral samples were biomechanically tested in indentation geometry. Young's modulus at equilibrium, dynamic modulus at 1 Hz and the ratios of these moduli values between Sites 1 and 2 were calculated. Age and site effects were evaluated statistically using ANOVA tests. The level of significance was set at P<0.05. RESULTS: Fetal cartilage was significantly thicker compared to the other ages with no further age-dependent differences in cartilage thickness from age 5 months onwards. Young's modulus stayed constant at Site 1, whereas at Site 2 there was a gradual, statistically significant increase in modulus during maturation. Values of dynamic modulus at both Sites 1 and 2 were significantly higher in the fetus and decreased after birth. Values for both moduli were significantly different between Sites 1 and 2 from age 18 months onwards. The ratio of values between Sites 1 and 2 for Young's modulus and dynamic modulus showed a gradual decrease from approximately 1.0 at birth to 0.5-0.6 in the mature horse. At age 18 months, all values were comparable to those in the mature horse. CONCLUSIONS: In line with the concept of functional adaptation, the neonate is born with biomechanically 'blank' or homogeneous cartilage. Functional adaptation of biomechanical properties takes place early in life, resulting in cartilage with a distinct heterogeneity in functional characteristics. At age 18 months, functional adaptation, as assessed by the biomechanical characteristics, has progressed to a level comparable to the mature horse and, after this age, no major adaptations seem to occur. POTENTIAL RELEVANCE: Throughout life, different areas of articular cartilage are subjected to different types of loading. Differences in loading can adequately be met only when the tissue is biomechanically adapted to withstand these different loading conditions without injury. This process of functional adaptation starts immediately after birth and is completed well before maturity. This makes the factor of loading at a young age a crucial variable, and emphasises the necessity to optimise joint loading during early life in order to create an optimal biomechanical quality of articular cartilage, which may well turn out to be the best prevention for joint injury later in life.

High hydrostatic pressure induces ERK and PI3 kinase phosphorylation in human HCS-2/8 chondrosarcoma cells.

High continuous hydrostatic pressure has been shown to affect many cellular functions within the pressurised cells, for instance, accumulation of heat shock protein 70 occurs during pressurisation. Various signal transduction pathways are likely to mediate these changes, however, at the present time our knowledge of the pathways involved is rather limited. The aim of this study was to investigate whether some of the well known transduction pathways are activated by the exposure of human chondrosarcoma cells to 15-30 MPa hydrostatic pressure. The results showed an increased presence of the active, phosphorylated forms of extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K) in cells exposed to 15 and 30 MPa continuous hydrostatic pressure, while 0.5 Hz cyclic loading had weaker effects. Inhibition of ERK-pathway with UO126 did not prevent the accumulation of heat shock protein 70. No activation of c-Jun N-terminal protein kinase (JNK) or p38 could be noticed in pressurised cells. In conclusion, we could identify at least two different signal transduction pathways that are activated under high continuous hydrostatic pressure. Accumulation of heat shock protein 70 was independent of ERK-activation.

Subchondral bone reaction associated with chondral defect and attempted cartilage repair in goats.

Repair of cartilage damage with autologous chondrocyte transplantation (ACT) has become popular in clinical use during the past few years. Although clinical results have mostly been successful, several unanswered questions remain regarding the biological mechanism of the repair process. The aim of this study was to develop a goat model for ACT. The repair was not successful due to the graft delamination, but we characterize the subchondral changes seen after the procedure. A chondral lesion was created in 14 goat knees, operated on 1 month later with ACT, and covered with periosteum or a bioabsorbable poly-L/D-lactide scaffold. After 3 months, only two of the five lesions repaired with ACT showed partly hyaline-like repair tissue, and all lesions (n = 4) with the scaffold failed. Even though the lesions did not extend through the calcified cartilage, the bone volume and collagen organization of bone structure were decreased when assessed by quantitative polarized light microscopy. There was a significant loss of bone matrix and distortion of the trabecular structure of subchondral bone, which extended several millimeters into the bone. The subchondral bone demonstrated strong hyaluronan staining in the bone marrow and cartilaginous areas with signs of endochondral ossification, suggesting structural remodeling of the bone. The goat model used here proved not to be an optimal model for ACT. The changes in subchondral bone may alter the biomechanical properties of the subchondral plate and thus the long-term survival of the repair tissue after ACT.

Analysis of lapine cartilage matrix after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate.

OBJECTIVE: To study the short and long term effects of radiosynovectomy on articular cartilage in growing and mature rabbits. METHODS: The articular cartilage of the distal femurs of rabbits was examined four days, two months, and one year after radiosynovectomy with holmium-166 ferric hydroxide macroaggregate ([(166)Ho]FHMA). Arthritic changes were evaluated from histological sections by conventional and polarised light microscopy, and glycosaminoglycan measurements using safranin O staining, digital densitometry, and uronic acid determination. Proteoglycan synthesis was studied by metabolic [(35)]sulphate labelling followed by autoradiography, and electrophoretic analysis of extracted proteoglycans. Northern analyses were performed to determine the mRNA levels of type II collagen, aggrecan, and Sox9 in cartilage samples. RESULTS: Radiosynovectomy had no major effect on the histological appearance of articular cartilage in mature rabbits, whereas more fibrillation was seen in [(166)Ho]FHMA radiosynovectomised knee joints of growing rabbits two months after treatment, but not after one year. Radiosynovectomy did not cause changes in the glycosaminoglycan content of cartilage or in the synthesis or chemical structure of proteoglycans. No radiosynovectomy related changes were seen in the mRNA levels of type II collagen, whereas a transient down regulation of aggrecan and Sox9 mRNA levels was seen in young rabbits two months after [(166)Ho]FHMA radiosynovectomy. CONCLUSIONS: [(166)Ho]FHMA radiosynovectomy caused no obvious chondrocyte damage or osteoarthritic changes in mature rabbits, but in growing rabbits some transient radiation induced effects were seen--for example, mild cartilage fibrillation and down regulation of cartilage-specific genes.

Lifelong voluntary joint loading increases osteoarthritis in mice housing a deletion mutation in type II procollagen gene, and slightly also in non-transgenic mice.

OBJECTIVES: To investigate the effects of voluntary running on the incidence and severity of osteoarthritis (OA) and associated changes in cartilage matrix and subchondral bone in a transgenic Del1 mouse model for OA. METHODS: Del1 mice and their non-transgenic littermate controls were housed from the age of 5-6 weeks to 15 months in individual cages with running wheels. The running activity of each mouse was monitored for the entire 12 month period. Additional Del1 and control mice were housed in individual cages without running wheels. At the end of the experiment the severity of OA was evaluated by light microscopy, and the articular cartilage matrix changes by digital densitometry and quantitative polarised light microscopy. RESULTS: Lifelong voluntary running increased the incidence and severity of OA significantly in Del1 mice (transgenic runners), and slightly also in non-transgenic runners. Severe OA changes increased from 39% in transgenic non-runners to 90% in transgenic runners (p=0.006) in lateral tibial condyles, and from 24% to 80% (p=0.013) in lateral femoral condyles, respectively. The proteoglycan content of articular cartilage was reduced in transgenic runners in comparison with transgenic non-runners (p=0.0167), but a similar effect was not seen in non-transgenic runners compared with non-transgenic non-runners. No attributable differences were seen in the collagen network of articular cartilage or in the subchondral bone between any of the groups. CONCLUSION: The Del1 mutation has earlier been shown to disturb the assembly of the cartilage collagen network and thereby increase the incidence and severity of OA with age. In this study, voluntary running was shown to increase further cartilage damage in the lateral compartments of the knee. This suggests that articular cartilage in Del1 mice is less resistant to physical loading than in control mice. Despite severe OA lesions in the knee joint at the age of 15 months, Del1 mice continued to run voluntarily 2-3 km every night.

Transgenic mouse models for studying the role of cartilage macromolecules in osteoarthritis.

The development of transgenic technology has made possible the generation of targeted gene-mutated mouse lines suitable for use in experimental osteoarthritis (OA) research. Transgenic mice harbouring mutations in cartilage collagen types II and IX develop early-onset OA and are therefore promising models of age-related OA, even though the mice often show signs of chondrodysplasia. Also, mouse lines harbouring other engineered mutations of the extracellular molecules have given rise to early OA. The molecular background of a few spontaneous mutations in mice has also been clarified and the characterization of the OA phenotype is now in progress. These mutations cause severe chondrodysplasia and death in homozygous mice, but the heterozygous offspring develop the early-onset OA phenotype.

Ultrasonic characterization of articular cartilage.

Osteoarthrosis is the most important joint disease that threatens health of the musculoskeletal system of elderly people. Today, there is a need for sensitive, quantitative diagnostic methods for successful and early diagnosis of the disorder. In the present study, we aimed at evaluating the applicability of ultrasound for quantitative assessment of cartilage structure and properties. Bovine articular cartilage was investigated both in vitro and in situ using high frequency ultrasound. Cartilage samples were also tested mechanically in vitro to reveal relationships between acoustic and mechanical parameters of the tissue. The collagen organization and proteoglycan content of cartilage samples were mapped, using quantitative polarized light microscopy and digital densitometry, respectively, to reveal their effect on the acoustic properties of tissue. The high frequency pulse-echo ultrasound (20-30 MHz) technique proved to be sensitive in detecting the degeneration of the superficial collagen-rich cartilage zone. In addition, ultrasound was found to be a potential tool for measuring cartilage thickness. When the results from biomechanical indentation measurements and ultrasound measurements of normal and enzymatically degraded articular cartilage were combined, collagen or proteoglycan degradation in the tissue could be sensitively and specifically differentiated from each other. To conclude, high frequency ultrasound is a useful tool for evaluation of the quality of superficial articular cartilage as well as for the measurement of cartilage thickness. Therefore, ultrasound appears to be a valuable supplement to the mechanical measurements of articular cartilage stiffness.

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.